Radioimmunoassay in The Clinical Chemistry Laboratory

RADIOIMMUNOASSAY IN THE CLINICAL CHEMISTRY LABORATORY

. .

J P Felber Division de Biochimie Clinique. Departement de M6decine. Centre Hospitalier Universitaire Vaudois. Switzerland

1. Introduction ........................................................... 1.1. Principle and Scope of Application of Radioimmunoassays . . . . . . . . . . References ............................................................ 2. Antigen ............................................................... Reference ............................................................. 3 Antibody . . . . . . . . . . . . . . ............................................ 3.1. Antigen as Immuno ......................... 3.2. Immunization . . . . . . . . ......................... 3.3. Assessment of the Antisera ........................................ References ............................................................ 4 . Labeled Antigen ........................... ....................... 4.1. Specific Activity of the Labeled Antigen ...................... 4.2. Iodination Procedures ............................................ 4.3. Purification of the Labeled Antigen ................................ 4.4. Assessment of the Labeled Antigen ration .............. 4.5. Storage of the Labeled Antigen . . . .................... References ............................................................ 5. Incubation ............................................................ Reference ............................................................. 6. Separation Procedures ................................................. 6.1. Methods for Separating the Antibody-Bound Antigen from the Incubation Mixture ............................................... 6.2. Methods for Separating the Free Antigen from the Incubation Mixture References ....................................................... 7. Measurement of Radioactivity .......................................... 8. Calculation of Results .................................................. References ............................................................ 9. Quality Control ..................... ............................... 10. Application of Radioimmunoassay ....................................... 10.1. Radioimmunoassay of Small Peptide Hormones . . . . . . . . . . . . . . . . . . . 10.2. Radioimmunoassay of Larger Protei mones . . . . . . . . . . . . . . . . . . . 10.3. Radioimmunoassay of Steroids . . . . .......................... 10.4. Radioimmunoassay of Enzymes .................................. References ............................................................

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130 130 133 133 134 134 135 136 137 141 142 143 143 145 146 146 147 148 150 150 151 157 163 165 165 168 168 169 169 173 175 176 177

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J. P. FELBER 1.

Introduction

1.1. PRINCIPLE AND SCOPE O F APPLICATION OF RADIOIMMUNOAS SAYs The radioimmunological method is based on the principle of the competition between an unlabeled and a labeled antigen for a specific antibody in limited concentration. It was first developed b y Yalow and Berson (Y 1) for the measurement of insulin concentration in plasma, on the basis of previous studies ( B l ) on the quantitative aspects of the reaction between insulin and insulin-binding antibody. Later it was extended first to other polypeptide hormones and then to many other substances which, as antigens or haptens, can produce antibodies and bind to these antibodies. In the radioimmunoassay system, the antigen and its specific antibody form a soluble antigen-antibody complex. The process is reversible. Ag+Ab

(1)

e AgAb

where Ag represents antigen, Ab antibody, and AgAb the complex of antigen with antibody. A similar reaction is obtained by using a labeled antigen (Ag*) Ag*

+ Ab

e Ag*Ab

(2)

In this case, the Ag*Ab complex (complex of labeled antigen with antibody) possesses the radioactivity of the labeled antigen bound to the antibody. The addition of unlabeled antigen (Ag) to this last reaction (2) produces competition between the unlabeled (Ag) and the labeled antigen (Ag*) for the binding sites of the antibody (Ab) if the antibody is in limited concentration.

\

(3)

concen (limit twtion)

AgAb

The principle of radioimmunoassay is that of competitive inhibition of the binding of labeled antigen (Ag*)to a specific antibody (Ab) by an unlabeled antigen (Ag). The higher the concentration of the unlabeled antigen (Ag), the lower will be the radioactivity of the antigen-antibody complex (Ag*Ab) and the higher that of the free labeled antigen (Ag").

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In the radioimmunoassay system, the unlabeled antigen is represented by the known standard solution or by unknown samples. The concentration of antigen in an unknown sample is determined by comparing the degree of inhibition produced by this unknown sample concentration with that produced by a known concentration of the same substance used as a standard. The radioactivity of the antibody-bound labeled antigen must be separated from that of the free labeled antigen and counted for radioactivity. In a graph where the radioactivity of both free and antibody-bound labeled antigens are measured in function of the concentration of the unlabeled antigen, the radioactivity of the antibodybound antigen decreases while that of the free antigen increases as the concentration of the unlabeled antigen increases (Fig. 1).The two curves are complementary, since the addition of the radioactivity of the free and the antibody-bound antigens is constant and represents the total radioactivity. The principle of the radioimmunoassay is in fact that of saturation analyses. Competition depends directly on the mass-law action. The higher the concentration of the unlabeled antigen, the lower the radioactivity of the labeled antigen-antibody complex. This is done by choosing the dilution of the antiserum for competition for the sites of the antibodies to exist between the unlabeled and the labeled antigen. In other words, antigens (labeled and unlabeled) are in excess over the antibodies, thus allowing competition between them for the sites of the antibodies. Any increase in the concentration of the unlabeled antigen will decrease the possibility for the labeled antigen to bind to the antibodies. The great advantage of the radioimmunological method resides in its high sensitivity, its high specificity, and in the possibility it offers to perform simultaneously a large number of determinations. The high sensitivity of the assay depends mainly on the high avidity of the antibody which can be chosen and, for a minor part, on the high specific activity of the labeled antigen. It is important to realize that the specificity of the assay is of immunological order. It is in no way related to the biological activity of the antigen. The measurement is based on the binding capacity of the antigen to the antibody independent of its biological activity. This is particularly important in the case of hormone or enzyme measurements, since the assay may measure inactive molecules if the molecular structure that is recognized by the antibody is identical with that of the active antigen. Over the past years, the use of the radioimmunological method has been extended to almost any type of molecule for which specific an-

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S A Q ~0 0 0 0 0 0 OAgO

6Ag' 2Ag"

0 0 0 0 0 0 0 0

- QQ -

+

0

0 0

2

4

6

8

I0

0 0

12

[A0"]

FIG. 1. Radioactivity of the free-labeled antigen (Ag*) and of the antibody-bound labeled antigen (Ag*Ab) as a function of the concentration of the unlabeled antigen (Ag").

tibodies can be produced. Since almost any substance is or can be rendered immunogenic the field of application of radioimmunology is IikeIy to reach broad extension. It is already applied to measurements of polypeptide or steroid hormones, enzymes, tumor antigens, circulating antibodies, viruses, and drugs.

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The development of a radioimmunoassay requires antigen in a pure state, specific antisera, and a labeled antigen used as tracer. Separation methods are needed to separate the free from the antibody-bound antigen. The radioactivity of one or the other of these two species is measured independently. REFERENCES B1. Berson, S. A., and Yalow, R. S., Quantitative aspects of the reaction between insulin

and insulin-binding antibody. J. Clin. Inoest. 38, 1996-2016 (1959). Y1. Yalow, R. S . , and Berson, S. A., Ininiunoassay ofendogenous plasma insillin in man. J. Clin.Invest. 39, 1157-1175 (1960).

2.

Antigen

The antigen may be any substance which can bind to specific antisera. It may be small or large polypeptides, proteins with or without hormonal or enzymic activity, steroids, prostaglandins, drugs, etc. Differing from biological assays, which can make use of impure substances as long as they possess biological activity, a basic requirement for radioimmunoassays is the need of pure substances to use as a standard and labeled compound. Any impurity is liable to interfere with the assay if the antibodies used were raised with the same impure antigen. When antigens are not commercially available in the pure state, chromatographic steps are needed in order to obtain pure antigens from less pure preparations. The purity of the antigens must be verified, and repurification performed if necessary. Several small peptide hormones are now obtained b y chemical synthesis. This has been an asset in the development of the radioimmunoassay, since it provides rare antigens in sufficient amounts. However, synthesis does not mean that the peptide is pure. Impurities, which are often closely related “ error” peptides, may cause major interference in the assay determination. It is essential for the antigen used as a standard to be identical with the unknown antigen to be measured. Differences in the molecular structure would produce differences between the standard and the unknown in the binding to the antibody, and therefore yield erroneous results. This is the case when the standard used in the measurement does not originate from the same animal species as the unknown. For instance, the binding constant between antisera raised against porcine ACTH is usually higher with porcine ACTH used as a standard than with human ACTH to be determined for clinical purposes. The binding constants, however, should be identical when the immunological determinants of the antiserum bind only to the amino acid sequence

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J. P. FELBER

which is common to ACTH from both species (amino acid sequence

1-24).

Often there is no cross-reaction between biochemical substances from one animal species and antisera raised against the same substance from another species, This is the case, for instance, for human growth hormone or human FSH which can be determined only b y using antisera raised against human hormones. This specificity may also exist in the case of enzymes from different organs from the same individual. For example, rabbit muscle fructose 1,8diphosphatase was shown not to cross-react with the homologous rabbit liver enzyme for the antiserum to liver fructose 176-diphosphatase(Kl). The problem of specificity is acute for the determination of substances which are closely related in structure, as for instance, glucoproteic hormones (TSH, LH, FSH, HCG) which share a similar a-subunit or hormones with identical amino acid sequences, such as a-MSH, P-MSH, and ACTH, or gastrointestinal hormones from one or another “family.” The specificity of the assay, in these cases, depends essentially on the specificity of the antiserum. Often, steroids can be specifically determined only after prior chromatography, when antisera for one particular steroid do not exist.

REFERENCE K1. Kolb, H. J., and Grodsky, G. M., Biological and immunological activity of fructose 1,6diphosphatase. Application of a quantitative displacement radioimmunoassay. Biochemistry 9, 4900-4906 (1970).

3.

Antibody

The antibody is the key of the radioimmunoassay. On the antibody depend both sensitivity and specificity of the assay. It is at the center of the competition between unlabeled and labeled antigen for its binding sites. The sensitivity of the assay depends above all on the avidity (or affinity) of the antibody for the antigen. Avidity (or affinity) is an expression of the energy of binding of the antibody-antigen reaction. The law of mass action can be applied to the reaction between the combining sites of an antibody and its specific antigen. The term “avidity” (or affinity) is used to denote the energy of the reaction and is essentially the same as the equilibrium constant of association (K)in physical chemistry with K

= [AbAgl/[Ab][Agl

(4)

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The energy of binding for any individual antibody is determined b y the complementary relationship between the antigenic determinant of the hormone and the combining site of the antibody molecule. One must realize that antisera usually contain a population of various antibodies. The avidity measured is therefore an average of a large number of values. The specificity of the radioimmunoassay depends on the specificity of the antiserum used in the assay. The specificity of the antibodies contained in the antiserum is based on the reaction between the combining sites of the antibody and the antigenic determinants of the antigen. A specific antiserum should recognize the antigenic determinant, i.e., an amino acid sequence and a tertiary structure proper to one specific antigen, with the exclusion of the antigenic determinants of all other substances, even those with close structural similarities. In the radioimmunoassay, there is usually no formation of precipitating antigen-antibody complexes as in other immunological methods. The antigen-antibody complex is soluble, since the high dilution of the constituents prevents the formation of a precipitating network. Antibodies are formed in the lymphoid tissue. At the beginning of immunization, macroglobulins (IgM) are formed, whereas immunogamma globulins (IgG) are characteristic of secondary responses. 3.1 ANTIGENAS

IMMUNOGEN

The immunogenicity of a peptide molecule depends on its size and on the difference in molecular structure with the molecules naturally occurring in the injected animal. In contradistinction to the antigen used as a standard or for labeling, there is usually no need for the immunogen to be absolutely pure. A degree of purity of from 5 to 10%is sufficient. It has even been shown that impure substances are often better immunogens than pure products, since impurities may have an adjuvant effect (B2). However, pure antigens are required when there is need to produce highly specific antisera to distinguish closely related substances, or when a high degree of purity is desired in synthetic materials because of possible contamination with closely related error peptides. Polypeptides of more than 10 amino acids are generally injected as such for immunization. Small polypeptides, or nonimmunogenic substances such as steroids, drugs, etc., have to be coupled in order to become immunogenic. They are usually coupled to a substance which is immunogenic as such. Bovine serum albumin, bovine thyroglobulin, human gamma globulin or synthetic poly-(L) lysin are most often

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J. P. FELBER

used for this purpose. Even when the antigen exists as a substance occurring naturally in the immunized animal, antibody production is possible when powerful adjuvants and coupling are used. Several coupling reagents have been proposed to couple non- or poorly immunogenic substances to a protein support. Among them, carbodiimide (Gl)and glutaraldehyde (Rl)are most commonly used. The specificity of the antiserum depends in part on the position taken by the coupled antigen in the complex formed with the immunogenic support. The antiserum is usually specific for the portion of the antigen which is in opposition to the site bound to the proteic support. This is particularly true for steroids. Carbodiimide coupling can be performed as follows: 0.25 ml of a solution containing 100-200 mg l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, freshly dissolved in distilled water, is added to a mixture of 20 mg of the hapten and 10 mg of bovine serum albumin dissolved in 0.5 ml HzO.The reaction is allowed to proceed with gentle agitation at room temperature for 5 to 30 minutes and is terminated by dialysis for 24 to 72 hours to remove the reactants. Glutaraldehyde coupling has been first proposed for the conjugation of ACTH to bovine serum albumin (Rl).Six mg porcine ACTH and 20 mg bovine serum albumin (Armour Fraction V) are first dissolved in 2 ml 0.1M phosphate buffer, pH 7.0. One ml glutaraldehyde solution, 0.021 M ,is then added in drops with constant stirring. The solution is diluted with isotonic saline.

