[27] Development and use of ultrasensitive enzyme immunoassays

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409

[2 7] D e v e l o p m e n t a n d U s e of U l t r a s e n s i t i v e Enzyme Immunoassays

By G L E N N W O O D E.

TRIVERS, CURTIS C . HARRIS, C A T H E R I N E R O U G E O T , and FERNANO DRaY

Enzyme immunoassays (EIAs) have been the most successful of the nonisotopic immunoassays that have been developed to supplement or replace radioimmunoassay (RIA). Because of their safety, low cost, simplicity, and comparable sensitivity, EIAs have undergone rapid and continuous growth in their varieties and their widespread applications for measuring small quantities of molecules of medical and scientific interest. Both laboratory and clinical studies have shown their usefulness in the fields of cancer research 1-6 and infectious disease. 7-9 Although EIAs of polypeptide hormones are still being developed and are not as yet available for routine clinical application, experience in other fields suggests that this will soon become a reality. In this chapter we describe the basic principles of EIAs and provide examples of hormones measured by EIAs. These assays employ reagents immobilized in the wells of Microtiter plates and are usually quantitated by measuring visible, fluorescent, or radioactive products of enzyme substrates. When highly sensitive EIAs were needed, ultrasensitive enzyme radioimmunoassays (USERIA) were developed.9a The sensitivity of these EIAs is in the femtomole range or less. History and Development of the EIA EIAs and RIAs are formulated on similar immunological principles. EIAs are primarily different in that enzyme activity is substituted for 1 I. C. Hsu, M. C. Poirier, S. H. Yuspa, D. Grunberg, I. B. Weinstein, R. H. Yolken, and C. C. Harris, Cancer Res. 41, 1091 (1981). z I. C. Hsu, M. C. Poirier, S. H. Yuspa, R. H. Yolken, and C. C. Harris, Carcinogenesis 1, 455 (1980). 3 j. A. Roth and R. A. Wesley, Cancer Res. 42, 3978 (1982). 4 F. P. Perera, M. C. Poirier, S. H. Yuspa, J. Nakayama, A. Jaretzki, M. Curnen, D. M. Knowles, and I. B. Weinstein, Carcinogenesis 3, 1405 (1982). 5 C. C. Harris, R. H. Yolken, and I. C. Hsu, Methods Cancer Res. 20, 213 (1982). 6 A. Haugen, J. D. Groopman, I. C. Hsu, G. R. Goodrich, G. N. Wogan, and C. C. Harris, Proc. Natl. Acad. Sci. U.S.A. 78, 4124 (1981). 7 A. Voller, D. E. Bidwell, G. Huldt, and E. Engvall, Bull. W. H. O. 51, 209 (1974). 8 I. Ljungstrom, E. Engvall, and E. I. Ruitenberg, Proc. Int. Congr. Parasitol. 3rd, p. 1204 (1974). 9 R. H. Yolken, Yale J. Biol. Med. 53, 85 (1980). 9~ C. C. Harris, R. H. Yolken, and I. C. Hsu, Proc. Natl. Acad. Sci. U.S.A. 76, 5336 (1979).

METHODSIN ENZYMOLOGY.VOL. 103

Copyright© 1983by AcademicPress, Inc. All fightsof reproductionin any formreserved. 1SBN0-12-182003-3

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QUANTITATION OF NEUROENDOCRINE SUBSTANCES

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radioactivity as marker for the presence and quantity of an immunoreacrant. The enzyme is coupled to one of the reactants and amplifies the signal from the antigen-antibody reaction by rapidly converting enzymic substrates to product. In 1969, Avrameas ~° reported the conjugation of several different enzymes to antibodies, using sodium periodate treatment for horseradish peroxidase and glutaraldehyde for alkaline phosphatase and several others. Avrameas used these new reagents to detect solubilized antigens 1~ and antigens localized in tissue. 1° Engvall and Perlmann ~2 combined the radioimmunosorbent technique (RIST) for insolubilizing antibody by coupling to cellulose and the Avrameas method for enzyme-antibody conjugation by glutaraldehyde treatment. The result was a fluid-phase (F-P) EIA for measuring alkaline phosphatase-labeled human IgG, and the introduction of the phrase "enzyme-linked immunosorbent assay" with the acronym ELISA. Soon after, they modified an RIA method used by Catt and Tregear ~3 in which antibody was immobilized on the inner surfaces of polystyrene tubes. This "solid-phase" (S-P) modification 14 allowed the separation o f " f r e e " reactants by decantation and eliminated the necessity for time-consuming centrifugations required in their first study. Van Weeman and Schuurs ~5reported the first EIA for a hormone in 1971. However, it was not until 1975 that Dray e t al., ~6 designed an EIA for a hormone that achieved the sensitivity of the RIA. Often since its evolution, EIA has progressed in parallel with the RIA. However, a departure occurred in 1974 when, Voller et al. 7 designed an assay using polystyrene Microtiter plates coated overnight with solubilized malaria antigen for the purpose of monitoring malaria infections in the field. Microtiter plate assays have subsequently become one of the more popular and important S-P methods. In addition, methods have also been developed for coupling antigens and antibodies to other solid phases including agarose, 17 cellulose, 18 silicone rubber rods, 19 polyacrylamide and glass beads. 2° More recently, magnetic polyacrylamide agarose 10A. Avrameas, Immunochemistry 6, 43 (1969). H S. Avrameas, Int. Rev. Cytol. 27, 349 (1970). 12 E. Engvall and P. Perlmann, Immunochemistry 8, 871 (1971). 13 K. Catt and G. W. Tregear, Science 158, 1570 (1967). ,4 E. Engvall, K. Jonsson, and P. Perlmann, Biochem. Biophys. Acta 251, 427 (1971). 15 B. K. Van Weeman and A. H. W. M. Schuurs, FEBS Left. 15, 232 (1971). ~6F. Dray, J. M. Andrieu, and F. Renard, Biochim. Biophys. Acta 403, 131 (1975). J7 j. G. Streefkerk and A. M. Deelder, J. lmmunol. Methods 7, 225 (1975). Js B. Ferrua, R. Maiolini, and R. Masseyeff, J. lmmunol. Methods 25, 49 (1979). J9 S. Ohtaki and Y. Endo, in "Enzyme Immunoassay" (E. Ishikawa, T. Kawai, and K. Miyia, eds.), p. 198. Igaka Shoin, Tokyo, 1981. 2o F. Dray and C. Gros, in "Enzyme lmmunoassay" (E. Ishikawa, T. Kawai, and K. Miyia, eds.), p. 146. Igaka Shoin, Tokyo, 1981.

