Chapter 2 Immunodiffusion in gels

CHAPTER 2

Immunodiffusion in gels

During the process of gelation of agar, a micelle structure is formed. Molecules with a molecular weight below 200,000 can pass relatively freely through the micelles; but those of a larger size, such as fibrinogen, a-2-1ipoproteir1, a-2-macroglobulin and TgM-globulin are retarded because of friction between the surface of the protein molecules and the channels of the micelles (Wieme 1959). The immunoprecipitates formed between antigen and antibody during the phase of immunodiffusion are usually of a molecular size above 200,000 and cannot diffuse further or be washed out of the gel once they have been formed. On the other hand, un-reacted antigen and antibodies may easily be washed out if they have a molecular weight below 200,000. This is the basis for the identification of immunoprecipitates with protein stains in both immunodiffusion and immunoelectrophoresis.

2. I . Diflusion in one dimension 2.1.1. Single (simple) difusion in one dimension (Oudin’s technique) The process of diffusion of an antigen in an antibody-containing gel, or of an antibody in an antigen-containing gel (fig. 2.1) is in agreement with Fick’s law (see p. 410). The experimental technique based on this fact (single diffusion) was first described in detail byOudin( 1946).It may be used for qualitative evaluation of mixtures of antigens, but its main application has been in the field of quantitative estimation of protein 423

Subjecf indrx p. 557

424

IMMUNOCHEMICAL IDENTIRCATION OF MACROMOLECULES

antigens in biological fluids (see review by Oudin 1952; Storiko 1965). The Oudin technique is based on the fact that in gels, proteins are physicochemically independent of each other (Oudin 1952; BuchananDavidson and Oudin 1958) and so the diffusion of mixtures of antigens into an antibody-containing gel will produce independent precipitates from each antigen-antibody pair. It is not possiblc to apply this method to polyvalent reactants. Thus, the diffusion of a complex antigen mixture into a gel containing a polyvalent antiserum will give rise to multiple precipitin lines partially overlapping each other. However, using a mono-specific antiserum, the corresponding antigen can easily be identified in different samples by the presence or absence of an immunoprecipitate in the gel. With specific antisera it is also possible to reveal specific antigenic groups in similar proteins of different biological origin, thus for instance, the allotypic groups in, serum protein antigens (Oudin 1956). Since the distance of diffusion after a certain time is proportional to the logarithm of the concentration of the antigen in question, this technique may be used for quantitative purposes by comparing the distance of migration of the test sample of antigen with migration of the same antigen at known concentration, the same antibody concentration being used throughout. However, the mathematical equations outlined are valid only if the other antigens present are nonrelated antigens. If the sample contains two or more related antigens, the relationship between the length of diffusion and the concentration of the reactants is more complex (Buchanan-Davidson and Oudin 1958). In practice (see pp. 41 1412,451-453) quantitative results are in all cases based upon determination of distances or areas of diffusion (vide infra). The diffusion time ( t ) determines the migration distance of the precipitate ( x ) according to the equation: x = k l J t , kl a constant. Details of the use of this technique are described in appendix 8. 2.1.2. Single radial dirusion (Petrie plate technique)

I

In single radial diffusion one of the components in the antigen-antibody system (usually the antibody) is incorporated in an agar-gel spread on a flat surface, whereas the other 'component is applied in a hole in the

IMMUNODIFFUSION IN GELS

425

gel and allowed to diffuse radially. Ring-shaped precipitation bands will form and migrate concentrically around the holes; after some time the migration ceases, owing to the fact that the amount of antigen applied in the holes is not in such excess over the antibody in the gel as in the case of the Oudin tube technique. After a certain time at a fixed temperature, the areas covered by the immunoprecipitates are examined either directly or after staining with protein stains (Mancini et al. 1965). This technique was originally developed by Petrie (1932) for qualitative comparative purposes; if two antigens applied in neighbouring wells are immunologically identical, a complete fusion of the boundaries of the zones containing immunoprecipitates occurs. In cases of non-identity, the rings around each well are not influenced by the neighbouring rings. Where there is partial antigenic relationship, the rings coalesce. This method was developed by Mancini et al. (1964) for quantitative purposes. The authors demonstrated empirically that a straight-line relationship exists between the antigen concentration and the area or diameter of the terminal immunoprecipitate, provided the diffusion is allowed to proceed until all the antigen has been combined: Sw+Sp= S O + K C A where ~, S , is the area of the well, Spthe area of the immunoprecipitate, SOthe intersection with the abscissa. A rectilinear relationship was found over a wide range of antigen concentration. A series of dilutions of standard antigen solution is set up as a reference on the test plate. This method has recently been used widely (review Schwick and Storiko 1965; Storiko 1965; Becker et al. 1968) for estimation of serum proteins, but it is a relatively slow technique compared with the Laurell electrophoresis in antibody-containing gel (p. 437). The method is not limited to proteins with low isoelectric point as is the Laurell technique which does not allow determination of immunoglobulins possessing a low electrophoretic mobility (vide infra). Details of the use of this technique are described in appendix 9.