3.2. IMMUNIZATION Immunization is most commonly performed in rabbits or guinea pigs, unless larger animals, usually goats, are required to produce large batches of antisera. The immunogen, either the pure or impure product, or the coupled material, is dissolved in buffer and emulsified with 2 to 3 parts of complete Freund's adjuvant. Emulsion is done by using a small mixer or more simply by rapidly filling and emptying a syringe (with the needle) with the mixture of aqueous and oily components. As a rule, small amounts of substance are used for immunization, usually between 10 p g and 1 mg per animal, and the injections are repeated at low frequency, not more than once a month. In the rabbit, the immunogen is injected as a freshly prepared emulsion in F r e u n d s adjuvant, in the upper part of the four legs (1-3 ml altogether). The following injections are carried out at one month's intervals alternately in the front and the rear legs. In the guinea pig, the immunogen is injected subcutaneously, 0.25-0.5ml altogether, in

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the upper part of the legs or in the abdominal wall. Injections in the foot pads should be avoided because they are painful to the animal. Bleeding is performed 10-14 days after the last injection, to test or harvest the antiserum. In the rabbit, blood is removed from the ear veins or by cardiac puncture (up to 40 ml). In the guinea pig, it is taken from the paws, at the root of the nails, or b y cardiac puncture (4-8 ml). AAer the first 3 months, injections and bleeding can continue at 2- to 3-month intervals. A simple and successful procedure has been suggested b y Vaitukaitis et al. (Vl). After shaving the rabbit’s fur on the back and lateral surfaces, the immunogen emulsion is injected intradermally into from 40 to 50 sites over a considerable part of the body surface. Booster injections can be given after 40 days, but usually bring no further improvement. The animals develop extensive skin ulceration, but appear to remain in good condition. Usually antisera with high titer are obtained after 60 days from the first and usually unique multiple-site injection. In some cases, it is possible to improve the avidity of the antisera by separating the highly avid antibodies by means of affinity chromatography ( C l , S l ) .

3.3. ASSESSMENT OF

THE

ANTISERA

Except in the case where the immunogen is coupled to a protein in a chosen position, there are few possibilities, during immunization, to direct the specificity and avidity of the future antisera. The major task, in raising antisera, resides in their assessment. Once harvested, the antisera must be tested for their titer, avidity, and specificity in order to select the most suitable one. The antibody titer is usually defined as the final dilution of an antiserum in the incubation mixture required to bind the appropriate amount of labeled antigen in the absence of unlabeled antigen (Hl).It is estimated by an antibody titration curve, obtained by varying the antiserum dilutions in the radioimmunoassay in the presence of a constant concentration of labeled antigen and in the absence of any added unlabeled antigen. In practice, the antiserum dilution is successively doubled, staiting from an initial 1/2OO or 1/1OOO dilution ofthe collected antiserum. The labeled antigen is added to the incubation medium. The unlabeled antigen is replaced by buffer. After the end of incubation, the antibody-bound labeled antigen is separated from the free labeled antigen and measured for radioactivity. The radioactivity plot of the antibody-bound fraction in ordinate versus the antiserum dilution in the abscissa forms an S-shaped curve (Fig. 2).

138

J. P. FELBER 'lo BINDING

0 , (/lo0

l/200

'

l/EOD

l/JOO

1/3200 I

I

l/t;OO

1/12800 I

1/51200 1

l/&lOO V2:SOO ANTISERUM DILUTION

FIG.2. Antiserum titration curve. Radioactivity of the antibody-bound labeled antigen as a function of the dilution of the antiserum, in the absence of unlabeled antigen.

This S-shaped curve is useful to indicate the dilution at which the antiserum should be employed to get the best sensitivity of the radioimmunoassay. In the upper part of the curve, there is an excess of antibody which results in low sensitivity, as all the sites of the antibody are occupied by the labeled antigen. High sensitivity resides within the range of from 20 to 70% binding. Within this range, the limited concentration of the antibody allows competition to take place between the unlabeled and the labeled antigens for the binding sites of the antibody. The avidity of the antiserum is a measure of the sensitivity of the assay system. It is reflected by the steepness of the descending part of the antibody titration curve (Bl), although this is by no means a good criterion (Hl). A practical way to assess the avidity of an antiserum is to compare, in the same assay, two antibody titration curves, one with the addition of a small concentration of unlabeled antigen, the other without. Antisera with high avidity show a wide separation between the

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two curves (Fig. 3). The best antiserum can be retested by means of

standard curves, the lowest detectable concentration representing the limit of sensitivity of the antiserum (Fig. 4). The speci$city of the antiserum derives from the structure of the molecules that have served as immunogen. However, the nature of the combining sites may vary from one antibody to another, even though the same antigen may have served for immunization. The specificity of the antiserum may be directed toward one or another segment of the immunogen molecule. This is of importance if a structure similar to that recognized by the antibody exists in other closely related antigens. Antisera are usually heterogeneous, since they often contain a population of different antibodies. The specificity of the antiserum is therefore a resultant of the antibodies it contains. Affinity chromatography may be used to enhance the specificity of some antisera by selecting the specific antibody ( S l ) . The specificity of an antiserum is tested b y performing crossreaction studies with related materials. This is of major importance in %total bound 100

75-

50-

25.

01

I

111112

1heoeo

vzwaoo

antiserum dilution

FIG.3. Antiserum dilution curve, in the absence (-,

antiserum alone) and in the presence (---, antiserum with added unlabeled antigen) of a fixed concentration of unlabeled antigen.

140

J. P. FELBER

01 0

D

Added Antigen Conc.

FIG.4. Standard curve. In ordinate: ratio ot‘the radioactivity of the antibody-bound labeled antigen in the presence of unlabeled antigen ( B ) over the radioactivity of the antibody-bound labeled antigen in the absence of unlabeled antigen ( B u )x 100. In abscissa, an increasing concentration of added unlabeled antigen. The sensitivity of the method is related to the error.

the case, for instance, of the different glucoproteic hormones (TSH, LH, FSH, HCG) which possess, apart from the specific P-chain, an cr-chain which is analogous for all four hormones. A similar situation exists in regard to gastrointestinal hormones which belong to one or another structural “family,” or to the different steroids which possess the same basic structure. Cross-reactivity is tested by means of standard curves in which the antigen to be assayed is compared with the other substances to be tested for cross-reactivity. A specific antiserum should show full displacement for the correct antigen and no displacement for the other substances (Fig. 5). The percentage of crossreactivity of the related material is calculated by the ratio of the mass of this related material required to displace 50% total binding (binding in the absence of added unlabeled antigen) over the mass of the specific material, to achieve the same displacement.

RADIOIMMUNOASSAY IN THE LABORATORY

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50

40 CII

t

\

D C

-

n 30

;r

20

10

I

20

,

40

I

1

I

60

80

100

ng/mi

FIG.5 . Search for the specificity of the assay. Standard curve for the antigen to be measured ( X-x , trypsin) with no cross-reaction with related antigens (chymotrypsin 0---0,and chymotrypsinogen A 0---0).

REFERENCES €31. Berson, S. A., and Yalow, R. S., Inimunoassay of protein hormone. In “The Hormones” (G. Pincus, K. V. Thimann, and E. B. Astwood, eds.), Vol. 4, pp 557-630. Academic Press, New York, 1964. B2. Berson, S. A., and Yalow, R. S., Radioinimunoassay. I n “Methods in Investigative and Diagnostic Endocrinology” (S. A. Berson and R. S. Yalow, eds.), Vol. 2A, pp. 84-135. North-Holland Publ., Amsterdam and Am. Elsevier, New York, 1973. C1. Cuatrecasas, P., Insulin-Sepharose: Iinmunoreactivity and use in the purification of antibody. Biochem. Biophys. Res. Commun. 35, 531-537 (1969). G1. Goodfriend, T. L., Levine, L., and Fasman, G. D., Antibodies to bradykinin and angiotensin: Ause ofcarbodiimides in immunology.Science 144,1344-1346( 1964). H1. Hum, B. A. L., and Landon, J., Antisera for radioimmunoassay. In “Radioimmunoassay Methods” (K. E. Kirkham and W. M. Hunter, eds.), pp. 121-147. Churchill Livingstone, Edinburgh and London, 1971. R1. Reichlin, M., Schnure, J. J., and Vance, V. K., Induction of antibodies to porcine

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ACTH in rabbits with nonsteroidogenic polymers of BSA and ACTH. Proc. Soc. E x p . Biol. Med. 128, 347-350 (1968). S1. Sato, N., and Cargille, C. M., Separation of specific from nonspecific anti-FSH antibody by affinity chromatography on sepharose-HCG. Endocrinology 90,302306 (1972). V1. Vaitukaitis, J., Robbins, J. B., Nieschlag, E., and Ross, G. T., A method for producing specific antisera with small doses of immun0gen.J. Clin. Endocrinol. 33,988991 (1971).

4. Labeled Antigen

The third component of the reaction that takes place in the radioimmunoassay is the labeled antigen, often called “the tracer.” The principle of radioimmunoassay is such that the concentration of labeled antigen has to be of the same order of magnitude (plus or minus tenfold) as that of the antigen to be measured, in order to allow competition between labeled and unlabeled antigen to take place for the combining sites of the antibody. The quality of labeling is of greai importance. The antigen used for labeling must be chemically pure, as the antigen serving as the standard. It also has to keep, after labeling, its antigenic properties to bind to the antibody. However, there is no need for complete identity with the antigen used as the standard as according to the general principle of the radioimmunoassay method, comparison is made between the unknown and the standard for the inhibition of labeled antigen to antibody. This is of basic importance in radioimmunology, since labeling modifies the molecule of the antigen when the radioisotope is added. The isotopes most commonly used are 12jI, I3lI, ’%, and SH. 3H is used mainly in radioimmunoassays of steroids and of some drugs. ItaI of having a much higher and “‘I offer the advantage, over “H and degree of specific activity. Radioiodine is generally used for labeling polypeptides and proteins. The theoretical specific activity of l3’I is higher than that of 12‘1 (125 vs 17.4 mCi/pg). However, 12‘1 is generally preferred to la’I for its longer half-life (60 days vs. 8 days), its higher counting efficiency (90%vs 45% in a NaI (Ti) Well detector) and its higher isotopic abundance in commercial supplies (95%vs less than 25% for I3’I). Labeled iodine is generally placed on the tyrosine residues of the various antigens. When antigens lack tyrosine, tyrosine or a larger molecule containing tyrosine or histamine is conjugated to the antigen before or after labeling. For instance, Goodfriend and Ball (G2) conjugated a desaminotyrosyl group directly to the N-terminal arginyl group of bradykinin. Newton et al. (N2) conjugated the gastrin tetrapeptide moiety to a random copolymer of tyrosine, alanine, and

RADIOIMMUNOASSAY IN THE LABORATORY

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glucamic acid, and used this conjugate for radioiodination. When synthetic polypeptides are used, a tyrosine molecule is often added during synthesis. I n the case of steroids, steroid protein conjugates have been proposed, particularly human serum albumin conjugates, which can be labeled by any standard iodination techniques (Jl).

4.1 SPECIFIC ACTIVITY OF THE LABELEDANTIGEN One atom per mole of I2’I yields a specific activity of approximately 1000 pCi/pg when the molecular weight of the antigen is 2000 and, therefore of approximately 100pCi/pg when it is 20,000.As mentioned

below, enrichment of the specific radioactivity is possible with very small polypeptides when separation of the labeled from the unlabeled peptide can be achieved by means of chromatography. A high specific activity is chosen for very sensitive assays, which helps decrease statistical errors in the counting rate. However, if an increase in the iodination of the molecule raises the specific activity, overiodination, on the contrary, decreases the affinity of the molecule for the antibody. The increase in iodination is limited b y the necessity for the antigen molecules not to accept more than one radioactive iodine atom per molecule ( B l ) , since overiodination leads to degradation of the labeled molecule ( B l ) . Because all molecules are not equally labeled, labeling with less than a 0.5 atom per mole of protein is needed to prevent overiodination.

4.2. IODINATION PROCEDURES Iodination includes oxidation of the labeled iodine, which is supplied as Na+I-, with binding of the positively charged labeled iodine to the antigen. It is immediately followed b y purification of the labeled product from unreacted iodine and from fractions of the labeled antigen damaged during oxidation.