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beads 2~ have been employed in order to eliminate the centrifugation normally required for washing and for separating "free" reagents. One class of EIA can be performed in a single step. The "homogeneous" EIA (one antibody and one reaction mixture) developed by Rubenstein e t al. z2 is based upon covalent coupling of a hapten to an enzyme in such a way that, either by steric hindrance or conformational changes, complexes of hapten and anti-hapten antibody inhibit the enzyme substrate interactions. Therefore, "free" haptens in a mixture of enzymelabeled hapten and antihapten antibodies cause an increase in enzyme activity, giving evidence of the specific hapten in a standard preparation or an unknown sample. Most current EIA methods are of the heterogeneous class (requiring one or more wash steps to separate "free" reactants and using one or more antibodies from different species). They are of several different types, including S-P assays in which the unlabeled target antigen or antibody is immobilized on insoluble surfaces, primarily plastic. As in RIAs, assays are designed for noncompetition (NC), competition (C), or competitive inhibition (immunometric23), with either the subject antigen or an antibody as the enzyme-labeled component. In competitive and noncompetitive assays the resulting measure may be direct or indirect with respect to the relationship of the enzyme marker to the molecule being assayed. In noncompetitive assays the antigen (Ag) or the primary antibody (Ab0 is immobilized, and the Ag, Abl, or a heterologous antibody (ABE)is conjugated to the enzyme marker (see Table I). An example of a noncompetitive design is the "sandwich" EIA 24 (variation No. 6, Table I). Immobilized, unlabeled Abl binds Ag from the fluid phase or unknown sample, and the addition of enzyme-labeled Abl results in the direct quantitation of the number of Ag molecules bound in the S-P reaction complex. Performed in reverse, i.e., with labeled antigen, the assay works as well for the detection of antibody. Competitive assays have the basic design of noncompetitive assays except that the unbound specific reactant (either antibody or antigen, labeled or unlabeled) is mixed in F-P with known amounts of the bound reactant as standard or with unknown samples for identification. If neither F-P reactant is labeled, as in the USERIA, the measure is indirect and an additional step is required to introduce an enzyme-labeled heterologous reactant, usually antibody (variation No. 4, Table I). 21 J. L. Guesdon and S. Avrameas, Immunochemistry 14, 443 (1977). 22 K. E. Rubenstein, R. S. Schneider and E. F. Ullman, Biochern. Biophys. Res. Commun. 47, 846 (1972). 23 R. Elkins, Nature (London) 284, 14 (1980). 24 R. Maiglini and F. R. Masseye, J. lmmunol. Methods 8, 223 (1975).

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TABLE I VARIATIONS IN SOLID-PHASE EIA PROCEDURES FOR NONCOMPETITION (NC), COMPETITION (C), AND COMPETITIVE INHIBITION (CI) MODELS USING LABELED (*) AND UNLABELED ANTIGEN (Ag) OR ANTIBODY (Ab) FOR DIRECT (D)

AND INDIRECT (ID) MEASUREMENTS Addition of reagents in sequential steps 1-5 after washing the immunosorbent (Assay type/ method)

1

2

3

Immobilized antigen I. (NC/D) *AbT

Substrate

2. (NC/ID) ~

Abl

*Ab2

Measure hydrolysis Substrate

3. (CI/D)

*Ab~ + Ag

Substrate

4. (CI/ID)"

Ab~ + Ag

*Ab2

5. (C/D)

*Abl + Abl

Substrate

Measure hydrolysis

Immobilized antibody 6. (NC/D) b Ag

*Abl

Substrate

7. (NC/1D) c

Ag

Abj

*Ab2

8. (CI/D)

*Ag + Abl

Substrate

9. (C/D)

*Ag + Ag

Substrate

Measure hydrolysis Measure hydrolysis

Measure hydrolysis Substrate

4

5

Measure hydrolysis

Measure hydrolysis

Measure hydrolysis Substrate

Measure hydrolysis

a Indirect "sandwich"; the indirect "sandwich" (Nos. 2 and 4) is the variation used in the USERIA. b Direct "sandwich." c Amplified direct "sandwich."

The EIA literature is extensive and constantly expanding. Descriptions of many potentially applicable and new methods can be found in several excellent monographs, reviews, and proceedings of symposia j8'24-31 now available. Accessing the literature via computer searches 25 E. Engvall, in "Biomedical Applications of Immobilized Enzymes and Proteins" (T. M. S. Chang, ed.), Vol. 2, p. 87. Plenum, New York, 1977. 26 D. Watson, Lancet 2, 570 (1976). 27 A. Voller, D. E. Bridwell, and A. Bartlett, "The Enzyme Linked Immunosorbent Assay (ELISA)." Flowing Pub., Guernsey, England, 1977. 28 E. T. Maggio, ed., "Enzyme Immunoassay." CRC Press, Boca Raton, Florida, 1980.

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can be extremely difficult, yielding primarily RIA references, owing perhaps to the lack of differentiating nomenclature. In the remaining discussion we will emphasize the development of the USERIAs.9a General Considerations Antibody Production Methods for antibody production can be found in basic texts and in special publications on immunology techniques. This is an area that abounds with variations in method and success. Antibodies for heterologous EIAs are prepared by the procedures used for any other purpose. In our laboratory, as in most, rabbits are routinely used for polyclonal sera and mice for high-specificity monoclonal preparations. Most proteins are immunogenic and can be given in their natural forms for antibody responses. Smaller molecules, such as polypeptides and other haptens, may require a carrier, an immunogenic larger protein, in the immunization mixture. Covalent and electrostatic coupling between molecules such as keyhold limpet hemocyanin (KLH) or bovine serum albumin (BSA) is frequently used with molecules of 5000 daltons or less. The value of an antiserum from conjugated immunogens is based upon the ratio of antihapten antibodies to those directed against the carrier-hapten "linker" or "bridge," which can exist as a part of the carrier molecule. It is also important that the enzyme-labeled antibody demonstrates good and stable affinity (avidity) for Abl, its target antigen. Enzyme-Labeled Antibodies (Conjugates) The most common procedures for coupling enzyme to antibody involve glutaraldehyde or sodium m-periodate; the former for a host of different enzymes, the latter for peroxidase conjugation. 1 Noncovalent coupling is also done using the avidin-biotin system. 31 Avidin has four sites for biotin to attach. If biotinylated Abl is allowed to react with immobilized Ag, and avidin is subsequently added, the result is a potential amplification of the signal when biotinylated enzyme is introduced as the last reactant. This is a within-assay antibody-conjugation through avidin 29 S. B. Pal, ed., "Enzyme Labelled lmmunoassay of Hormones and Drugs." de Gruyter, Berlin, 1978. 3o R. M. Nakamura, W. R. Dito, and E. S. Tucker III, eds., Lab. Res. Methods Biol. Med. 4, (1980). 31 A. Voller, A. Bartlett, and D. Bidwell (eds.), "Immunoassays for the 80's." University Park Press, Baltimore, Maryland, 1981.