Subject index p. 557

426

IMMUNOCHEMICAL IDENTIFICATION OF MACROMOLECULES

I I

agai-+'antibody

I

agar i- antigen ant i body

I$=

antigen

I

a!&E 5.

aritlbody

antigen

I

I

A!:

arItibOd:

Fig. 2.1. Single and double immunodiffusion. The relationship between the concentration of an antigen or an antibody diffusing from a lateral well through an agar gel and the distance of migration at a certain time (Oudin 1952). Abscissa: distance from the trough. Ordinate: concentration (C) of the diffusing reactant (antigen: ag, antibody: ab) at a certain time. The point of equivalence is indicated by eq. A : diffusion curve for an antigen diffusing through agar gel mixed with corresponding specific antibody; B: diffusion curve for an antibody diffusing through agar gel mixed with the corresponding antigen; C and D: diffusion curves in double diffusion of both antigen and antibody through an intermediate block of agar; D : shows the change in the point of equivalence between the two reactants with change in antigen concentration. The ordinate on the right represents the concentration of antibody. The curve on the right represents the diffusion curve of specific antibody in the intermediate agar block. The ordinate on the left represents antigen concentration. The two diffusion curves on the left represent curves corresponding to two different concentrations of antigen applied in the lateral antigen well. (Continued on page 427.)

IMMUNODlFFUSlON IN GELS

421

2.2. Double diflusion 2.2.1. Double difusion in one dimension This method was developed from the Oudin technique by Oakley and Fulthorpe (1953). It involves the presence of antigen and antibody compartments situated in a glass tube on each side of the common gel compartment. After application of the reactants in their respective compartments, the antigen and the antibody will diffuse towards each other in the common gel. A precipitate will be formed at the place of equivalence. The mathematical basis of this method has been mentioned previously (pp. 410-41 1). Comparison of single linear diffusion with double diffusion (fig. 2.1) shows that the distance travelled under identical experimental conditions is greater in the former method. This means that single diffusion determines antigens more accurately than double diffusion. The method has the same range of application as the Oudin technique (Ouchterlony 1962). 2.2.2. Double difusion in two dimensions This method, which was developed simultaneously by Ouchterlony (1949) and Elek (1948), involves the use of agar plates with wells for both antigens and antibodies. The two reactants diffuse into the gel,

The change in point of equivalence with increased concentration of antigen is shown as A,. Had this been a single diffusion experiment (e.g. A), the point of equivalence for the right hand curve would have been more to the right; its change in position with change in antigen Concentration is indicated as A I . From these changes in position of point of equivalence in single and double diffusion, it is seen that single diffusion gives a more pronounced change in the point of equivalence with change in antigen concentration than does double diffusion. This argues for superiority of single diffusion over double diffusion in quantitative estimation of antigen or antibody. The dotted lines continuing the diffusion curves, indicate that the exact shape and the mathematical equation explaining these extremities are unknown because of the unknown consumption of the reactants at the point of equivalence. Subject index p. 557

428

IMMUNOCHEMICAL IDENTIFICATION OF MACROMOLECULES A

0

0 O O

0

0

O O

C

0 0

D

0

0

0 0

0

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0

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0

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P 0

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Fig. 2.2. Examples of basic set-ups for double immunodiffusion. A : The circular well method; B : the (quantitative) agar plate method; C: quadrangular set up; D : the basin set-up.

in which the immunoprecipitatesare formed at the point of equivalence for each antigen-antibody pair. Each precipitate acts as an immunospecific barrier for the particular pair of reactants and prevents their further diffusion, but does not hinder diffusion of other reactants. Thus, if the concentrations of antigen and antibody are reasonably balanced, the precipitate does not migrate, but grows peripherally in lines or arcs at constant angles to the line joining the two wells. In an unbalanced mixture, the excess of one reactant might give rise to migrating or multiple precipitates through alternate solubilization and reprecipitation. Petrie dishes or microscope slides may be used; the wells are situated either circularly around a central hole, in triangles or quadrangles, or alongside a central trough (figs. 2.2 and 2.3). The method is simple to carry out and is widely used for examination of antigenic compositions of unknown samples as well as for the evaluation of the antigenic relationship between different protein antigens. The method may also be applied to a semi-quantitative evaluation of the titre of one of the reactants compared with known concentrations in other wells (Fein-