4.2.1. Chloramin T Method The most widely used method for labeling is that using Chloramin T, first described by Hunter and Greenwood ( H l ) and Greenwood et ul. (G3). Chloramin Tis the sodium salt of the N-monochloro derivative

of p-toluene sulfonamide which, in an aqueous solution, slowly forms hypochlorous acid, a mild oxidant. The reaction is optimal at p H 7.5. It allows iodination of veiy small quantities of polypeptides or proteins (1-5 pg). The total volume of the reaction must be kept to a minimum, since the degree of incorporation of radioiodine into the antigen de-

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J. P. FELBER

pends on the concentration of the reactants. A typical procedure is given below. Solutions of antigen, Chloramin T, and sodium metabisulfite are prepared in a 0.05 M sodium phosphate buffer, pH 7.5. The reagents are added into a small reaction vial, in the following order: Na""1, 0.8-2 mCi (or Na""1, 2-5 mCi), in 5-20 p1 of buffer; 0.5 M Na phosphate buffer, pH. 7.5, 10-20 pl;2-5 p g antigen in 10 pl of buffer; and 50 p g Chloramin T, in 10 pl of buffer. Wait 10-60 seconds. Then add quickly in order to stop the oxidation reaction sodium metabisulfite, 100 p g in 100 pl. When a milder oxidation is required, as in the case of labeling of human prolactin, the quantity of Chloramin T can be de*' creased to 20 p g and the reaction time reduced to 3-5 seconds. Since in some cases the Chloramin T method results in a reduction of immunoreactivity, several modifications have been tried to reduce the damaging effect caused by the conditions of oxidation. Redshaw and Lynch ( R l ) have proposed to replace Chloramin T by an aqueous solution of chlorine or sodium hypochlorite. The authors were able to improve the binding to antisera when compared with antigens iodinated in parallel with Chloramin T. The method is similar to that of Chloramin T. It requires initial optimization of the oxidant concentration.

4.2.2. Lactoperoxidase Method Enzymic oxidation with the use of lactoperoxidase has been pro-

posed by Thorell and Johansson ( T l ) to specifically oxidize iodide without otherwise affecting the antigen. This method has allowed radioiodination of polypeptide hormones to take place with no significant changes in their immunological reactivity. Iodination can b e performed at room temperature in a small test tube with slow mixing, at pH between 3 and 8. The reagents diluted in buffer are added, in the following order: Na"51, 1 mCi into 10 pl; 5 p g of antigen into 25 pl; 4 p g of lactoperoxidase into 4 pl; and 0.88 mM H20, into 1p1. After avariable time ofreaction, the reaction is stopped b y dilution with buffer. The mixture is submitted to purification.

4.2.3. Conjugution Lubeling In order to avoid the difficulties associated with iodination of labile antigens, Rudinger and Ruegg (R2) and Bolton and Hunter (B2) have proposed to complex a labeled and purified intermediate with the antigen. The procedure includes preparation of a '2JI-labeled ester by the Chloramin T method, its purification from all oxidizing

RADIOIMMUNOASSAY IN THE LABORATORY

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and reducing agents, and conjugation of the ester to any free amino group of protein or polypeptide molecule. Iodinated p hydroxyphenylpropionic acid N-hydroxysuccinimide ester (the Bolton-Hunter reagent) is commercially available. It is convenient for labeling peptides or proteins whose immunologic reactivity is altered by iodination of tyrosyl groups, and for peptides lacking tyrosine. Conjugation labeling with an iodinated intermediate is also a general procedure used for steroid labeling. It offers the advantage over "H of producing tracers with a higher affinity for antibodies (Jl).The method is rather simple and precise. Coupling of ";'I-histamine has given good results (Jl).Histamine is first iodinated, subsequently conjugated to the steroid and the tracer is then purified by thin-layer Chromatography, thus insuring high specific activities. The tracer can be stored at +4"C in ethanol and remains stable for several months. Detailed procedures have been described for the preparation of a '""I-histamine-estradiol-oxime conjugate by Nars and Hunter (N 1)and Hunter et al. (H2). The method has been extended to the labeling of progestagens ( C l ) .

4.3. PURIFICATION

OF THE

LABELEDANTIGEN

Purification generally takes place immediately after iodination. The purpose of this step is to separate the pure labeled material from any damaged fraction during the iodination procedure, and from free labeled iodide. When little or no damaged material is anticipated, purification is carried out on a small Sephadex G-25 or G-50 column, 1 x 10 cm, or even smaller. The column is equilibrated with 0.05 M phosphate buffer, pH 7.5, containing 1 : 100 sheep or horse serum, or with 0.1 or 0.2% bovine or human seiuni albumin in order to minimize adsorption of the '2"I-labeled protein. The same buffer is used for elution. When the labeled antigen must be purified from damaged components or when in impure material has been used for labeling, requiring further purification, longer columns (for instance 90 x 1 cm) should be used. In the case of small polypeptides such as angiotensin I or angiotensin 11, purification by means of gel chromatography ( G l ) or high-voltage electrophoresis ( V l ) is capable of separating the nionoiodinated antigen from the unlabeled one, thus increasing the specificity of the tracer. This is possible because of the rather high atomic weight of iodine in comparison with the weight of the peptide molecule. In some cases, when the antigen strongly adsorbs to silica, precipitated silica, such as QUSO-32 (Philadelphia Quartz Co.,

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J. P. FELBER

Philadelphia, Pennsylvania) is used for purification. Yalow and Berson ( Y l ) have proposed this simple method to purify labeled ACTH or parathyroid hormone. 4.4. ASSESSMENTOF THE LABELED ANTIGEN PREPARATION The labeled material has to be assessed for both its purity and antigenic properties. Chromatoelectrophoresis, as proposed by Yalow and Berson (Y I), has been widely used as an indication of the purity of the labeled antigen. The details of the procedure have been given by the authors. In short, 10-200 p1 of labeled antigen are placed on a strip of Whatman No, 3 MM filter paper. Chromatoelectrophoresis is carried out in 0.1 M Verona1 buffer, pH 8.6, with a constant voltage (20-25 V/cm), the lid of the apparatus remaining open to allow evaporation. With a 25-cm length of paper strip between the two buffer vessels, at 600 V, separation usually takes place within 45-60 minutes. The band is then analyzed for radioactivity either on a scanner or after it has been cut into several successive pieces which are placed in test tubes and measured separately on a gamma counter. The pure labeled protein or peptide usually remains at the origin, where it strongly binds to the cellulose of the paper strip (Fig. 6). Damaged components follow either as a shoulder of the first peak or as a second peak. Free iodide moves with the front. The presence of a second peak or shoulder indicates poor quality of the labeled material. However, its absence is no proof of quality, since the procedure is based on adsorption and migration qualities of the labeled antigen, but not on its antigenic qualities. To measure whether or not the labeled antigen has kept its antigenic properties after iodination, a good test consists in setting up a short standard curve in which the labeled antigen is incubated with the antiserum in the absence and in the presence of a few different concentrations of the unlabeled antigen. The dilution of the antibody is chosen so as to yield about 50% binding of the tracer alone. After a short incubation, separation and counting are performed as for normal assays. This short assay allows to compare the affinity of the labeled material with that of previous labelings. The addition of several different concentrations of the unlabeled antigen yields an indication on the sensitivity which can be achieved by using the tracer.

4.5. STORAGE OF

THE LABELED ANTIGEN

Labeled antigen is usually stored at +4"C. The material is partially diluted to decrease the effects of radiation damage. This is done in a

RADIOIMMUNOASSAY IN THE LABORATORY

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147

Before purification

FIG.6. Chromatoelectrophoretogram of labeled antigen before and after purification. The shoulder, just after the first peak, represents “damaged” labeled antigen, and the fast moving peak, unreacted iodide.

buffer containing human or bovine serum albumin to prevent loss of the material through adsorption on the wall of the container. It can be lyophilized for shipment. Degradation often occurs during storage, depending on the nature of the antigen and on the quality of the labeling. The stored material often has to be reassessed both for purity and binding capacity, before further use. When necessary, the material can be repurified on gel filtration or, when possible, by adsorption chromatography.

REFERENCES B1. Berson, S. A., and Yalow, R. S., Quantitative aspects of the reaction between insulin and insulin-binding antibody.]. Clin. Znoest. 38, 1966-2016 (1959). B2. Bolton, A. E., and Hunter, W. M., The labelling of proteins to high specific radioactivity by conjugation to a ““I-containing acylating agent. Biochem. J . 133,529-538 (1973). C1. Cameron, E. H. D., Scarisbrick, J . J., Morris, S. E., and Read, G., “‘I-iodohistamin

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derivatives as tracers for the radioiinmunoassay of progestagens. In “Steroid Immunoassay,” Proc. Tenovus Workshop, Sth, Cardiff, 1974 (E. H. D. Cameron, S. G. Hillier, and K. Griffiths,eds.), pp. 153-164. Alpha Omega Publ., Cardiff, Wdes, U.K., 1975. G1. Gandolfi, C., Malvano, R., and Rosa, U., Preparation and immunoreactive properties of monoiodinated angiotensin labelled at high specific activity. Bwchim. Biophys. Acta 251,254-261 (1971). G2. Goodfriend, T. L., and Ball, D. L., Radioimmunoassay of bradykinin: Chemical modification to enable use of radioactive i0dine.J. Lab. Clin. Med. 73, 501-511 (1969). G3. Greenwood, F. C., Hunter, W. M., and Glover, J. S., The preparation of ‘:L’I-labelled human growth hormone of high specific radioactivity. Biochem. J . 89, 114-123 ( 1963). H I . Hunter, W. M., and Greenwood, F. C., Preparation of iodine-131-labelled growth hormone of high specific activity. Nature (London) 194,495-496 (1962). H2. Hunter, W. M., Nars, P. W., and Rutherford, F. J., Preparation and behaviour of ‘2,71-labelled radioligands for phenolic and neutral steroids. In “Steroid Immunoassay,” Proc. Tenovus Workshop, 5th, Cardiff, 1974 (E. H. D. Cameron, S. G. Hillier, and K. Griffiths, eds.), pp. 141-152. Alpha Omega Publ., Cardiff, Wales, U.K. 1975. J1. Jeffcoate, S. L., Use of (’H) and (“‘I) tracers in steroid radioimmunoassays. Pathol. Biol. 23, 903-905 (1975). N1. Nars, P. W., and Hunter, W. M., A method for labelling oestradiol-17 with radioiodine for radioimmunoassays. J. Endocrinol. 57,47-48 (1973). N2. Newton, W. T., McGuigan, J . E., and Jaffe, B. M., Radioimmunoassay of peptides lacking tyr0sine.J. Lab. Clin. Med. 75, 886-892 (1970). R1. Redshaw, M. R., and Lynch, S . S . , An improved method for the prepaixtion of iodinated antigen for radioiinmunoassay. J. Endocrinol. 60, 527-528 (1974). R2. Rudinger, J., and Ruegg, U., Preparation of N-Succinimyl 3-(4-hydroxyphenyl) proponiate. Biochern. J. 133, 538-539 (1973). T1. Thorell, J. I., and Johansson, B. G . , High-specific activity labelling of glycoprotein hormones by means of lactoperoxidase (LPO). In “Structure-Activity Relationships of Protein and Polypeptide Hormone” (M. Margoulies and F. C . Greenwood, eds.), Int. Congr. Ser. No. 241, pp. 531-535. Excerpta Med. Found., Amsterdam, 1972. V1. Callotton, M. B., Parallel radioiinmunoassays of angiotensin I and of angiotensin 11 for measurement of renin activity and of circulating active hormone in human plasma. In “Immunological Methods in Endocrinology” (R. Levine and E. F. Pfeiffer, eds.), pp. 94-100. Thieme Verlag, Stuttgart, Academic Press, New York, 1971. y ofendogenous plasma insulin in man. Y 1. Yalow, R. S., and Berson, S . A., Immuno J. Clin. Invest. 39, 1157-1175 (1960). 5.

Incubation

In the radioimmunological system, the different constituents are incubated together at a chosen temperature and for a given time. Both the antiserum dilution and the concentration of the labeled antigen are identical in all tubes. The only variable is the concentration of the unlabeled antigen, either in the standard curve or in the unknown. The antiserum dilution has been previously chosen b y means of a dilution curve so that from 20 to 70% of the labeled antigen is antibody-bound in the absence of the labeled antigen (see Section 3 ) .