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QUANTITATION OF NEUROENDOCRINE SUBSTANCES

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in a biotin sandwich. Whatever the conjugation method is, the conjugate must have high titer and good specificity, both of which must be confirmed by experiment. The conjugate must also be stable for long-term storage and free of both unbound enzyme and antibody. This objective is apparently difficult to achieve, but the advantage in heterogeneous EIAs is that unbound enzyme in the conjugate is removed in the wash step and does not contribute to erroneous results. The most popular enzymes in use with EIAs are alkaline phosphatase (AP), horseradish peroxidase (HRP), glucose oxidase (GO), and fl-galactosidase (BGS). These enzymes satisfy the much desired criteria of availability--low cost and easy conjug a t i o n - a n d , moreover, they have a chromogenic, fluorogenic, or radioactive substrate that is stable and easily measured. In addition, their substrates are cheap, safe, and soluble: p-nitrophenylphosphate (PNPP) for AP; o-phenylenediamine (OPD) + H202 for peroxidase; o-dianisidine for GO; fl-D-galactoside for BGS. In a comparative study 32 of these four enzymes, AP and BGS were shown to be superior to GO and HRP in reproducibility and sensitivity, which were judged to be comparable to RIA. The difference seems largely due to the greater stability of the substrates used with AP and BGS. The substrates of GO and HRP are apparently particularly unstable in EIAs with extended incubation periods. USERIA The USERIA is a S-P EIA modified in the final step by substituting a radiolabeled substrate, i.e., tritiated adenosine 5-monophosphate ([3H]AMP), tritiated p-nitrophenylphosphate ([3H]PNPP), or tritiated onitrophenyl-/3-D-galactoside ([3H]BGS) for nonradiolabeled enzyme substrates. This method (see Models 2 and 4, Table I) has been used to develop assays for several antigens including cholera toxin and rotavirus, 9a carcinogen-DNA adducts, 1,6 and epidermal growth factor (EGF). 33 We describe the method using results that include our experiences with several new antigens. Materials and Methods

Reagents Antigens. Among the antigens used in the assay development studies are hormones or biochemical molecules with hormonal-type effects in in 32 j. L. Guesdon, T. Ternynck, and S. Avrameas, J. Histochern. Cytochern. 27, 1131 (1979), 33 G. E. Trivers, C. C. Harris, M. Yamaguchi, I. C. Hsu, and R. Yolken, J. CellBiol. 87, 230 0980).

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ULTRASENSITIVE ENZYME IMMUNOASSAYS

415

vitro and in vivo human and animal systems. They include adrenocorticotropic hormone (ACTH), using the synthetic tetracosapeptide (1-24) (Bachem), human chorionic gonadotropin (/3 subunit) (HCGb), prostaglandin (PG), thymosin a-1 (t~-l) (from Dr. A. L. Goldstein, GWU, Washington, D.C.), and EGF (from Dr. H. Haigler, UCLA). The data obtained with ACTH will be used to describe the method. Antisera. Two antisera were used in each EIA: one a rabbit antiserum or IgG specific for the antigen (Abl); the other a commercial preparation of alkaline phosphatase-conjugated goat IgG raised against rabbit IgG (Ab2)(Miles Laboratories). Goat anti-rabbit IgG (URIA, Pasteur Institute) was used for immunoprecipitation in the competitive RIA, and sheep antirabbit IgG (URIA) in the insolubilized RIA. Radioiodinated Labels

1. 125I-labeledACTH. Radiolabeling of ACTH was performed by the Iodogen method. 34 Two micrograms of Iodogen (Pierce), 1,3,4,6-tetrachloro-3a-6a-diphenylglycoluril, in 20/zl of dichloromethane was added to an Eppendorf tube, and the organic solvent was evaporated under N2. Twenty microliters of 0.5 M phosphate buffer (pH 7.4) and 5/zl of 0.1 N acetic acid containing 1/zg or 0.4 nmol of ACTH were added successively and allowed to react for 5 min. Five microliters of Na125I (=750 Ci; Radiochemical Centre, Amersham) was added; after 5 min the reaction mixture was chromatographed on CM-52 cellulose (Whatman). Pure monoiodinated ACTH (immunoreactively stable for at least 6 months) was eluted with 0.5 M ammonium acetate buffer (pH 5.2), collected in 0.05 M PBSBSA, fractionated, and stored at 80° or after lyophilization. The specific activity was -~1500 Ci/mmol. 2. [12SI]Abz.Purified sheep IgG (Ab2) was also labeled by the Iodogen method (10/xg + 1 mCi 1251)and purified by gel filtration on AcA-34 (IBF). The specific activity was =5000 Ci/mmol. Substrates. PNPP was obtained from Sigma; [3n]AMP was from Amersham and [3H]PNPP from New England Nuclear (specific activity 15 and 30 Ci/mmol, respectively). Equipment Microtiter Plates. Solid-phase EIAs provide the much desired ease of handling, simplicity, and reductions in the quantities of valuable reagents. However, it should be noted that only relatively small quantities of a reagent attach to the plates, and denaturation and detachment of mole-

34S. Kochwa, (1967).

M. Brownell, R. E. Rosenfield, and L. R. Wasserman, J. Immunol. 99, 981

416

QUANTITATION OF NEUROENDOCRINE SUBSTANCES

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cules may occur. Most important is the fact that the quantity of molecules absorbed can vary from lot to lot and, occasionally, from well to well of a Microtiter plate. In order to limit such occurrences, it is necessary to test a number of different lots or batches of the available products, then store sufficient stock to accommodate the assay for extended periods of time. For the assays described below we used several lots of Dynatec polyvinyl (PV) plates, each pretested for the efficiency of binding a particular antigen to be studied. Polystyrene (PS) plates were also tested but were in no instance chosen for an assay. Instruments. The specialized equipment used included an automatic plate washer, and an EIA reader, but both processes are achievable manually.

Antigen Coating in Microtiter Plates Antigens were coated in 100-/zl volumes of carbonate buffer, pH 9.6, and 1 × or 10× PBS, pH 7.4. Plates were (a) sealed with Parafilm, covered with a top and incubated at 4° at room temperature or at 37° for 16 hr or longer; (b) evaporated to dryness in uncovered plates at 37°. Coated plates were then stored at - 2 0 ° until used. Procedure: EIA/USERIA 1. Antigen-coated (central 60 wells), PV Microtiter plates are washed two or three times with phosphate-buffered saline, pH 7.4 (PBS). Then 200/zl of 1-2% horse serum (HS) in PBS containing 0.05% Tween 20 (PBS-Tween) is added to each well to coat residual binding sites and reduce nonspecific binding (NSB). The plates are covered with Parafilm and a plate top and are incubated for 1 hr at 37° in a humidified incubator or a moisture chamber in a convection incubator. 2. Plates are washed five times with PBS-Tween (5× wash) and I00/zl of Ab~ diluted in 1% HS is added per well according to the protocol. HS is added to the outside wells. The plates are incubated for 1-2 hr as described above. 3. The 5× wash is repeated, 100/zl of Ab2 diluted in 1% HS is added to the test wells, HS is added to the periphery, and the plates are incubated for 1 hr at 37°. 4. The 5x wash is repeated, then three washes are performed with 0.1 M diethanolamine (DEA) buffer, pH 9.8, to remove phosphates remaining from the PBS: substrate is added in 100/A volumes containing 1 mg/ml PNPP in 1 M DEA buffer, pH 9.8 (EIA); or 30-100 pmol of [3H]AMP (specific activity of 15-19 Ci/mmol) or [3H]PNPP (29.9 Ci/mmol) in 0.02 M DEA buffer, pH 9.0 (USERIA).