IMMUNODIFFUSION IN GELS

429

Fig. 2.3. The double immunodiffusion method based upon triangular plate technique for qualitative and quantitative analysis. (I) Antibody reservoir; (2) reservoir for standard (reference) antigen; (3) reservoir for unknown (sample) antigen ; the distances A and Bare a function of the distances travelled by the end point of the immunoprecipitate formed by the unknown reactant and the standard indicator respectively. The ratio A:B is called the diffusivity ratio. This ratio is linearly related to the logarithm of concentration of the unknown sample (see p. 453).

berg 1959). By applying serial dilutions of samples in circularly placed wells, the immunoprecipitates around each well will fuse with each other. The end point of this immunoprecipitate indirectly defines the antigen concentration as the reciprocal value of the dilution of the antigen present in the corresponding well. As shown in fig. 2.1 a direct quantitative estimation of one of the reactants is possible by measurement of the distance of immunodiffusion at a certain time, but since the distance is small, it is not recommended for accuracy. Full instructions for double diffusion using the Ouchterlony technique are given in appendix 11.

2.3. Determination of difusion constants During immunodiffusion (double diffusion) the point of first appearance of the precipitate along the axis joining the two wells depends on Subject index p . 557

430

I MMUNOCHEMICAL IDENTIFICATION OF MACROMOLECULES

the diffusion coefficient of the two reactants, expressed by the equation : where XAg and X A are ~ the migration distances from the antigen and the antibody well respectively, and D refers to the diffusion coefficient of the two reactants (Polson 1958). This equation has been used to estimate the diffusion coefficients of the antigen or antibody (Van Oss and Heck 1961). In a two-dimensional double diffusion, the initial precipitate will be formed medially at a certain point between the two wells and will extend as an arc with a radius determined by the distances from the two wells r = X A b . X A g / ( X A b - X A g ) . The ratio between the two migration distances is proportional to the square root of the ratio between the two diffusion coefficients as indicated above. The diffusion coefficient of one of the reactants can be determined on the basis of this equation by knowledge of the diffusion coefficient of the other reactant and by estimation of the position of the precipitate in the gel between the respective wells when the two reactants are present in equivalent proportions (Preer 1956). Thus, the diffusion coefficient for hemocyanins, poliomyelitis virus, diphtheria antitoxin and the turnip yellow mosaic virus have been estimated by this method. Another experimental set-up with two rectangular troughs placed at right angles has been recommended by Ouchterlony (1962) and by Allison and Humphrey (1959). If the two reactants are employed in equivalent amounts, the precipitin arc is a straight line and the angle ( a ) between the antigen well and the precipitin arc depends only on the diffusion coefficients: tg(a) = D A g / D A b . Working with one reactant with a known diffusion coefficient, it is possible to determine the diffusion coefficient of the other. XAg/XAb= J ( D A g / D A b ) ,

2.4. Immunoelectrophoresis 2.4.I . General principles Immunoelectrophoresis consists of a combination of electrophoresis and radial immunodiffusion in gels. It is based on the fact that in agar gel, the movement of molecules in an electric field is similar to that in a liquid medium, with the advantage that free diffusion during and

43 1

IMMUNODIFFUSION IN GELS

after electrophoresis is lessened. The constituents of a mixture are then defined both by their electrophoretic mobilities and by their antigenic specificities. IMMUNOELECTROPHORESIS Centra1,hole

Agarplate

A Protein sample placed in central hole

t

B -

Antibody %reservoir

Paper connection

Electrophoresis

I

C

Protein fractions obtained Antiserum placed in antibody reservoir An\tiserum D Diffusion

E

Precipitation

Precipitation arc

Fig. 2.4. Method for micro-immunoelectrophoresis. A: The microscope slide is covered with buffered agar gel. The central hole (for insertion of the antigen) is dug out of the agar gel. B: Electrophoresis is performed for 14 h (10 v/cm). C: The electrophoretic pattern is obtained ; antiserum solution is placed in the antibody reservoir which is dug out of gel after electrophoresis. D:Diffusion is allowed to proceed in a moist chamber. E: As the result of diffusion, antigen-antibody reactions occur between corresponding fractions, and precipitation arcs or lines appear; these are specific for each antigen. The precipitation arc for albumin is illustrated here (Clausen 1960a). Subject index p . 557