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The concentration of the labeled antigen has been chosen in order to present enough counts to allow detection and to be of the same order

of magnitude as the mean concentration of the unlabeled antigen, to allow competition with it for the combining sites of the antibody. As a rule, all reagents are added successively to the test tubes and incubated for the same length of time. The volume of the different components depends on the separation procedure chosen, which will terminate incubation. Blanks have to be introduced to check the nonspecific binding of the labeled antigen. In these blanks, the antiserum is replaced in some tubes by buffer, and in others by plasma. The components are added in the following order: (1) standard or unknown plasma, (2)antiserum, and ( 3 )labeled antigen. The unknowns take the place of the standards in the assay, with which they are compared. Each assay should also contain two or more plasmas whose values are known in order to make between-assay comparison. Incubation is usually carried on until equilibrium is reached. In some cases, however, separation can be performed before this step. It is then important for the time of incubation to be identical in all tubes. Delayed addition of the tracer has been suggested to increase the sensitivity of the assay. The unlabeled antigen (standard or unknown) is incubated first alone with the antiserum, to allow longer contact of the antibodies with the unlabeled antigen than with the tracer, which is added after 2/3 or %I of the total incubation time. This can increase the sensitivity of the assay only b y a factor of 1.5 to 2. A better improvement of the sensitivity of the assay is obtained by using, when possible, a more avid antiserum. The temperature at which incubation is carried out is of importance. Equilibrium is reached faster at room temperature or at 37°C than at 4°C. However, higher temperatures may increase the damage to labeled and unlabeled antigens during incubation, thus decreasing their binding capacities. The buffers that are most often used are: 0.04 or 0.05 M phosphate, pH 7.4; 0.04 or 0.05M Tris, pH 7.4; 0.01 or 0.02 M Veronal, pH 8.6; and 0.1 M borate, pH 8.4.As most antigens have the tendency to bind to any surface (glassware, tubes, pipettes, etc.), thus disappearing from the solutions, a given proportion of protein is always added to the buffer. This is done b y adding 0.1 or 0.2% of bovine or human serum albumin. In the case of some small peptide hoimones, even high albumin concentrations do not prevent adsorption. The assay, therefore, must b e carried out in plasma. The standard is prepared with the hormone diluted in hoimone-free plasma. Hormone-free plasma can be obtained b y taking blood from subjects with no circulating hormone, or after removing the hoimone from nor-

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ma1 plasma. In the case of ACTH, plasma is taken from hypophysectomized patients, or from subjects treated with dexamethasone. For other hormones, such as glucagon or PTH, charcoal is used to extract the hormone from the plasma (1 g for 20 ml plasma). Small peptide hormones are often sensitive to proteolytic enzymes. This is again the case for ACTH and glucagon, which are degraded by the proteolytic enzymes of the plasma. Therefore, the plasma has to b e collected into tubes containing Trasylol. The whole assay of glucagon is performed in the presence of Trasylol. For ACTH, the assay can be performed either in the presence of mercaptoethanol or after extraction of the plasma by silicic acid. The presence of nonspecific constituents of the plasma or of other biological media such as proteins or salts, may interfere with the assay. Proteins, in particular, interfere with the separation procedure when methods based on adsorption of the free antigen are used. It decreases the binding capacity of the free antigen to the adsorbent. In this case, it is necessary to dilute the plasma or the other biological media and to have the same protein concentration in the standard as in the unknown, to allow comparison to be made. High osmolarity in the solutions may also lead to fallacious results (Gl). Dilution or desalting may therefore be required in this case. The various problems due to the presence of nonspecific constituents are avoided when the plasma or the other biological media are sufficiently diluted ( l / l O for plasma), or when the assay is preceded by an extraction procedure, as is often the case in the radioimmunoassay of steroids.

REFERENCE G1. Girard, J., and Greenwood, F. C., Radioimmunoassay for human growth hormone in urine. Aspecific factors imitating the presence of growth hormone. In “Protein and Polypeptide Hormones” (M. Margoulies, ed.), Part 2 , Int. Congr. Ser. No. 161, pp. 332-334. Excerpta Med. Found., Amsterdam, 1968. 6.

Separation Procedures

Separation of the antibody-bound labeled from the free labeled antigen is required to measure their radioactivity separately in order to indicate the proportion of the labeled antigen bound to the antibody as a result of the competitive effect of the concentration of unlabeled antigen. Displacement in the radioactivity of either species (free or antibody-bound labeled antigen) is compared with that of the standards forming the standard curve. Separation is performed at the end of the incubation period, which it terminates. The methods used for that purpose can be divided into two categories, one separating the antibody-bound and the other the free antigen from the incubation mixture.

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6.1. METHODS FOR SEPARATING THE ANTIBODY-BOUND ANTIGEN FROM THE INCUBATION MIXTURE These methods either precipitate the antibodies together with the antigen fixed to them or make use of antibodies previously bound to solid material (solid phase radioimmunoassay). In contradistinction to the methods based on separation of the free antigen, which depend on the qualities of the antigen itself, these methods can be applied to the radioimmunoassay of any substance, since they act on the antibody instead of on the antigen.

6.1.1. Double-Antibody Technique (Zmmunoprecipitation) In this procedure, the antibody (first antibody) together with the antigen fixed to it is specifically precipitated by an anti-gamma globulin (second antibody) with which it forms a large precipitating complex. The procedure has been developed mainly by Morgan and Lazarow (M2), Hales and Randle (Hl), and Sonksen (S2) on the basis of studies made by Skom and Talmage ( S l ) . It can be summarized by the following scheme: (first ab)

Antigen

+ antibody * [Antigen-antibody] +

soluble complex

Anti-gamma globulin (second all)

.1

[Antigen-antibody-anti-gamma globulin]

precipitating complex

The anti-gamma globulin (second antibody) serum is commercially available. It is prepared by immunization of the animal with IgG from the animal species providing the first antibody. For example, antiguinea pig gamma globulin is prepared in the rabbit by immunization with guinea pig IgG. It specifically precipitates antisera prepared in the guinea pig. Similarly, antirabbit gamma globulin can be prepared in the goat or in other large animals to precipitate antibodies prepared in the rabbit. Anti-gamma globulin sera are species-specific and do not precipitate IgG from an animal species other than that with which they are prepared. The preparation of IgG is given by Hales and Randle (Hl). Anti-gamma globulin has to be tested for optimal dilution before use in an assay. For that purpose, titration is performed: increasing dilutions of anti-gamma globulin serum are added to a series of tubes at the end of the incubation period, all tubes containing the same dilution of the first antiserum and the same concentration of the labeled antigen, without any addition of unlabeled antigen. A plateau is usually obtained where maximum precipitation is reached. Precipitation is often

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J. P. FELBER

decreased at a low dilution (prozone phenomenon) as well as at a too high one. Anti-gamma globulin sera presenting a peak rather than a plateau should be discarded because they present the risk of variations in the degree of precipitation. The anti-gamma globulin serum is added at the end of the incubation period. Incubation is then continued for a second period to allow immunoprecipitation to take place. It is generally performed for 16 to 24 hours either at +4"C or at room temperature, depending on the conditions required for the first incubation. It is terminated by centrifugation (or b y microfiltration) followed by counting either the precipitate or the supernatant, or both, in a gamma counter. An example is given in Table 1. The presence of complement in plasma or sera has been shown to interfere with the precipitation by delaying it (M3). For this reason, it is important to allow sufficient time for the second incubation (16 to 24 hours). Some authors add EDTA (0.005or 0.01 M ) to the diluent of the anti-gamma globulin serum, to accelerate precipitation. The effect of EDTA seems to b e most marked with low-avidity antisera. It is important to use as second antibody an antiserum prepared against pure gamma globulin rather than against whole serum in order to prevent cross-reactions with proteins contained in human plasma (Wl). When the titer of the first antibody is high, either noimal guinea pig serum or normal rabbit serum, depending on the origin of the first antiserum, is added to the dilution of the first antiserum as nonimmune carrier serum. This increases the bulk of the first IgG to give maximal precipitation with optimal dilution of the second antibody. Two major variations of the double-antibody technique are preprecipitation and the double-antibody solid phase. Preprecipitation has been proposed by Hales and Randle ( H l ) . The first and second antibodies are first incubated to form a complex that is added to the assay as a complete binding reagent. The other steps of the assay are unchanged. The pui-pose of preprecipitation is to achieve maximal precipitation in the absence of interfering substances contained in the plasma of the unknowns. The duration of the assay is shortened by eliminating the second incubation period. However, the sensitivity is lower than that obtained in the usual postprecipitation system, possibly because the avidity of the first antibody is altered by the binding to precipitated antibody (Rl). Double-Antibody Solid Phase (DASP) has been developed by Hollander and Schuur (H6). In this method, the second antibody is covalently linked to Sepharose beads. This immunoadsorbent (antirabbit gamma globulin Sepharose) is added at the end of the first incubation

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period and allowed to react with the first antibody complexes. Then it can be separated b y centrifugation or sedimentation. This immunoadsorbent is commercially available. The major drawback of the method is that it requires constant or frequent mixing during the second incubation period.

6.1.2. Fractional Precipitation Fractional precipitation of the antigen-antibody complex with neutral salts or organic solvents has been proposed as an inexpensive and rapid method of separation. It is based on the precipitation of immunoglobulin at a critical concentration of the precipitate while leaving the free antigen in solution. This method is delicate to use, for its applicability depends on the properties of the free antigen. In each case optimal conditions have to be determined for immunoglobulin to b e precipitated while the free antigen remains in solution. Ammonium sulfate has been proposed for the assay of small peptides such as oxytocin, arginine-vasopressin and lysine-vasopressin (C4). Ethanol has been successfully used in the separation of a wider range of peptides: oxytocin, arginine-vasopressin, lysine-vasopressin, insulin, and human placental lactogen (HPL) (C4). Heding proposed the use of ethanol for the radioinimunoassay of insulin (H2) and of glucagon (H3). A final ethanol concentration of from 80 to 81% is used. The precipitate remains stable for at least 2 hours. Dioxan has been proposed at a final dilution of 66% for the assay of HCG (Tl) and FSH (Ll). After addition of aqueous dioxan, the tubes are left in the cold for 30 minutes before centrifugation. T h e supernatant contains the free hormone while the precipitate represents the antibody-bound hormone. Polyethylene glycol (PEG) was proved useful for the separation of free from antibody-bound antigen in the assays of insulin, parathyroid hormone, growth hormone and arginine-vasopressin. PEG-6000 is added as an aqueous solution to the incubation mixtures at the end of incubation (D2). Optimal concentration was shown to be 12% (w/v) (Dl), in the presence of plasma. PEG causes negligible coprecipitation of the free hormone with insulin, vasopressin, and angiotensin, but a larger fraction of the free hormone is coprecipitated with parathyroid and growth hormones, probably because of the larger size of these hormones. 6.1.3. Solid-Phase Radioimmunoassay In solid-phase radioimmunoassays, the antibody is coupled to a solid-phase matrix, while retaining its specific immunological proper-

TABLE 1

SAMPLE WORKSHEET FOR INSULIN FhDIOIMMUNOASSAY BY THE DOUBLE-ANTIBODY

r ul 4

Tube number

Standard or unknown (0.1ml)"

Antiserum or NRS (0.1Id)*

Buffer Buffer Buffer Buffer Buffer Buffer Buffer Buffer Insulin, 2 pU/ml Insulin, 2 pU/ml Insulin, 4 pU/ml Insulin, 4 pU/ml

Buffer Buffer Buffer Buffer NRS 1/600 NRS 1/600 Antiserum 1/1OO,OOO+ NRS 1/600 Antiserum 1/1OO,OOO + NRS 1/600 Antiserum l/lOO,OOO + NRS 1/600 Antiserum 1/1OO,OOO + NRS 1/600 Antiserum 11100,OOO+ NRS 1/600 Antiserum UlOO,OOO + NRS 1/600

METHOD Goat antirabbit gamma globulin '251-Insulin serum 1/80 (0.1ml)d.' (0.1 ml)'

10 pU/ml 10 pU/ml 10 pU/ml 10 pU/ml 10 p U / d 10 pU/ml 10 pU/ml

10 pU/ml 10 pU/ml 10 pU/ml 10 pU/ml 10 pU/ml

1/80 1/80 1/80 1/80 1/80 1/80 1/80 1/80 1/80 1/80 1/80 1/80

26

Plasma blank

29

Reference plasmas

30 31

34

r

Unknown plasmas

Plasma Plasma Reference plasma 1 (low) Reference plasma 1 (low) Reference plasma 2 (high) Reference plasma 2 (high) Unknown plasma 1 Unknown plasma 1 Unknown plasma 2 Unknown plasma 2

NRS 1/600 NRS 1/600 Antiserum 1/1OO,OOO + NRS 11600 Antiserum 1/1OO,OOO + NRS 1/600 Antiserum 1/1OO,OOO + NRS 11600 Antiserum 1/1OO,OOO + NRS 1/600 Antiserum 1/1OO,OOO + NRS 1/600 Antiserum 1/1OO,OOO + NRS 1/600 Antiserum 1/1OO,OOO + NRS 1/600 Antiserum 1/1OO,OOO + NRS 1/600

10 pU/ml 10 pU/ml 10 pU/ml 10 pU/ml 10 pU/ml 10 pU/ml 10 pU/ml 10 pU/ml 10 pU/ml 10 pU/ml

1/80 1/80 1/80 1/80 1/80 1/80 1/80 1/80 1/80 1/80

" Buffer: 0.05 M phosphate buffer, p H 7.4. Standard curve: Insulin diluted in 0.05 M phosphate buffer, pH 7.4, containing 0.1 g/liter merthiolate (to prevent bacterial growth), and 2 g/liter human or bovine serum albumin (to prevent adsorption of insulin to glassware). * Antiserum: 1/1OO,OOO diluted in 0.05M phosphate buffer, pH 7.4, containing 0.1 gAiter merthiolate + 1/600 NRS (normal rabbit serum) as nonimmune carrier serum. Followed by first incubation for 24 hours at 4°C. Anti-gamma globulin serum: Antirabbit, prepared in the goat, diluted i n 0.05 M phosphate buffer, p H 7.4, containing 0.1 g/liter merthiolate + 0.01 M EDTA. ' Followed by second incubation for 16-24 hours at 4 T , and centrifugation for 20 minutes at 35004000 g .