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5. (a) Hydrolyzed PNPP yields p-nitrophenol that is measured in the EIA reader at 405 nm. (b) Hydrolyzed [3H]AMP releases [3H]adenosine that is measured in 20-~1 volumes from each well eluted with three washes of 1 ml each of 1 : 20 PBS from an 0.8-ml column of D E A E Sephadex (A-25). Unhydrolyzed, charged [3H]AMP is retained on the column. The eluates containing uncharged [3H]adenosine are collected in 7-ml vials, 3 ml of scintillation fluid (with detergent) is added, and the radioactivity is counted. (c) Hydrolysis of [3H]PNPP releases [3H]PNP that is measured by adding 20/A of sample volumes directly to scintillation vials containing 2 ml of PBS, pH 7.4. Three milliliters of a nondetergent scintillation fluid is added, and the resulting biphasic mixture separates the hydrolyzate by dissolution into the scintillation layer for counting. Unhydrolyzed [3H]PNPP remains in the aqueous layer. All tests are performed in duplicate or triplicate and each assay includes the following controls. I. Noncompetitive EIA and USERIA Background: nonspecific binding of heterologous proteins and plastic by A. Abl: wells containing all reagents except Ag. B. Ab2 : wells containing all reagents except Abl (zero control for calculating specific reactions). II. Competitive EIA and USERIA 1. Negative controls: same as I, B above. 2. Positive controls--uninhibited, I00% controls: wells containing all reagents except inhibitor (pure antigen or sample). III. Standard USERIA controls 1. Spontaneous hydrolysis control: counts from an eluate of 20 ~1 of chromatographed stock [3H]AMP or unchromatographed stock [3H]PNPP collected at the time (zero) the substrate is added to the plate(s). 2. Total count control: counts from 20 /.d of unchromatographed stock [3H]AMP or unincubated [3H]PNPP. Some controls are procedural checks that measure the stability of the immunosorbent and the antibody performances with respect to nonspecific activity, and are not used to calculate results. Calculations

Arithmetic means and standard deviations are calculated for each duplicate or triplicate set. In the EIA, net values for noncompetitive test wells are obtained by the formula: (Test mean) - (zero control mean) = net test value

418

QUANT1TATION OF NEUROENDOCRINE SUBSTANCES

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Noncompetitive EIAs are done primarily to establish conditions for competitive assays. The noncompetitive USERIA is also used primarily to design the competitive USERIA. However, some differences do exist. The background control in the Ag-coated wells of competitive USERIA usually doubles spontaneous hydrolysis. However, in the noncompetitive USERIA, which does not have Ag in the control wells, the control can be as high as four to five times the spontaneous hydrolysis controls, which provides an evaluation of substrate stability with time in storage. In competitive assays (a) all test values are corrected for the controls without Abe, and (b) percentage of inhibition is computed by the following formula: 1.0 -

(test mean) - (control mean) ] (100-%oie-~-m-ea~) ~ [co-nir-~ m-ean)J 100

Biological Extract Preparation Tissue. Anterior pituitary lobes of decapitated adult male rats were dissected immediately (on ice) and homogenized in 5-10 ml of cold 2.5 N acetic acid. The homogenate was heated to 95° for 15 min, then centrifuged at 9800 g for 5 min. The supernatant was diluted with an equal volume of BSA (1 mg/ml of buffer) and lyophilized. Peripheral Plasma. Blood samples (rat and human) were collected into chilled tubes containing anticoagulant and centrifuged immediately for 15 min at 3000 g and 4 °. The plasma was separated and stored at - 2 0 °. Chromatographic Purification. Plasma samples were chromatographed on Sep-Pak reversed-phase C~8 cartridges (Waters Associates), selected because of the high percentage (>95%) of the hormone recovered from pretested biological samples (silicic acid (Mallinckrodt, 100 mesh) and gel filtration AcA-202 (IBF) were less efficient). With the top of the cartridge attached to a 3-ml disposable plastic syringe, the Cj8 reversed phase was activated and the sample (in 2 ml of methanol) was added, followed by 5 ml of distilled water. Two successive applications of the sample were made to the column. The silicic matrix was then washed with 5 ml of 1% trifluoroacetic acid (TFA) (Fluka), and the peptides (adsorbed on the surface of the matrix), were eluted with 5 ml of M e O H H20-TFA (80 : 19: 1, v/v/v). The eluate was evaporated to dryness under N2, and the extract was dissolved in the appropriate buffer for the selected immunoassay and lyophilized.

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Results Microtiter Plates

The decision to immobilize the antigen eliminates the necessity for having to label it. This is of particular importance if, as in our laboratory, (a) most of the antigens of importance are in short supply and difficult to make or (b) the antigen exists as a part of a large host molecule, making specific labeling difficult. The binding of proteins to polymers is dependent upon the nature of both the protein and the polymer. 34,35 Binding to polystyrene tubes is apparently independent of concentration up to approximately 1 mg (the upper limit of the "region of independence" or the maximum amount of protein that can bind in a single monolayer) and increases proportionately with temperature and time of incubation) 6 However, depending upon the polymer and the protein, a maximum of absorbed molecules can be reached, which then will decline with further incubation. 35 This may be due to dissociation in protein-to-protein attachments that occur beyond the "region of independence." These "bonds" are perhaps weaker than protein-to-plastic bonds. For that reason, it is important not to exceed the maximum amount of protein in the region of independent binding or I000 ng if one is using polystyrene tubes (6.5 cm 3 of effective coating surface). The limits for plates and especially PV U-bottom plates are not known, but we have not generally found it necessary to exceed 1/xg in establishing acceptable levels of the immobilized proteins. Proteins may bind PS best at their isoelectric p H . 37 Molecular size appears to affect the efficiency of binding; i.e., it has been shown that the amount that binds is inversely proportional to the molecular weight. 36 Moreover, proteins absorbed at very low concentrations can react immunologically like denatured material) 4 We tested both PV and PS plates with each new antigen-antibody system. In addition, we examined different "coating" conditions, i.e., antigen concentrations, Ab dilutions, pH, ionic strengths, temperature, and incubation time. The results of these tests depended most upon the characteristics of the individual antigens. However, purified polypeptides and proteins bound very poorly, if at all, to U-bottom PS plates (Fig. 1). Smaller molecules had lower binding efficiencies and greater molecular or immunogenic instability when bound. For example, EGF (Mr 6400) and prostaglandin E2 (Mr 500) did not bind to PS in our hands and Alpha 1 (Mr 35 R. G. Lee, C. Adamson, and S. W. Kim, Thrombosis Res. 4, 485 (1974). 36 L. A. Cantarero, J. E. Butler, and J. W. Osborne, Anal. Biochem. 105, 375 (1980). 37 I. Oreskes and J. M. Singer, J. Immunol. 86, 338 (1961).

420

QUANTITATION

OF NEUROENDOCRINE

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SUBSTANCES

uJ n,0CI)

0.1i

PS

t

I

0.010

5

10 20 pg ACTH/well

50

100

Fro. 1. ACTH coating efficiencyin Microtiter plates of different plastics. EIA dose responses were performed in (1) polyvinylplates from Dynatechor (2) polystyreneplates from Nunc. 3000) did so very poorly. periods without loss of ACTH and Alpha I were coated fresh before each

Moreover, while DNA stores well for indefinite immunoreactivity when bound to PV plates, used within a week after coating, and EGF was assay.

Determining Optimal Assay Conditions Our S-P Microtiter plate EIAs use Ag as the immobilized immunosorbent, rabbit antibodies (Ab]) specific for the Ag, and commercial goat anti-rabbit IgG-alkaline phosphatase conjugate (Ab2). Each component (the Ag and both Abs) must be optimized individually and in combination in order to obtain the quantities of each required for the most sensitive assay. Titration of Ag and Abl. Optimization for the EIA was accomplished via a series of "checkerboard" titrations of the reagents, beginning with decreasing amounts of the Ag (e.g., 1000 to 1 ng/well in four 10-fold serial dilutions) versus one or more reasonably low dilutions (1 : l0 s, 1 : 10 4, etc.) of Abi, and a proven high concentration of the conjugate (I : 103).

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ULTRASENSITIVE ENZYME IMMUNOASSAYS

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There were four major conditions used for coating the antigens (see Methods). Based upon prejudgments of available information, drying was used for the initial test of ACTH. ACTH (Fig. 2) was tested dry and gave acceptable readings from 0.2 ng to 1 ng using between 1 : 250 and 1 : 10,000 of Abe. This two-way titration (Ag vs Abl) established an acceptable low range of immobilized Ag, as immunosorbent, that was measurable by a reasonably high dilution of Abl. (Acceptable low Ag levels produce substrate conversions that are high enough for statistical accuracy and provide accurate and significant differences when decreased by the activity of an inhibiting reactant.) After refrigerated coating, 1 ng of HCGB per well was measurable between 1 : 2000 and 1 : 10,000 dilution of Ab~ (data not shown, DNS). Ag and Abl Titrations for Plate-Coating Conditions. Optimum conditions for binding the lower range of the Ag to Microtiter plates were tested. PS and PV plates were tested using various diluents (see Methods)

2.0

1.8 1.6 1.4 I,M

1.2 o

1.0 0.8

0.6 0.4 0.2

10

50

100

200

400

1000

pg ACTH ADDED/WELL

FIG. 2. Enzyme immunoassay "checkerboard" dose response of increasing amounts of ACTH-bound (37° to dryness) in polyvinyl plates versus decreasing concentrations of rabbit ACTH antibody: 1 : 250 (O O), 1 : 2500 (© 0), 1 : 5000 (A A), and 1 : 10,000 (A .ZX).

422

QUANTITATION OF NEUROENDOCRINE SUBSTANCES

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1.8

1.6 1.4

m•1.2 U Z m 1.0 O o~ <~ 0.8 0.6

o.41 i o.21 10-2

I I 10-3 10-4 CONCENTRATION OF ACTH ANTIBODY

FIG. 3. Titration of anti-ACTH antibody in polyvinyl plates with 200 pg of ACTH added per well and incubated at 4° (O------O) or 37° (© O) in Parafilm-covered plates at 37° until dry (A A) for approximately 16 hr.

with different ionic strengths and pH values, and each combination at varying temperatures and for different periods of incubation. Alpha 1 (3000 Mr) coated best when dried at 37° (DNS). In addition, as have all Ags tested to date, a-1 attached better when dissolved in PBS, pH 7.4, than it did in carbonate buffer, pH 9.8. PG coated when hydrated at room temperature dissolved in 0.001 ng of bovine IgG. The most resistant of any of the molecules we tested for PV coating, PG was not detectable when dissolved in 0.01% gelatin, 0.001% BSA, or the standard buffers and incubated in any manner described. Contrary to experience with PG, HCGb (Mr 22,000) was the most efficient of the molecules tested for binding PV; i.e., it attached almost equally well under the major coating conditions in PBS, and drying was only slightly better than hydration (DNS). ACTH attached to PV plates whether at 4°, at 37 °, or dried. However, drying was the most effective (Fig. 3). EGF, however, is not detected

[27]

423

ULTRASENSITIVE ENZYME IMMUNOASSAYS

T A B L E II DETERMINATION OF OPTIMUM CONJUGATE CONCENTRATION FOR ACTH EIA Conjugate dilution factor ACTH/well (pg)

NR c 0 10 30 100 1000 NR 0 10 30 100 1000

8000 (1 : 200,000 0.006 0.006 ± 0.001 0.005 ± 0.003 0.012 ± 0.000 0.061 ± 0.006 0.660 -+ 0.050 (1 : 100,000 0.000 ± 0.009 ± 0.001 0.010 -+ 0.001 0.014 ± 0.001 0.035 ± 0.006 0.338 ± 0.059

4000

2000

anti-ACTH antiserum) 0.007 ± 0.014 0.014 ± 0.001 0.024 ± 0.021 ± 0.000 0.030 ± 0.032 ± 0.000 0.059 ± 0.120 -+ 0.010 0.193 ± 1.232 ± 0.047 1.918 ± anti-ACTH antiserum) 0.005 ± 0.011 ± 0.014 -+ 0.005 0.020 ± 0.022 ± 0.010 0.029 ± 0.026 -+ 0.007 0.036 ± 0.074 ± 0.016 0.108 ± 0.628 ± 0.014 1.260 ±

100(F~

0.002 0.008 0.003 0.006 0.013

0.024 0.054 0.062 0.110 0.408 2.000

± 0.006 ± 0.011 ± 0.001 _+ 0.024 ± 0.000

0.002 0.003 0.002 0.022 0.149

0.038 0.044 0.044 0.064 0.204 1.930

± ± 0.001 ± 0.004 ± 0.004 _+ 0.016 ± 0.100

a Absorbance at 405 mm. b Optimum conjugate dilution. NR, normal rabbit seium.

when dried on PV plates, and could not be stored after hydrated attachment. or-1 was immunologically stable for long periods when dried and stored at - 2 0 °. On the other hand, DNA, ACTH (Mr 37,000), and a-1 coated best when dried at 37° . Conjugate Titration. In the second titration we used the acceptable low antigen levels, in combination with one or more high dilutions of Abl, to titrate the conjugate. This titration examined the feasibility of higher dilutions of Ab2 efficiently reacting with the lower range of acceptable amounts of Ag and Abl (Table II). Decreases in substrate hydrolysis were directly related to the decreases in reactant concentrations. The uncorrected data (not adjusted for normal rabbit serum control) shows that within the dose response, differences between ACTH levels appeared best at the highest Ab2 concentration (l : 1000). However, decreases in the control values (normal rabbit serum) between conjugate amounts were directly proportional to decreases in conjugate concentrations. A plot of the data for 1 : 200,000 Abl (Fig. 4) shows that although Ab2 at 1 : 1000 was superior to the 1 : 2000 level in total activity, the latter was in the beginning of the linear response and produced acceptable substrate conversion. In addition, when the data were corrected for the control values (Table III), the result was higher test to control ratios at the lower Ab2