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J. P. FELBER

ties toward the antigen. Its major advantage is to facilitate separation of the free from the antibody-bound antigen at the e n d of the incubation period. This is done essentially by washing the solid phase. TWO methods are most commonly used: antibody covalently coupled to solid material is added to the test tubes together with the other constituents of the incubation mixture, and plastic tubes are precoated with physical adsorption of antibody. Antibody Coupled to Activated Particles (Immunosorbent). Polysaccharide polymers and cellulose particles previously activated by treatment with cyanogen bromide (W3) are made to react with the amino groups of specific antisera. This produces a stable conjugate which possesses the immunological properties of the antibody. Such conjugates have been developed with ultrafine Sephadex particles, as the matrix and antisera to LH (luteinizing hormone), FSH (folliclestimulating hormone), and hCG (human chorionic gonadotropin) (W2). Commercial kits have been developed on this principle for the radioimmunoassay of several hormonal and nonhormonal substances. For the radioimmunoassay of FSH and LH in serum, Wide et ul. (W4) proposed the following procedure: a sample of 0.1 ml of the serum sample to be assayed is put into a plastic tube. Reference standards and controls are run in parallel. The immunosorbent suspension, containing the specific antibody coupled to the Sephadex support is added in 0.5 ml aliquots with a syringe to which the metal holder of a Cornwall pipette is added. The test tubes are shaken, then covered with a plastic film and left to incubate at room temperature for 2-3 hours. An aliquot of 0.1 ml of the solution with the labeled hormone (40,000 cpm or about 100 pg) is added to the test tubes which are then capped with a plastic stopper. The test tube racks are slowly and vertically rotated for about 24 hours by means of a rotating apparatus. Then the test tubes are centrifuged and decapped with a pricker. Two milliliters of a washing solution of saline with 0.5% Tween 20 is added, using an automatic dispenser. After centrifugation the supernatant is removed b y suction. The immunosorbent at the bottom of the 12 test tubes can be prevented from being suctioned off by a plastic guard attached to the needle. The washing procedure is then repeated three times. The test tubes are capped again and placed in an automatic gamma counter. This method has been adapted for the radioimmunoassays of other hormones [insulin, HCS (human chorionic somatotropin) also called HPL (human placental lactogen), hTSH (human thyroid-stimulating hormone)] and nonhormonal substances in plasma, and for the assays of FSH and LH in urine. In the case of urine, 0.1 ml urine is placed in the test tube, and 0.1 ml aliquot ofthe immunosorbent are used, instead

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of 0.5 ml for the serum. This method, commercially available, is easy to use, but the repeated washings are time consuming, unless special, inexpensive equipment is used to suction off the washing liquid of several tubes simultaneously . Antibody-Coated Plastic Tubes is a simple method, proposed by Catt and Tregear (C2), in which the antibody is coated directly on the inner wall of the test tubes. It is based on the observation of the ability of many polymers to adsorb small quantities of gamma globulin from diluted solutions of antisera. In practice, polystyrene tubes are exposed to an appropriate dilution of antiserum for several hours and then thoroughly washed and employed for radioimmunoassay by incubation with the labeled antigen, and standards and samples containing the antigen to b e measured (Cl). The antibody coating is performed at room temperature by adding uniform aliquots (0.5-5.0 ml) of antiserum to each tube. The antiserum is diluted in 0.1 M carbonate-bicarbonate buffer, pH 8.2-9.6. The dilution varies with the titer and the avidity of the antiserum, and the antibody-coating solution can be reused for further batches of tubes. The tubes may b e stored, freeze-dried, for several months at low or room temperature ( C 3 ) . For the assay itself, the following procedure has been proposed by Ceska et al. (C3) for the radioimmunoassay of insulin: to the incubation tubes, previously coated under or over the final incubation volume, 500 pl of incubation buffer (0.05M phosphate, p H 7.4, containing 0.15 M sodium chloride, 0.05% sodium azide and 0.05% Tween-20) are first added, followed either by 100 pl of insulin standard or 100 p1 of plasma sample, 100 pl of ""I-insulin diluted with the incubation buffer, and finally another 500 p1 of the incubation buffer, to give a total volume of 1200 pl. The tubes are incubated overnight at room temperature. At the end of incubation, the tubes are washed twice with distilled water or tap water and then counted in a gamma counter. This procedure can be extended to other polypeptide hormones, in particular hGH, hCS and hLH. The coated-tube assay method requires a fairly large quantity of high-titer antiserum. For antisera with lower titers, coupling to activated polymers (immunosorbent) is preferred. 6.2. METHODS FOR SEPARATING THE FREE ANTIGEN FROM THE INCUBATION MIXTURE In these methods, removal of the free antigen from the incubation mixture is based on the adsorption qualities of the antigen. Free antigen is adsorbed on solid particles which precipitate, while the

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J. P. FELBER

antibody-bound antigen remains in the solution. After centrifugation, as compared to separation methods where the antibody-bound antigen is removed, the free antigen is found in the precipitate and the antibody-bound antigen in the supernatant. These methods are useful for the radioimmunoassay of small polypeptides and steroids which present high affinity to adsorbents such as charcoal, silica, or some ion-exchange resins, This is particularly the case for angiotensin I and 11, glucagon, ACTH, insulin, gastrin, and parathyroid hormone. They cannot b e used for larger proteins which do not present a sufficient affinity for the adsorbents, and which are easily displaced by the plasma proteins. Adsorption of antigens to the surface of the adsorbent depends on many factors, particularly the relative surface area of the adsorbent, the size and charge of the antigen, and the nature and concentration of the competing proteins. The surface area of the adsorbent is related to the size of the particles, small particles being more effective for the same mass. The adsorbent concentration must b e adapted to each antigen, a larger concentration being necessary for antigens with low affinity. The concentration of the competing proteins, such as plasma or albumin, is of great importance, since proteins decrease the affinity of the antigen to the adsorbent. Lack of competing proteins, due for instance to a high plasma dilution, may allow the binding of the antibodybound antigen together with the free antigen, whereas a too small plasma dilution may prevent adsorption of the free antigen to the adsorbent. The concentrations of the different constituents have to be tested for optimal conditions. They can be kept as long as the same adsorbent is used. However, they will have to be readjusted when a new batch of adsorbent is introduced. 6.2.1. Charcoal-Dextran Method This method has been introduced by Herbert et al. (H5) for the radioimmunoassay of insulin. It is based on the binding capacities of charcoal. The binding of large proteins is prevented by presaturation of charcoal with dextran. A nonspecific adsorbent such as charcoal may be made specific by soaking in a solution of dextran of appropriate molecular size and configuration (H4). It has been shown that charcoal coated with dextran of average molecular weight 10,000 (Dextran 10) adsorbs angiotensin but rejects insulin; with dextran of average molecular weight 80,000 (Dextran 80) charcoal adsorbs insulin but rejects growth hormone, whereas with dextran of molecular weight 250,000 (Dextran 250) charcoal adsorbs growth hormone. These coatings exclude hormone bound to antibody. The most useful charcoal

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was shown to be charcoal made of wood (Norit-A neutral pharmaceutical grade decolorizing carbon, NFX from Amend Drug and Chemical Company, Irvington, New Jersey). Dextran is supplied by Pharmacia, Fine Chemicals, Piscataway, New Jersey or Uppsala, Sweden. For the radioimmunoassay of insulin, a charcoal suspension and a dextran solution are prepared separately. Five grams Norit-A charcoal are suspended in 100 mlO.01 M phosphate or Veronal-acetate buffer, pH 7.4, containing 0.15 M NaCl, and 0.5 g Dextran 80 is dissolved in 100 ml of the same buffer. Other grades of dextran may be used, depending on the molecular weight of the antigen to be measured, Dextran can be replaced by blood fractions such as bovine or human serum albumin, gamma globulin, Ficoll (Pharmacia), etc. (H4). The dextran-coated charcoal suspension is prepared by mixing equal volumes of the two compounds. The mixture is briefly shaken and then stored at 4°C. Just before use it is resuspended by mixing and is kept in suspension during use with the help of a magnetic stirrer. To keep the plasma concentration equal in all test tubes, neutral plasma (any normal plasma) is usually added to the tubes containing the standard curve (and the maximal activity), just before the separation step so that it does not have time to react with the other components of the incubation mixture. Some authors prefer to add plasma free of antigen from the beginning of the incubation. They use either plasma previously treated with charcoal (1g Norit A for 20 ml plasma) or plasma from patients who are not secreting the hormone to b e tested. A given volume of the dextran-coated charcoal is added to all the tubes. They are capped, quickly mixed by repeated inversion for approximately 10 seconds, and centrifuged for 15 minutes at 2500 g. Centrifugation should be started within 10 minutes after mixing, to avoid possible dissociation of the free antigen from charcoal. After centrifugation, charcoal forms a solid button at the bottom of the tubes. The supernatant liquid can be counted after decantation into counting tubes. It contains the antibody-bound antigen. Or, more simply, the incubation tubes with the charcoal are counted. They contain the free antigen, fixed to the charcoal (Fig. 7). Table 2 shows an example of insulin assay, using the charcoal-dextran method of separation. The conditions of the assay have to be tested for each type of antigen to b e measured, and even for each batch of charcoal. It is important to get optimal conditions where the free antigen is bound to charcoal while the antibody-bound antigen remains excluded. The concentration of proteins (dilution of plasma, presence of serum albumin) is critical. In each assay, a plasma blank containing no antiserum has to

SAMPLE WORKSHEET FOR

Tube number

TABLE 2 INSULIN hDIOIMMUNOASSAY

BY THE

CHARCOAL-DEXTRAN METHOD

Albumin buffer (3.5mg/ml) (0.5 ml)"

Standard or unknown plasma (0.1ml)

Antiserum or NRS (0.1 ml)"

""I-Insulin (0.1 ml)'

Buffer Buffer

Buffer Buffer Buffer Buffer Buffer Buffer Buffer Buffer Insulin 2 pU/ml Insulin 2 pU/ml Insulin 4 pU/ml Insulin 4 pU/ml

Buffer Buffer Buffer Buffer NRS 1/40,000 NRS 1/40,000 Antiserum 1/40,000 Antiserum 1/40,000 Antiserum 1/40,000 Antiserum 1/40,000 Antiserum 1/40,000 Antiserum 1/40,000

20 pUlml 20 pU/ml 20 pU/ml 20 pU/ml 20 pU/ml 20 pU/ml 20 pU/ml 20 pU/ml 20 pU/ml 20 pU/ml 20 pU/ml 20 pU/ml

Buffer Buffer Buffer Buffer Buffer Buffer Buffer Buffer Buffer

Neutral buffer or plasma (0.1 ml)"

Charcoaldextran (C-D) suspension (0.5 mly

-

Plasma Plasma Plasma Plasma Plasma Plasma Plasma Plasma Plasma

C-D C-D C-D C-D C-D C-D C-D C-D C-D

suspension suspension suspension suspension suspension suspension suspension suspension suspension

28 Refer. 29 plasmas 271 30

Plasma Plasma Reference plasma 1 (low) Reference plasma 1 (low) Reference plasma 2 (high) Reference plasma 2 (high) Unknown 1 Unknown 1 Unknown 2 Unknown 2

NRS 1/40,000 NRS 1/40,000 Antiserum 1/40,000 Antiserum 1/40,000 Antiserum 1/40,000 Antiserum 1/40,000 Antiserum 1140,000 Antiserum 1/40,000 Antiserum 1/40,000 Antiserum 1/40,000

20 pU/ml 20 pU/ml 20 pU/ml 20 pU/ml 20 pU/ml 20 wU/ml 20 pU/ml 20 pU/ml 20 pUlml 20 pU/ml

Buffer Buffer Buffer Buffer Buffer Buffer Buffer Buffer Buffer Buffer

C-D C-D C-D C-D C-D C-D C-D C-D C-D C-D

suspension suspension suspension suspension suspension suspension suspension suspension suspension suspension

Buffer: 0.01 M phosphate or Veronal-acetate buffer, pH 7.4, containing 0.15 M NaCI. For albumin buffer: add 3.5 mg/ml human or bovine serum albumin. " NRS: normal rabbit serum. Followed by incubation for 16-24 hours at 4°C. " Neutral plasma is added to the incubation mixture of the standard cuwe just before separation by charcoal-dextran to keep the protein concentration equal to that of the unknown plasmas. Followed by mixing and centrifugation, and counting of the supernatant or precipitate. 'I

r

Buffer Buffer Buffer Buffer Buffer Buffer Buffer Buffer Buffer Buffer

162

J. P. FELBER C?

25C CHARCOAL

20(

150

2.5 is

50

100

3

200 UUlml

FIG.7. Standard curve for insulin, using the dextran-charcoal method. Free labeled insulin is found in the precipitated charcoal (ascending curve), and the antibody-bound labeled insulin, in the supernatant (descending curve).

be included in order to verify the interference of plasma proteins. The

charcoal-dextran technique has been shown to be useful mainly for the assays of antigens that strongly adsorb to charcoal, such as small peptide hormones (insulin, glucagon, gastrin, ACTH, angiotensin I and 11, calcitonin, and parathyroid hormone) or steroids. The method has been critically studied b y Palmieri, Yalow, and Berson (Pl), and by Binoux and Ode11 (Bl).