424

QUANTITATION

OF NEUROENDOCRINE

[27]

SUBSTANCES

1.8 1.6 1.4 1.2 z<

,~ et,

1.0

o

',,,'3

0.8 0.6 0.4 0.2

~o

lo 2

103 10

102

103

pg ACTH ADDED/WELL

FI6.4. Four-hour(left)and 24-hour(right) enzymeimmunoassay"Checkerboard"titration of bound ACTHand 1:200,000 dilutionof Ab~ versus decreasingconcentrationsof the alkaline phosphatase-labeled goat anti-rabbit IgG antibody: 1:1O00 (A A); 1:2000 (A A); 1 : 40o0 (O - O); 1: 800o (O 0% concentrations (2 and 4 × 10-3) and lowering of the optimum reaction concentrations. Moreover, in 1:200,000 Abl, the optimum Ab2 was placed at 1 : 2000 for a 4-hr incubation with the substrate. This combination also gave the best differentials between amounts of S-P ACTH. For the 24-hr assay, 1 : 4000 Ab2 became the optimal for both dilutions ofAb~. However, when the Abj concentration was increased (10-5), the optimum Ab2 concentration decreased (1:4000). These results provided optimum levels of Abl (i.e., 2 x 10-5) for specific amounts of Ab2 (1 : 2 x 10-3) to be used with a specific and acceptable range of S-P Ag (200-500 pg/well). Similar conditions are required for sensitive immunometric assay of the Ag in fluid-phase competition for Abe. Optimization of USERIA. Optimization did not end with achievement of specifications for an EIA. The USERIA could be performed with the conditions derived but would not be optimal. Rather, owing to the higher signal amplification generated by USERIA, the assay could, in some in-

[27]

425

ULTRASENSITIVE ENZYME IMMUNOASSAYS T A B L E III DETERMINING OPTIMUM CONJUGATE CONCENTRATION FOR A C T H E I A RATIO OF TEST CONTROL MEAN A405 Conjugate dilution factor 8000

ACTH/well (pg)

4 Hr

0 10 30 1~ 1000

1.0 1.2 2.8 9.8 110.0

0 10 30 100 1000

1.0 .83 2.0 10.2 110.0

4000

24 H r

4 Hr

24 Hr

2000 4 H:

1:2~,000 anti-ACTH antiserum 1.0 1.0 1.0" 1.0a 1.2 1.8 5.& 2.5 a 2.0 2.3 6.3" 3.6" 7.2 7.8 28.6 a 15.6" 83.0 78.0 267.0" 208.0 a l : 1 0 0 , 0 0 0 a n t i - A C T H antiserum 1.0 1.0 ~ 1.0~ 1.0 1.0 1.8 a 1.8 a 1.1 1.8 3.1" 3.6" 3.0 8.2 12.6" 16.~ 11.4 86.0 135.0" 176.0 a 118.0

1000

24 Hr

4 Hr

24 H r

1.0 2.2 3.5 14.8 197.0

1.0 1.0 2.0 9.0 95.0

1.0 1.1 2.7 14.0 122.0

1.0 .7 2.6 11.0 90.3

1.0 1.0 1.7 11.6 60.0

1.0 1.0 2.2 12.2 48.8

° O p t i m u m conjugate and Ab~ dilution for short and prolonged incubation periods.

stances, be overweighted with reagents and less sensitive than otherwise possible, with careful repetition of the procedure described, using [3H]AMP as substrate. For example, in Fig. 5 the sensitivity of a competitive USERIA for HCGB is shown to increase with the decrease in the amount of immobilized antigen. The 200 pg previously used in the HCGB EIA was therefore reduced to 100 pg per well vs 1 : 50,000 Abl for USERIA. Similarly, in determining conditions for a competitive USERIA for ACTH, we retested in USERIA the results achieved in the EIA. Figure 6 shows results of such a test of Ab2 concentrations in the USERIA for ACTH, beginning below the EIA optimums and incubating with the [3H]AMP for only 1 hr. These tests for USERIA conditions resulted in specifics for competitive USERIA that were considerably different from those we established for the competitive EIA. An important influence on these differences derived directly from concerns for validation and reproducibility of the assay in practice. The conditions used are shown in Fig. 7, and include three major modifications of the standard protocol: (a) In the second step, 0.05% bovine y-globulin (gG) or 0.1% HS was used instead of 1% FCS. Serum contains both ACTH and other molecules (i.e., BSA) that will bind ACTH and could interfere with the assay. (b) Also in step 2, the time and temperature of the immunological reaction was changed from 1 hr at 37 °

426

QUANTITATION OF NEUROENDOCRINE SUBSTANCES

[27]

100

90 80 7O

Z

o

60

I

_z 50

~ 4o u.l o_

30 20

/ s i /

s

.

10

1.0

10 pg HCG8

102

500 103

FIG. 5. Results of competitive USERIA for HCGa showing increasing sensitivity when 200 pg (O Q), 100 pg (O---O), and 50 pg (A A) of the solid-phase hormone was added to the plate. After the coupling of alkaline phosphatase-labeled Abe, the plate was incubated for 3 hr with [SH]AMP.

to 16-24 hr at +4 °. In the first trial of the new assay, conditions were altered to favor molecular integrity of the hormone and maximum sensitivity for detection of normally occurring very low concentrations of ACTH. (c) In step 4, incubation with the substrate was 24 hr instead of the 4 hr suggested by earlier preliminary tests. In order to improve reproducibility, we used the upper level of the acceptable low amounts of S-P Ag (200-500 pg). As a result, the optimum Abl concentration was decreased to 1 : 2.4 x 10-5, which gave very low kinetics in these conditions (+4 °, 0.05% gG) and required longer incubation. The initial test of the USERIA for ACTH was a comparative study (Fig. 8 and Table IV) including both EIA and RIA competitive measure of purified ACTH in standard curve determinations. The solubilized RIA (RIAs) was performed with the double Ab method using 125I-labeled ACTH in the F-P and propylene glycol to facilitate the precipitation of the labeled complex. In addition, an insolubilized RIA (RIAt) was done with

[27]

ULTRASENSITIVE

ENZYME

427

IMMUNOASSAYS

1(]

9 g.I

z

8

e,,

7

',r

6

x

5

r-,

4

I

10

I

I

10

L

I

30

I

I

100

I

I

300

1000

pg ACTH ADDED/WELL

FIG. 6. A USERIA "checkerboard" titration of increasing solid-phase ACTH versus 1 : 50,000 Abt and decreasing concentrations of alkaline phosphatase-labeled Ab2: 1 : 1000 (A A); 1 : 2000 (O O), and 1 : 4000 (O O). The plate was incubated with [3H]AMP for 1 hr at 37°.