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163

6.2.2. Silicates (Talc, QUSO) Silica, which has a strong adsorption capacity for small peptides, has been demonstrated to b e useful in a separation method. Silica, with its large surface area, can be placed directly into test tubes in the form of powder or tablets. Adsorption occurs rapidly and the adsorbent packs well on centrifugation. Free antigen is bound to the pellet at the bottom of the tube, while the antibody-bound antigen remains in the supernatant. As with the charcoal-dextran method, the quantities of adsorbent and protein concentrations are critical and must b e adapted to each antigen. Talc and QUSO G-32 (Quartz Co., Philadelphia, Pennsylvania) have been used for the assay of ACTH and parathyroid hormone (R2). 6.2.3. Anion-Exchange Resins Anion-exchange resins, especially Dowex I and Amberlite CG-4 B, have also been used in a separation technique, particularly by Meade and Klitgaard ( M l ) for the assay of insulin, and by Yalow and Berson ( Y l ) for the assay of gastrin. Adsorption of free antigen is almost immediate. Owing to interference of heparin with the binding sites of anion-exchange resin, this anticoagulant has to be omitted during blood collection.

REFERENCES B1. Binoux, M. A,, and Odell, W. D. Use of dextran-coated charcoal to separate antibody-bound from free hormone: A critique. ]. Clin.EndocrinoL Metab. 36, 303-310 (1973). C1. Catt, K. J. Solid-phase radioimmunoassay of peptide and steroid hormones. In “Hormones in Human Blood. Detection and Assay” (H. N. Antoniades, ed.), pp. 139-154. Harvard Univ. Press, Cambridge, Massachusetts, 1976. C2. Catt, K., and Tregear, C. W., Solid-phase radioimmunoassay in antibody coated tubes. Science 158, 1570-1578 (1967). C3. Ceska, M., Grossmiiller, F., and Lundkvist, U., Solid-phase radioimmunoassay of insulin. Acta Endocrinol. (Copenhagen) 64, 111-125 (1970). C4. Chard, T., Martin, M., and Landon, J., The separation of antibody-bound from free peptides using ammonium sulphate and ethanol. In “Radioimmunoassay Methods” (K. E. Kirkham and W. M. Hunter, eds.), pp. 257-266. Churchill Livingstone, Edinburgh and London, 1971. D1. Desbuquois, B., and Aurbach, G. D., Use of polyethylene glycol to separate free and antibody-bound peptide hormones in radioimmunoassays.]. Clin. E n d o c r i d . 33,732-738 (1971). D2. Desbuquois, B., and Aurbach, C . D., Use of polyethylene glycol to separate free from antibody-bound hormones in radioimmunoassays. In “Hormones in Human Blood. Detection and Assay” (H. J. Antoniades, ed.), pp. 155-159. Harvard Univ. Press, Cambridge, Massachusetts, 1976.

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H1. Hales, C. N., and Randle, P. J., Immunoassay of insulin with insulin-antibody precipitate. Biochem. J . 88, 137-146 (1963). H2. Heding, L. G., A simplified insulin radioimmunoassay method. In “Labelled Proteins in Tracer Studies” (L. Donato,G. Milhaud, and J. Sirchis, eds.), pp. 345-350. Euratom, Brussels, 1966. H3. Heding, L. G., Radioimmunological determination of pancreatic and gut glucagon in plasma. Diabetologia 7, 10-19 (1971). H4. Herbert, V., and Bleicher, S. J.. Separation ofantibody-bound from free hormone by the coated-charcoal technique. In “Hormones in Human Blood. Detection and Assay” (H. N. Antoniades, ed.), pp. 115-120. Harvard Univ. Press, Cambridge, Massachusetts, 1976. H5. Herbert, V., Lau, K.-S., Gottlieb, C. W., and Bleicher, S. J., Coated charcoal immunoassay of insulin. J . Clin. Endocrinol. 25, 1375-1384 (1965). H6. Hollander, F. C., and Schuurs, A. H. W. M. In “Radioimmunoassay Methods” (K. E . Kirkham and W. M. Hunter, eds.), pp. 419422. Churchill Livingstone, Edinburgh and London, 1971. L1. Leyendecker, G., Saunders, D. M., and Saxena, B. B., Further improvements in the radioimmunoassay of human pituitary follicle-stimulating hormone (FSH). Klin. Wochenschr. 49,658-660 (1971). M1. Meade, R. C., and Klitgaard, H. M., A simplified method for immunoassay of human serum a1bumin.J. Nucl. Med. 3,407-416 (1962). M2. Morgan, C. R., and Lazarow, A., Immunoassay of insulin using a two-antibody system. Proc. Soc. E r p . Biol. Med. 110,29-32 (1962). M3. Morgan, C. R., Sorenson, R. L., and Lazarow, A,, Further studies of an inhibitor of the two-antibody immunoassay system. Diabetes 13,579-584 (1964). P1. Palmieri, G. M. A., Yalow, R. S., and Berson, S. A,, Adsorbent techniques for the separation of antibody-bound from free peptide hormones in radioimmunoassay. Horm. Metab. Res. 3,301-305 (1971). R1. Ratcliffe, J. G., Separation techniques in saturation analysis. Br. Med. Bull. 30, 32-37 (1974). R2. Rosselin, G., Assan, R., Yalow, R. S., and Berson, S . A., Separation of antibodybound and unbound peptide hormones labelled with iodine-131 by tzlcum powder and precipitated silica. Nature (London)212, 355-357 (1966). S1. Skom, J. H., and Talmage, D. W., Nonprecipitating insulin antihodies.]. Clin. Inoest. 37, 783-786 (1958). S2. Sonksen, P. H., Separation of antibody-bound from free hormone by the doubleantibody technique. In “Hormones in Human Blood. Detection and Assay’’ (H. N . Antoniades, ed.), pp. 121-138. Haivard Univ. Press, Cambridge, Massachusetts, 1976. T1. Thomas, K., and Ferin, J., A new rapid radioimmunoassay for HCG (LH, ICSH) in plasma using dioxan. J . CEin. Endocrinol. 28, 1667-1670 (1968). W1. Welborn, T. A., and Fraser, T. R., The double-antibody immunoassay of insulin. A standardized second antibody reaction that eliminates spurious results with human serum. Diabetologia 1,211-218 (1965). W2. Wide, L., Radioimmunoassay employing immunosorbents. Acta Endocrinol. {Copenhagen), Suppl. 142, 207-218 (1969). W3. Wide, L., A x h , R., and Porath, J., Radioimmunosorbent assay for proteins. Chemical coupling of antibodies to insoluble dextran. Immunochemistry 4, 381-386 (1967). W4. Wide, L., Nillius, S. J., Gemzell, C., and Roos, P., Radioimmunosorbent assay of

1IAI)IOIMMUNOASSAY IN THE LABORATORY

7.

165

Measurement of Radioactivity

For the measurement of I2'I or '"I-labeled antigens, a well-type gamma counter is generally used, whereas tritium or I4C-labeled antigens are counted in liquid-scintillation counters. These devices are usually adapted with an automatic sample changer. For radioiodinated antigens, counting can b e done directly in the test tubes that have served for incubation, when the precipitate is counted. In this case, it is important to choose ready-for-incubation test tubes that fit into the counting apparatus. When the supernatant or an aliquot of it has to be counted, it is transferred into other tubes serving this purpose. The counts measured in the whole supernatant are complementary to those of the precipitate, the addition of the two representing the whole radioactivity introduced in the assay. Therefore, it is not always necessary to measure both precipitate and supernatant. The precipitate is counted and the radioactivity of the supernatant is determined by subtracting it from the total counts. 8.

Calculation of Results

Results are calculated by comparing the values of the unknowns with a standard curve. This implies that all conditions of the assay have been identical for the unknowns and the standard curve. This is particularly important for the protein concentration when using separation methods based on adsorption of the free antigen. Several types of plots are currently used. The simplest is an arithmetic scale where the radioactivity in CPM of the antibody-bound fraction, placed on the ordinate, is related to the concentration of the unlabeled antigen from the standard curve, in abscissa (Fig. 8).The counts per minute (CPM) can be replaced either by the calculated percent of the total antibodybound labeled antigen, using as 100%the total activity introduced in each tube, or by the calculated percent of the maximum binding, using as 100% the maximal activity measured in the zero dose of the standard curve. Some authors place on the ordinate the ratio of antibody-bound ( B ) over free ( F ) radioactivity (BIF ratio) (Fig. 9). By using a logarithmic scale for the concentration of the unlabeled antigen, on semilog paper an S-shape curve is obtained.

J. P. FELBER

166 CPM

%total binding

binding

10000 40.

1500.

30.

5000. 20.

2500.

O

10.

L O

L OI,, , 0 25 M 125

100

200

400 PO/rnl

*

FIG.8. Arithmetic plot of a standard curve, using either the counts per minute (CPM),the percent of total binding (total binding being represented by the total activity introduced in each tube) or the percent maximal binding (maximal binding being represented by the radioactivity of zero dose).

Linearization of the standard curve in the radioimmunoassay system has been made possible by using the logit transformation proposed by Rodbard et al. (R3, R4). The logit of the BIBo ratio (ratio of the antibody-bound radioactivity, B, of each sample over the antibodybound radioactivity of the zero dose, B o ) is placed on the ordinate, on a logit scale, while the unlabeled antigen concentration is entered on a logarithmic scale on the abscissa (Fig. 10).This can easily be achieved with any commercially available logit-log paper. The values have first to be corrected for nonspecific counts, by subtracting the buffer blank from the values of the standard curve and the plasma blanks from the unknowns. In these blanks, the antiserum had been replaced by normal serum from the same animal species, for example, normal guinea pig serum (NGPS) or normal rabbit serum (NRS). In comparison with the semi-log plot, logit transformation produces a compression of the curve in the central area, while both ends are elongated. The 95% confidence limits are easily obtained for withinassay variations. This system may easily be adapted to programmable desktop calculators. A great advantage of this system resides in the

RADIOIMMUNOASSAY IN THE LABORATORY

5x

s

‘1 mlrPD $ 9080.

70. 60. 50.

40. 30. 20. 10. 5-

3. 1

167

168

J. P. FELBER

possibility of comparing the standard curves from different assays (between-assay variations), and of measuring the parallelism between plasma dilution and the standard curve. The slope and the intercept are easily measured and characterize each curve. Mathematical details on the logit-log method are given by Rodbard et aZ. (R3) and Rodbard

A drawback of the logit-log method resides in the elongation of both ends of the curve, with a corresponding expansion of the error. The four-parameter logistic model, which was initially proposed by Healy ( H l ) and developed by Rodbard and Hutt (R2) has the advantage of making use of all the points of the curve and of giving the best fit for both ends. However, it is a nonlinear equation. It can b e used for any radioimmunoassay, and computer programs are available.

REFERENCES H1. Healy, M. J. R., Statistical analysis of radioiinniunoassay data. Biochem. 1. 130, 207-210 (1972). R1. Rodbard, D., Statistical quality control and routine data processing for radioinimunoassay and inimunoradiometric assays. Clin. Chem. 20, 1255-1270 (1974). R2. Rodbard, D., and Hutt, D. M., Statistical analysis of radioimmunoassays and immunoradiometric (labeled antibody) assays. A generalized weighted iterative, least-squares method for logistic cuive fitting. In “Radioimmunoassay and Related Procedures in Medicine,” Vol. 1, pp. 165-191. IAEA, Vienna, 1974. R3. Rodbard, D., Bridson, W., and Rayford, P. L., Rapid calculation of radioimmunoassay results.]. Lab. C h . Med. 74, 770-781 (1969) R4. Rodbard, D., Rayford, P. L., Cooper, J. A., and Ross, G . T., Statistical quality control of radioimmunoassays. J . Clin. Endocrinol. 28, 1412-1418 (1968). 9.

Quality Control

The development of a quality control system is essential to evaluate the stability and reproducibility of the assay system. It is based first of all on running a few quality-control samples in duplicate (or triplicate, etc.) in each assay. These samples can either come from plasmas with known concentrations of the antigen to be measured, or they can be made b y adding a known quantity of the antigen to plasma previously stripped of endogenous antigen by charcoal treatment. This last procedure is mostly used for steroids. Quality control charts should be kept with results from each assay. They permit a rapid analysis of stability and reliability of the radioimmunoassay system. The use of the logit-log method allows one to coinpare for each assay the slope of the standard curve, the 50% intercept, the maximal binding at zero dose, and the nonspecific binding. Between-assay variations can be easily calculated by the Student’s t-test.

RADIOIMMUNOASSAY I N THE LABORATORY

169

Another important point for quality control is to verify the parallelism between successive dilutions of an unknown sample and the standard curve. Usually a plasma known to contain a high level of the substance to be measured is selected, thus allowing measurable levels at several dilutions. Parallelism can be checked by any type of plot, although the logit-log plot permits easier comparison (Fig. 11).Parallelism is a good indication, although not an absolute proof, of identity between the material to be measured and the antigen of the standard curve. Nonparallelism, on the contrary, indicates nonidentity and, therefore, that the assay is not reliable for measuring levels of the unknowns. 10.

Application of Radioimmunoassay

The radioimmunological method may be applied to almost any substance which can be obtained in the pure state and to which specific antibodies can be formed. The most common fields of application are those of peptide hormones, nonpeptide homiones (steroids, thyroid hormones, prostaglandins) and nonhormonal substances such as cyclic AMP, enzymes, specific antigens from infectious diseases, and drugs.

10.1. RADIOIMMUNOASSAY

OF

SMALLPEPTIDE HORMONES

Small peptide hormones raise major problems due to their low plasma concentration, of the order of a picogram per milliliter, and to their low immunogenicity related to their small molecular weight (Table 3). This makes the development of a radioimmunoassay very difficult since, on one hand, very avid antisera are needed to have sufficiently sensitive assays to detect their low plasma concentrations and, on the other, small peptides are poor immunogens. Every step of the radioimmunoassay needs to be optimized to get the maximum sensitivity, in particular the search for highly avid antisera and for high specific radioactivity labeling without overiodination. Specificity is also of great importance, since many small peptide hormones belong to structurally related “families” of hormones, such as the families of the gastrointestinal hormones and of the ACTH-related peptides. Moreover, there is often a need to prevent the small peptide hormones from being adsorbed to glassware, and from proteolytic degradation in the presence of plasma. Because of their tendency to nonspecific adsorption to solid material, separation can often be carried out by methods based on adsorption of the free antigen, although other methods, such as the double-antibody or the solid phase antibody

170

J. P. FELBER

-I

10-

STANDARD HUMAN TRYPSIN ng/ml

FIG. 11. Verification of the parallelism between the values of immunoreactive

human trypsin contained in successive plasma dilutions (0-O), Logit-log plot. cuwe (*---*).

and in a standard

TABLE 3 SMALL PEFTIDE HORMONES:APPROXIMATIVE MOLECULAR WEIGHT AND RANGE OF CIRCULATING PLASMA LEVELSIN BASALCONDITIONS Hormones Insulin Glucagon ACTH @-MSH Angiotensin I Angiotensin I1 Oxytocin Vasopressin Secretin Gastrin CCK-PZ

CIP

TRH LHRH Calcitonin

PTH

Nr amino acids

Approximate MW

Basal plasma concentration

51 29 39 22 10 8 8 8 27 17 33 43 3 10 32 84

6000 3500 4500 2500 1200 1000 1000 1000 3100 2100 3900 5100 350 1200 3500 9000

0.2-1.0 ng/ml 130-260 pg/ml 5-50 pg/ml 20-150 pg/ml

-

8-56 pg/ml 0-2 pg/ml 0.6-2 pg/ml 0-80 pg/ml 0-240 pg/ml 0-200 pg/ml 75-500 pg/ml <60 pg/ml 0-70 pg/ml 10-280 pg/ml 250-2000 pg/ml

RADIOIMMUNOASSAY IN THE LABORATORY

171

techniques, can be used. On account of the complexity of radioimmunoassays for small peptide hormones, only a few of them are used for routine determinations.

10.1.1. Insulin The most common of the radioimmunoassays for small peptide hormones is that of insulin. It can be performed by using any one of the known separation methods. Hales and Randle (H2) have developed a method using a double-antibody technique, while Herbert et al. (H6) and Bleicher and Herbert (B5) used dextran-coated charcoal. Details of a method using polyethylene-glycol for separation are given b y Desbuquois and Aurbach (D2),while a commercially available kit makes use of insulin antibody coated to Sephadex as irnmunosorbent. The major and almost absolute indication for the radioimmunoassay of insulin is the diagnosis of insulinoma. The diagnosis of insulinsecreting tumors is often difficult to establish. Several clinical tests have been suggested (F2), with stimulation of insulin secretion b y glucose, tolbutamide, leucine, or glucagon. The extent of the rise in immunoreactive insulin yields useful indications for diagnosis. However, one of the best tests is simultaneous measurement of insulin and glucose during prolonged fasting. The dissociation of the two curves, with insulin going up and glucose going down, is almost pathognomonic of a B-cell tumor. Insulin does not serve in the diagnosis of diabetes, since the concept of diabetes is associated with glucose intolerance, whatever its origin. However, the measurement of insulin together with glucose during an oral glucose tolerance test yields useful indications as to the type of diabetes and the pancreatic reserve available (F4). Prognosis and treatment of a diabetic form are not the same in the presence of endogenous insulin secretion or in its absence. 10.1.2. GZucagon The assay for pancreatic glucagon is a rather difficult one, mainly because of the scarcity of specific antisera. Most antisera raised by immunization with glucagon are not specific for pancreatic glucagon. They measure GLI (glucagon-like immunoreactivity) of intestinal origin. Only a few antisera specific for pancreatic glucagon exist in the world. They have permitted many interesting physiological and physiopathological studies, particularly in the field of diabetes (F3, J 1).One exceptional clinical condition is known where glucagon measurement is needed for clinical diagnosis, that of glucagonoma.

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10.1.3. ACTH The assay for ACTH is a fairly difficult one. Only very few antisera in

the world possess the necessary avidity to allow direct measurements of ACTH in plasma ( B l , Vl). However, a method has been developed by Ratcliffe and Edwards ( R l ) , which permits ACTH radioimmunoassay with more commonly available ACTH antisera, after extraction of ACTH from plasma b y means of calibrated glass powder to which the hormone has adsorbed. In most clinical conditions, the information given by ACTH determinations can be read by measuring plasma cortisol. However, ACTH determination is useful to distinguish the origin of the disease. In the case of Cushing’s syndrome (B2), ACTH is undetectable when the disease is due to an adrenal tumor, whereas it presents normal or high levels when it is of pituitary origin or d u e to an ectopic tumor. In cases of adrenal insufficiency (B4),ACTH measurements are also useful, since they allow distinction between primary adrenal insufficiency, with high plasma ACTH levels, and secondary insufficiency of pituitary or iatrogenic origin, with low plasma ACTH levels.

10.1.4. Gastrin The radioimmunoassay for gastrin presents fewer technical difficul-

ties than the two previous ones. It is most useful for the diagnosis of the Zollinger-Ellison syndrome, due to hypersecretion of gastrin by the pancreatic tumor. Gastrin is also elevated in cases of antral hyperplasia and in pernicious anemia (H4, Y 1).

10.1.5. Angiotensin Z The radioimmunoassay for angiotensin I is also aniong the assays

which present some technical difficulties, It is used for the determination of renin activity. The angiotensin I decapeptide is the product of the action of renin on its substrate, angiotensinogen. The determination of plasma renin activity is carried out by measuring the generation of angiotensin I during plasma incubation. The assay for plasma renin activity provides important information on hypertension and renal diseases. Details of the methodology are found in the papers of Haber et al. ( H l ) and of Sealey and Laragh (S5).

10.1.6. Parathyroid Hormone The assay for parathyroid hormone (PTH) is performed only in a

limited number of laboratories, because of the complexity of the method and the difficulty of obtaining adequate antisera. It is mainly used for the diagnosis of hyperparathyroidism. The method is given in detail by Deftos (Dl).

RADIOIMMUNOASSAY IN THE LABORATORY

10.2.

~ D I O I M M U N O A S S A Y OF

173

LARGERPROTEIN HORMONES

The larger protein hormones can be divided into two major groups, according to their molecular structures. Human growth hormone (hGH), human prolactin (hPRL) and human chorionic somatomammotrophin (hCS), also called “human placental lactogen (hPL)” present close structural similarities, and cross-reactions may be encountered with not fully specific antisera. Another family is that of the glucoprotein hormones, which includes human thyroid-stimulating hormone (hTSH), human chorionic gonadotrophin (hCG), human luteinizing hormone (hLH) and human follicle-stimulating hormone (hFSH). The last four hormones are made of two subunits with close structural similarities for a-subunits and less close similarities between @-subunits. Thus, the @-subunits confer immunological specificity. In contrast to small peptide hormones, these hormones are good immunogens. They do not need to be coupled to immunogenic supports for immunization. They circulate in plasma at a nanogram per milliliter concentration. The major problem does not, therefore, reside in the sensitivity ofthe assay, but rather in its specificity. Since these hormones adsorb rather poorly to solid surfaces, separation methods based on the removal from the incubation mixture of the antibody-bound antigen, should be preferred to those based on adsorption of the free antigen. The double-antibody method is often used, although other methods, such as those using polyethylene-glycol (PEG) or solid phase antibodies, give good results. These various hormones are species-specific, Usually, little or no immunological cross-reaction exists with hormones from other animal species. 10.2.1. Human Growth Hormone Several methods have been proposed for the assay of human growth hormone (hGH) of the anterior pituitary, on the basis of different separation techniques. Most laboratories use the double-antibody system, as first proposed by Schalch and Parker (S3) and further developed by Boden and Soeldner (B6). Precipitation b y ethanol-ammonium acetate has been proposed by Saito and Saxena (S 1). Solid-phase radioimmunoassay has been developed, either by using plastic discs (C4) or coated tubes (C3). Plasma hGH levels are increased in acromegaly. The hormone concentration is measured to check the results of therapy. The assay is particularly useful for the evaluation of the pituitary function, usually during functional tests, either following stimulation with insulin-induced hypoglycemia, arginine infusion, injection of pyrogens or glucagon, or after inhibition by a glucose load.

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J. P. FELBER

10.2.2. Human Chorionic Somatomammotropan or Human Placental Lactogen The placental hormone, human chorionic somatomammotropin (HCS) or human placental lactogen (HPL), presents a structure close to that of hGH or hPRL. Plasma HCS levels increase gradually throughout pregnancy, to reach very high plasma levels, of the order of micrograms per milliliter. The major indication of the assay is that of a screening test for fetal distress during pregnancy ( E l , G2, S2). The assay presents no particular difficulty, since there is no need for high sensitivity, the hormone circulating at high levels. However, as it is often used for emergency cases, the classical double-antibody method ( G l ) should be replaced by other quicker methods such as ethanol precipitation (L2), dioxan precipitation (HS), or solid-phase with immunosorbent (L1).

10.2.3. Human Prolactin Human prolactin is secreted b y the anterior pituitary. It plays a major role in fertility. Not only does it participate in lactation, but it seems also to be involved in the normal sexual function of both men and women. Increased PRL levels, often due to microadenoma of the anterior pituitary, are seen in cases of male or female infertility (B3, T2). PRL secretion is stimulated b y estrogens, TRH (thyrotropinreleasing hormone) and many psychotropic drugs, and inhibited by L-dopa and other pharmacological drugs, particularly bromocriptine. Details of a radioimmunoassay using the double-antibody technique for separation are given by Sinha et al. (S6).

10.2.4. Human Thyroid-S timulating Hormone The major clinical application of the TSH radioimmunoassay lies in

the diagnosis of hypothyroidism. In these cases, both basal levels and responses to synthetic TRH (thyrotropin-releasing hormone) are decreased. The difficulty of the assay resides mainly in the lack of pure TSH and in cross-reactions with the other glycoproteic hormones. Most authors, in particular Hall et al. (H3), Patelet al. (Pl), and Hershman et al. (H7), use the double-antibody technique for separation of the antibody-bound from the free antigen. Values are expressed in terms of units of a reference standard.