[125I]Ab2. The Ag, A b l , and Abz used in each assay were f r o m the same stock preparations; h o w e v e r , Ab2 for the E I A and U S E R I A was APconjugated (Table IV).

Characteristics of the Standard Curves The E I A optimized for the new conditions ( + 4 ° immunologic incubation in 0.05% gG) required 1000 pg in S-P, and 1 : 60,000 of A b l . The RIAI had a 50% competitive inhibition (CIs0) at 194 pg, a detection limit (DL) at 50 pg, and was the least sensitive of the four assays. The R I A s , E I A , and U S E R I A d e m o n s t r a t e d successively higher sensitivity. With a CIs0 of 5.6

428

QUANTITATION OF NEUROENDOCRINE SUBSTANCES

[27]

coat ACTH in PBS Dulbecco's overnight (37 °C until dry) wash incubate with horse serum (2%,1hr 37 C) ,~ W A S H

1

>--o

(16hr t o 2 4 h r , 4 C) in PBS Tween + 0.05% bovine globulin or 0.1% horse serum ~ WASH

incubate (2 hr 37' C)

WASH

+ Substrate

incubate 4 hr to overnight, 37 °C

DETECTION

FIG. 7. Procedure for performing the indirect sandwich solid phase competitive enzyme immunoassay or ultrasensitive enzyme radioimmunoassayfor ACTH: l, Ag immobilized in polyvinyl plates; 2, addition of free antigen in solution with Abt; 3, addition of AbE-enzyme conjugate; 4, addition of nonradioactive or radioactive substrate; 5, quantitation of enzyme activity. pg, and a D L of 1 pg, USER1A exhibited approximately 6-, 10-, and 50fold greater sensitivity than did EIA, RIAs, and RIA~, respectively, while using lower amounts of reagents. Moreover, the data show (a) that, as a measure of A C T H , the radioactive substrate in U S E R I A provided encouraging improvement over classical EIA (colorigenic substrate), and (b) that the RIA~ used was inferior to the RIAs.

Application to Biological Samples Based upon the results described above, the RIAI was considered to be too insensitive for inclusion in the comparative measure of A C T H in biological samples. As stated, each immunoassay for A C T H was optimized individually to compare the different capabilities for performing the standard curves. F o r this purpose, dilutions of Ab~ and the amount of coated antigen were selected for each SP-EIA based upon reasonable reaction times required to develop acceptable specific amounts of the

[27]

429

ULTRASENSITIVE ENZYME IMMUNOASSAYS TABLE IV ACTH USING THE SAME STOCK REAGENTS

COMPARISON OF IMMUNOASSAYS FOR

RIA Insolubilized (I) phase

Assay Coating (pg) Rabbit anti-ACTH antibody 50% Competitive inhibition (pg) Detection limit (pg)

Soluble (S) phase

EIA Solid phase

USERIA Solid phase

2000

0 (10)"

1000

500

1 : 30,000 194

1 : 90,000 76

1 : 60,000 24

1 : 240,000 5.6

50

10

6

1.0 Alkaline phosphatase (Miles)

Sheep or goat anti-rabbit IgG antibody (Ab2) Substrate a 125I_ACTH"

100

90 80 70

"

1 /zg/ml,

--

1 /xg/ml

0.1 /zg/ml

PNPP

[3H]AMP, [3H]PPNP

1:5I-Ab2

\\ .,,,,,

50 40 30 20 I

10

55.4

10

I

24

50 75100

2OO

500

pg ACTH FXG. 8. Comparison of RIA, EIA, and USERIA for ACTH. Standard curves were generated using the methods described. RIA was done with insolubilized (I) 125I-labeled Abl (A &) and solubilized (S) (© ©) Ab~ using ~25I-labeled ACTH. The difference between the EIA (O Q) and the USERIA (× x) is in the source of the signal only (colorimetric; radioactive). Based upon 50% inhibition, the sensitivity of the USERIA was greater than that of EIA, RIA~, and RIAs by 4-, 14-, and 37-fold, respectively. See Table IV for details.

430

QUANTITATION

lie

li4

OF NEUROENDOCRINE

li2

[27]

SUBSTANCES

1il

rat plasma dilutions

~0

~

~

1:1000

1:500 I rat

1:250 1;125 a t pituitary' dilutions

1:62.5 t

80

70 60 x

oN) 4O 3O

10 I

I:,40

I

lO

I

1:20

1:~0

li5

human plasma dilutions

lOO pg ACTH/well

1:2.5 I

lOOO

FIG. 9. Competitive RIAs for ACTH: (l) standard curve, (2) rat plasma after stress, (3) rat plasma in basal conditions, (4) human plasma after addition of 10 ng of synthetic ACTH (1-24), (5) human plasma after addition of I0 ng of synthetic ACTH and Sep-Pak purification, (6) acid extract of the anterior lobe rat pituitary. Sample dilutions are shown for each biological sample. different signals. The effects of increasing amounts of purified A C T H and plasma on the immunologic reaction (B/Bo × 100 vs log dose) in each method are shown in Figs. 9-11 for RIAs, EIA, and USERIA, respectively. Immunoassays without Sample Purification A C T H was measured directly in 2-fold, serial dilutions of peripheral plasma from resting and stressed male adult rats and after the addition of 10 ng of pure A C T H to 1 ml of plasma from adult men (Table V). Results in EIA were similar to competitive RIA but required a smaller volume o f plasma (12.5/~1 vs 100/~1 for RIA) to obtain 50% inhibition. H o w e v e r , as shown in Fig. 10 (2), E L I S A gave proportional values of A C T H only after the second dilution. On the other hand, due apparently to the interference o f unknown constituents of plasma, CI was not achieved in the U S E R I A

[27]

431

ULTRASENSITIVE ENZYME IMMUNOASSAYS 1:8 I~

1:4

L'~

7o

'~

1:2

1:1

1:2Q~)

1:1000

I:'~Q

~.®

1'.250

1:125

ELISA

6o x

"

/

10 ~

l:y I

I

[

5

10

2O

1:20 1:~0 1i5 I human plasma dilutions 4O

100 ACTH/well

pg

1'21.5 ~ I

1000

FIG. 10. ELISA for ACTH: (1) the standard curve, (2) rat plasma after stress, (3) rat plasma in basal conditions, (4) human plasma after addition of 10 ng of synthetic ACTH (1-24), and (6) acid extract of the anterior lobe of rat pituitary. Sample dilutions are shown for each biological preparation. Note that curves (1) and (2) are parallel only after the second dilution of the sample. a b o v e a concentration of 0.4% of unpurified rat or h u m a n p l a s m a (Fig. 11). Immunoassays after Purification