10.2.5. Human Luteinizing Hormone and Follicle S timu luting Hormone The measurement of plasma human luteinizing hormone (hLH) and follicle-stimulating hormone (hFSH) is of great importance in evaluat-

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ing the hypothalamic-pituitary-gonadal axis. Levels are low in some cases of hypogonadism and in patients with hypopituitarism. Stimulation with LH-RH (luteinizing hormone-releasing hormone) is a good test for evaluating the pituitary function, particularly in cases of infertility. As with TSH, no pure preparation is available for LH and FSH and the values are given in terms of units of a reference standard. Among the various radioimmunoassay methods, the most widely used are those using the double-antibody technique for the separation step ( F l , 0 1 , S4). Solid-phase antibody methods have also been proposed, using antibodies coupled on Sephadex as immunosorbent ( W l ) or antibody-coated discs and tubes (C2). 10.3.

m I O I M M U N O A S S A Y OF STEROIDS

The radioimmunological method is widely used for the measurement of steroids. It presents certain characteristics of its own, due to the fact that steroids are not immunogenic per se and that they are so closely related in structure that the different antisera are seldom totally specific. As a rule, the biological material to b e tested is first extracted and chromatographed before being measured b y radioimmunoassay. The radioimmunoassay essentially serves as a first measuring device, specificity being given by the chromatographic procedure. However, in some cases, antisera can be obtained which are specific enough to avoid the chromatographic step. Extraction can also be omitted and the assay carried out directly in plasma or urine when a measure of quenching is performed. In this case, counting is measured in DPM instead of CPM, when using tritium-labeled steroids. The production of specific antisera represents the most important step in steroid radioimmunoassay. Steroids are first covalently bound to a protein carrier such as bovine serum albumin. This bond usually occurs between a hydroxyl or a ketone group of the steroid and the amino or carboxyl group of the protein. The site of steroid linkage is of importance because steroid antibodies are most specific for the portion of the steroid molecule which protrudes out of the carrier protein, i.e., the portion opposite to that linked to the protein. Immunization is performed as with other immunogens. A great task is represented by the assessment of titer, affinity and specificity of the antisera. Crossreactivity is expected with structurally related steroids. As a tracer, either tritium-labeled steroids or '231-labeledsteroids are used. The specific activity of tritium-labeled steroids is of the order of 25 to 100 Ci/mM and that of '"I-labeled steroids, 2200 to 4400 Ci/mM. The respective advantages of lZaIand tritium-labeled steroids are dis-

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cussed by Abraham (A3). The assay is performed, in most cases, after solvent extraction (A2, M 1).Chromatographic purification may not be needed for routine assay of the steroids found in relatively large amounts, compared with the other interfering steroids. This is the case in particular for testosterone, progesterone, and estradiol. Antisera with high specificity are needed. However, no antiserum is fully specific, and chromatographic separation must be used when high specificity is required, as well as for the measurement of steroids whose proportion in circulating media is comparatively low in relation to other interfering steroids. Various chromatographic systems have been proposed. Among them, celite microcolumns have been shown most useful (Al). The radioimmunoassay itself does not present major difficulties. Incubation is carried out in the same way as for peptide hormones. Several separation techniques have been proposed: dextran-coated charcoal system, double-antibody or ammonium sulfate precipitation, or solid-phase radioimmunoassay. Depending on the tracer used, counting is performed in beta or gamma counters. Calculations are similar to those used for the radioimmunoassay of peptide hormones, taking into account however, possible losses during extraction and chromatographic purification. The general aspects as well as the detailed procedures on radioimmunoassay of steroid hormones are found in articles by Abraham ( A l , A2, A3), and by the different authors of books edited by Cameron et al. ( C l ) and Gupta

(G3). 10.4. UDIOIMMUNOASSAY OF ENZYMES

The radioimmunological method has also been applied to the measurement of enzymes. It oEers the great advantage, over catalytic methods, of measuring enzymes in terms of concentration instead of activity. The assay is independent of the factors interfering with the biological activity of the enzymes. It benefits from the high sensitivity of radioimmunology and from the specificity of the antigen-antibody reaction. As with other radioimmunoassays, the method requires purity of the antigen used as a standard, and requires antigens to originate from the same animal species as the unknown to b e measured (F5). Among the various assays that have been developed over the past years, the radioimmunoassay of human trypsin has been shown to present clinical interest because it is the only method allowing determination of plasma trypsin concentration (Tl),as enzymic methods for trypsin cannot be used on account of the presence of high concen-

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trations of circulating trypsin inhibitors. The method is specific for pancreatic trypsin. It is used mainly in the diagnosis of acute pancreatitis. REFERENCES A l . Abraham, G. E., Radioimmunoassay of plasma steroid hormones. I n “Modem Methods of Steroid Analysis” (E. Heftmann, ed.), pp. 451-469. Academic Press, New York, 1973. A2. Abraham, G. E., Radioimmunoassay of steroids in biological materials. Acta Endocrinol. S u p p l . (Copenhagen), Suppl. 183, 1-41 (1974). A3. Abraham, G. E., Radioimmunoassay of steroids in biological fluids. J. Steroid Biochem. 6, 261-270 (1975). B1. Berson, S. A., and Yalow, R. S., Radioimmunoassay of ACTH in plasma.]. Clin. Invest. 47,2725-2751 (1968). B2. Besser, G. M., Practical use of plasma inimunoreactive ACTH measurements. In “Immunoassay Methods in Endocrinology” ( R . Levine and E. F. Pfeiffer, ed.), Hoimone and Metabolic Research, SuppI. 2, pp. 78-81. Thieme, Stiittgart and Academic Press, New York, 1971. B3. Besser, G. M., and Thorner, M. O., Prolactin and gonadal functions. Pathol. B i d . 23, 779-782 (1975). B4. Besser, G. M., Cullen, D. R., Irvine, W. J., Ratcliffe, J. G., and Landon, J., Immunoreactive corticotrophin levels in adrenocortical insufficiency. Br. Med. J . i, 374-376 (1971). B5. Bleicher, S. J., and Herbert, V., Radioimniunoassay of insulin in human plasma or serum using dextran-coated charcoal. In “Hormones in Human Blood. Detection and Assay” (H. N . Antoniades, ed.), pp. 165-175. Harvard Univ. Press, Cambridge, Massachusetts, 1976. B6. Boden, G., and Soeldner, J. S., A sensitive double-antibody radioimmunoassay for human growth hormone (HGH):Levels of serum HGH following rapid tolbutaniide infusion. Dicihetologin 3, 413-421 (1967). C1. Cameron, E. H. D., Hillier, S. G., and GriEths, K., eds., “Steroid Immunoassay,” Proc. Tenovus Workshop, 5th, Cardiff, 1974. Alpha Omega Publ., College Bldgs., Cardiff, U. K., 1975. C2. Catt, K. J., Radioimmunoassay with antibody-coated discs and tubes. Acta Endocrinol. (Copenhagen), S u p p l . 142,222-246 (1969). C3. Catt, K., and Tregear, G. W., Solid-phase radioimmunoassay in antibody-coated tubes. Science 158, 1570-1572 (1967). C4. Catt, K., Niall, H. D., and Tregear, G . W., A solid-phase disc radioimmunoassay for human growth horni0ne.J. Lab. Clin. Med. 70, 820-830 (1967). D1. Deftos, L. J., Parathyroid hormone. In “Methods of Hormone Radioimmunoassay” (B. M. Jaffe and H. R. Behrman, eds.), pp. 231-247. Academic Press, New York, 1974. D2. Desbuquois, B., and Aurbach, G. D., Radioimmunoassay of insulin using polyethylene glycol. In “Hormones in Human Blood. Detection and Assay” (H. N. Antoniades, ed.), pp. 200-203. Harvard Univ. Press, Cambridge, Massachusetts, 1976. E l . England, P., Ferguson, J . C., Lorrimer, D., Moffatt, A. M., and Kelly, A. M., Human placental lactogen: The watchdog of‘ fetal distress. Lclncet i, 5-7 (1974). F1. Faiman, C., and Ryan, R. J., Radioinimunoassay for human follicle-stimulating hormone. J. Clin. Endocrinol. 27, 4 4 4 4 4 7 (1967).

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F2. Fajans, S. S., Diagnostic tests for functioning pancreatic islets cell tumors. Diabetes, Proc. Congr. lnt. Diabetes Assoc, 6th, Excerptn Med. Found. lnt. Congr. Ser. 172, 894-897 (1969). F3. Faloona, G. R., and Unger, R. H., Glucagon. In “Methods of Hormone Radioimmunoassay” (B. M. J&e and H. R. Behrman, eds.), pp. 317-330. Academic Press, New York, 1974. F4. Felber, J. P., Clinical use of radioimmunological plasma insulin assessment. Klin. Wochenschr. 51,63-67 (1973). F5. Felber, J. P., Radioimmunoassay of enzymes. Metab., Clin. E x p . 22, 1089-1095 ( 1973). G1. Genazzani, A. R., Casoli, M., Aubert, M. L., Fioretti, P., and Felber, J. P., Use of human placental lactogen radioimmunoassay to predict outcome in cases of threatened abortion. Lancet ii, 1385-1387 (1969). G2. Genazzani, A. R., Cocola, F., Neri, P., and Fioretti, P., Human chorionic somatomammotropin (HCS) plasma levels in normal and pathological pregnancies and their correlation with the placental function. Acta Endocrinol. (Copenhagen), Suppl. 167, 1-39 (1972). G3. Gupta, D., ed., “Radioimmunoassay of Steroid Hormones,” Verlag Chemie, Weinheim, 1975. H1. Haber, E., Koemer, T., Page, L. B., Kliman, B., and Purnode, A,, Application of radioimmunoassay for angiotensin I to the physiologic measurements of plasma renin activity in normal human subjects../. Clin. Endocrinol. 29,1349-1355 (1969). H2. Hales, C. N., and Randle, P. J., Immunoassay of insulin with insulin-antibody precipitate. Biochem. J. 88, 137-146 (1963). H3. Hall, R., Amos, J., and Ormston, B. J., Radioimmunoassay of human serum thyrotropin. Br. Med. J. i, 582-585 (1971). H4. Hansky, J., and Chain, M. D., Radioimmunoassay of gastrin in human seium. Lancet ii, 1388-1390 (1969). H5. Haour, F., A rapid radioimmunoassay of human chorionic somatomammotropin (hCS or hPL) using dioxan. Horm. Metab. Res. 3,131-132 (1971). H6. Herbert, V., Lau, K.-S., Gottlieb, C. W., and Bleicher, S. J., Coated charcoal immunoassay of insulin. J . Clin. Endocrinol. 25, 1375-1384 (1965). H7. Hershman, J. M., Kenima, J. G., Kojima, A., and Saunders, R. L., Assay of thyroidstimulating hormone. In “Hormones in Human Blood. Detection and Assay” (H. N. Antoniades, ed.), pp. 464487. Harvard Univ. Press, Cambridge, Massachusetts, 1976. J1. Jergensen, K. H., and Larsen, U . D., Purification of “‘I-glucagon by anion exchange chromatography. Hwm. Metab. Res. 4,223-224 (1971). L1. Lebech, P. E., and Borgaard, B., Serum levels ofhuman chorionic somatomammotrophin (HCS) in normal and abnormal pregnancies. Acta Endocrinol. (Copenhagen), Suppl. 1 8 2 , 3 5 4 3 (1974). L2. Letchworth, A. T., Boordman, R. J., Bristow, C., Landon, J., and Chard, T., A rapid semi-automated method for the measurement of human chorionic somatomammotrophin. The normal range in the third trimester and its relation to fetal weight.J. Obstet. Gynaecol. Br. Commonw. 78, 542 (1971). M1. Magrini, G., and Felber, J. P., Radioimmunoassay of steroid hormones. In “Clinical Biochemistry, Principles and Methods” (H. c. Curtius and M. Roth, eds.), pp. 784-790. W. d e Gruyter, Berlin, 1974. 0 1 . Odell, W. D., Ross, G. T., and Rayford, P. L., Radioimmunoassay for luteinizing hormone in human plasma or serum: Physiological studies. J. Clin. Inuest. 46, 248-255 (1967).

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PI. Patel, Y. C., Burger, H. G., and Hudson, B., Radioimmunoassay of seium thyrotropin: Sensitivity and specificity. J . Clin. Endocrinol. 33, 763-774 (1971). R1. Ratcliffe, J. G., and Edwards, C. R. W., The extraction of adrenocorticotrophin and arginin-vasopressin from human plasma by porous glass. In “Radioimmunoassay Methods” (K. E. Kirkham and W. M. Hunter, eds.), pp. 502-512. Churchill Livingstone, Edinburgh and London, 1971. S1. Saito, T., and Saxena, B. B., A sensitive, rapid, and economic radioimmunoassay of human growth hormone using ethanol-ammonium acetate. J . Lab. Clin. Med. 85, 497-504 (1975). S2. Saxena, B. N., Emerson, K., and Selenkow, H. A., Serum placental lactogen (HPL) levels as an index of placental function. N . Engl. J . Med. 281, 225-231 (1969). S3. Schalch, D. S., and Parker, M. L. A sensitive double immunoassay for human growth hormone in plasma. Nature (London) 203, 1141-1142 (1964). S4. Schalch, D. S., Parlow, A. F., Boon, R. C., and Reichlin, S., Measurement of human luteinizing hormone in plasma by radioimmunoassay. J. Clin. Inuest. 47,665-678 ( 1968). S5. Sealey, J. E., and Laragh, J. H., Radioimmunoassay of plasma renin activity. I n “Radioimmunoassay” (L. M. Freeman and M. D. Blaufox, eds.), pp. 65-78. Grune 61 Stratton, New York, 1975. S6. Sinha, Y. N., Selby, F. W., Lewis, U. J., and Vanderlaan, W. P., A homologous radioimmunoassay for human prolactin. J . Clin. Endocrinol. Metab. 36, 509-516 (1973). TI. Temler, R. S., and Felber, J. P., Radioimmunoassay of human plasma trypsin. Biochim. Biophys. Acta 445, 720-728 (1976). T2. Thorner, M. O . , McNeilly, A. S., Hagan, C., and Besser, G . M., Longterni treatment of galactorrhea and hypogonadism with bromocriptine. Br. Med. J. ii, 419-422 (1974). V1. Vague, P., Oliver, C., Jaquet, P., and Vague, J., L e dosage radioimmunologique d e I’ACTH plasmatique. Rhsultats chez les sujets normaux. Rev. Eur. Etud. Clin. Biol. 16,485-493 (1971). W1. Wide, L., and Porath, J., Radioimmunoassay of proteins with the use of Sephadexcoupled antibodies. Biochim. Biophys. Acta 130, 257-260 (1966). Y 1 . Yalow, R. S., and Berson, S . A,, Radioimmunoassay of gastrin. Gastroenterology 58, 1-14 (1970).