Tissue. Extracts of rat anterior pituitary lobes were used as a source of A C T H . The values m e a s u r e d (Table V) were similar in each of the three assays. H o w e v e r , c o m p a r e d to the R I A , the amount of tissue required for m e a s u r e m e n t in the E L I S A was reduced by a factor of 2, and in the U S E R I A by a factor of 8. Plasma. The results of A C T H m e a s u r e m e n t s with U S E R I A following p l a s m a purification on Sep Pak columns are shown in Table V and Fig. 11. After the c h r o m a t o g r a p h i c step, U S E R I A gave results that were interpretable and similar to those f r o m competitive R I A or E L I S A . Again, however, the v o l u m e of p l a s m a required for CIs0 in the R I A was reduced

432

QUANTITATION

1:22400 •

1:11200 & ~

OF

NEUROENDOCRINE

1:5600

90

SUBSTANCES

1:28~0 1:1400 1:700 • • __ & rat pituitary dilutions

8o

[27]

1:350 A

1:175 •

USERIA

70

6o

®

8 ×

50

3O 2010

,

1:1320 L~ i

1

2.5

1:660 1:500 , ," ~ _ _ " " ~ 'j j human plasma dilutions,

5

10 20 pg ACTH/well

40

1 20 :

1 :30, i

100

FIG. ] 1. USERIA for ACTH: (1) standard curve, (4) human plasma after addition of l0 ng of synthetic ACTH (1-24), (5) human plasma after addition of ]0 ng of synthetic ACTH and

Sep-Pak purification,(6) acid extract of the anterior lobe of rat pituitary. Sample dilutions are shown for each biological preparation. Note that there was no parallelism when USERIA was performedwithoutplasma purification. by a factor of 3 in the EIA and by a factor of 12 in the USERIA. These differences in volume are almost identical to the differences in sensitivity obtained in the standard curves. Discussion Microtiter plate EIAs require a good and reliable antibody, a good stock of the specific antigens, reagent (conjugate), and supplies (i.e., plates) vital to the ongoing ability to perform the assays. One of the important demonstrations in our experience is that PV U-bottom plates either bind antigens more efficiently (more in a shorter period) or, for geometric reasons, they retain more molecules than the flat-bottomed wells with fight-angled walls. (In U-shaped wells gravity sedimentation could induce molecules to accumulate from the center or lowest point in the well. Binding may then occur in a "brick-laying" progression, terminating with securely bound molecules extending by vertical stacking

[27]

ULTRASENSITIVE ENZYME IMMUNOASSAYS

433

TABLE V IMMUNOASSAYS FOR ACTH IN BIOLOGICAL SAMPLES

Sample Without treatment Rat plasma Basal conditions After stress Human plasma + 10 ng ACTH/ml After treatment Rat anterior pituitary lobes after acid extraction Human plasma + 10 ng ACTH/ml after Sep Pak column

Solubilized RIA

ELISA

103.0 ±- 11 pg/ml 762.0 ± 46 pg/ml 10.4 --- 0.6 pg/ml

88.0 +- 14 pg/ml 801.0 -+ 80 pg/ml 10.9 -+ 0.8 pg/ml

NI b NI NI

319.0 ± 19 pg/ml

338.0 --- 38 ng/ml

324 --- 48.0 ng/ml

11.9 ± 1.2 pg/ml

12.0 ± 1.7 ng/ml

10-+ 1.6 ng/ml

USERIA ~

a USERIA required 8- and 4-fold less tissue and 12- and 4-fold less plasma than the ELISA and the RIA, respectively, to achieve 50% inhibition. b NI, not interpretable.

along the walls of the wells.) Also important is the use of commercial conjugates that allows the investigator to concentrate on producing one good antibody for each system and more easily to standardize the assay systems. EIAs are quite easy to perform. However, each antigen-antibody system must be approached as a new challenge, and each should be studied for optimum activity when allowed to react in experimental conditions. Only then can the maximum sensitivity of an EIA system be achieved. As suggested by our ACTH data, the EIAs, especially USERIA, may potentially replace RIA for certain immunoassays of medical interest. In each case, however, the decision will require experimental evidence for the assay of choice. The sensitivity limits of the EIAs are restricted primarily by the quality of the antibody and the efficiency of the immunosorbent to immobilize on surfaces conducive to the performance of the reactions required. As seen in these data, the success of any amplification procedure in enzyme immunoassays will depend on the last step, application to a given biological sample. In earlier studies, Dray et al., failed in their effort to apply another ultrasensitive and novel immunoassay, i.e., viro-immunoassay, for measuring steroids in plasma directly or after solvent extrac-

434

QUANTITATION OF NEUROENDOCRINE SUBSTANCES

[27]

tion) 8 Similar failures were experienced with USERIA for plasma ACTH when the assay was performed without purification. Moreover, it was after correction for "blank" interference (the amount of substrate converted in the absence of the test antigen) following chromatographic purification of plasma that USERIA was not only operative but shown to be more sensitive than ELISA. In addition, plasma purification for ACTH measurement gave higher values of the hormone after purification than found before purification (Table V). The moderate increase observed could be explained by the release of ACTH bound to macromolecules such as serum albumin. 39 For this reason, the requirement for an appropriate purification step, such as used to obtain USERIA measurements in plasma, becomes a general constraint for any immunoassay, whether directly measurable or not, because of the potential for the binding of peptides to macromolecules and the presence in plasma of related compounds (prohormones, metabolic products, etc.) that can recognize the hormone-specific antibody. Establishing Microtiter plate S-P USERIA requires essentially the same preliminary efforts as for the EIA. However, USERIA requires additional optimization for the highly amplified radio signals that constitute the increased sensitivity of the method. The use of radioactive reagents may seem a reversal of the process of simplification and affordability we have stressed in earlier discussion of the EIA. However, this shift achieves increased sensitivity and a significant extension of the basic EIA principles described. Whereas Engvall et al. combined enzymes, antibodies, and plastics to achieve a safe and simple amplified marker of Ag-Ab reactions, Harris and co-workers have combined enzymes and radiolabeled substrates to achieve the magnification of the enzyme-amplified antigen-antibody reaction signal. Moreover, it is now generally accepted 23'4°'41 that radiolabeled substrates provide valuable potential for effectively increasing the sensitivity of immunoassays. Developments of this kind are essential to our ability to investigate the more difficult problems in medical research, i.e., at the level of the gene and its interactions with nongenetic molecules. The greater sensitivity of USERIA justifies its additional requirements and will provide increased capabilities in many fields of investigation. 3s F. Dray, E. Maron, S. A. Tillson, and M. Sela, Anal. Biochem. 50, 399 (1972). 39 j. E. Stouffer and I. C. Hsu, Biochemistry 5, 1195 (1966). 4o E. Engvall, in "Enzyme Immunoassay" (E. Ishikawa, T. Kawai, and K. Miyai, eds.), p. 1. Igaku Shoin, Tokyo, 1981. 4t S. Avrameas in "Immunoassays for the 80's" (A. Voller, A. Bartlett, and D, Bidwell, eds.), p. 85. University Park Press, Baltimore, Maryland, 1981.