432

IMMUNOCHEMICAL IDENTIFICATION OF MACROMOLECULES

This technique may be performed on a macro scale, as originally demonstrated by Grabar and Williams (1953) and Poulik (1952,1958), or as amicromethod (Scheidegger 1955). The principle of this technique is illustrated in fig. 2.4. The antigen sample is applied to a hole placed in the middle of a glass plate coated with buffered gel. Electrophoresis separates the antigen mixture into various zones. A longitudinal trough parallel to the long edge of the plate is then made in the gel and is filled with antiserum against the antigen mixture. A double diffusion takes place; the antiserum diffuses into the gel and the antigens diffuse radially in all directions from the electrophoretic zones. The antigens eventually meet the antibodies and precipitates are formed at equivalence points; the number of precipitates formed corresponds to the number of independent antigens present (fig. 2.4, 2.5). Because of the previous electrophoretic separation the precipitates are dispersed in a characteristic system of arcs with precipitation lines located alongside the electrophoretic zones. Two- and three-dimensional immunoelectrophoresisor one-dimensional electrophoresis combined with two-dimensional chromato-immunodiffusion have been suggested (Blanc 1959, 1961 ; Peeters and Vuylsteke 1960).

Caption for page 433.

Fig. 2.5. Micro-immunoelectrophoresis of normal human serum. Serum was placed in the two holes, and after electrophoresis precipitation pattern was developed with a horse antiserum against pooled normal human serum (placed in central trough). The main precipitation arcs are marked. Experimental conditions - Antigen: 1.5 pl normal human serum (6.5 g protein/100 ml) have been applied in the two antigen wells. Electrophoresis: 10 V/cm for 70 min, biiffx: 0.05 M veronal buffer, pH 8.6. Antiserum: 80 pl polyvalent antiserum from a horse against normal pooled humanserum is applied in central trough. Immunodiffusion:16 hr at 24°C. Staining: After washing by standing for two days in 0.9 % (w/v) aqueous NaCl and for 1 hr in distilled water, the slide is dried in open air and stained for protein with Amido Black (appendix 18). Photography: See appendix 17.

434

IMMUNOCHEMICALIDENTIFICATION OF MACROMOLECULES

The advantage of immunoelectrophoresis over immunodiffusion is that complex antigenic mixtures may be separated because of the additional resolving power obtained through the electrophoretic step. Immunoelectrophoresis is, however, less sensitive owing to the dilution which occurs through spreading of materials during electrophoresis. Constituents in the mixture may be identified serologically in a complex spectrum of arcs by matching each individual arc by means of a marker arc produced by a monospecific reactant (antigen or antibody) added to the plate in a different location from the polyvalent serum. Alternatively the polyvalent antiserum may be absorbed by a pure antigen and then used for comparison with the unabsorbed serum. 2.4.2. Types of supporting media 2.4.2.1. Agar (agarose and agaro-pectin) (Araki 1937) Agar is a mixture of two linear polysaccharides, agarose and agaropectin. It is extracted from seaweeds (algae); chemically it consists mainly of galactose molecules linked together at C-1 and -3, and contains from 0.3 to 3.7% sulphur as sulphate (one sulphate per 8-50 galactose units). Ordinarily, the sulphate ions are combined with such ions as Na+, K+, Ca++and Mg++.These salts are thought to be necessary for gelation, because when agar is transformed to its acid form at pH 2.0, it cannot solidify (Jirgensons 1958). Gelation depends partly on formation of calcium bridges between sulphate groups and partly on hydrogen bonding. The content of inorganic ions, especially sulphate, determines the extent of electrostatic interaction with migrating colloids and of electroendosmosis and thus determines the electrophoretic properties of agar (Wieme 1959). Calcium ions are probably necessary for optimal migration of lipoprotein which will otherwise interact with the sulphate groups of the gel (Wieme 1959; Laurel1 1966). Agaro-pectin contains the sulphate (and carboxyl) groups of the agar, whereas agarose is a neutral polysaccharide. Therefore, agarose produces a less pronounced electroendosmosis than crude agar and agaro-pectin (for discussion of factors causing electroendosmosis, see Gordon 1968).

IMMUNODIFFUSION IN GELS

435

Agarose may be isolated as described by Araki (1937, 1956) and HjertCn (1962) (appendix 6). The use of an agarose gel instead of an ordinary agar gel has advantages as the gel is free from ionized groups and does not cause pronounced electroendosmosis or interaction with certain basic proteins such as lysozyme (Uriel et al. 1964). Agarose is more resistant to acids than agaro-pectin and therefore electrophoresis may be carried out at lower pH. On the other hand, the complicated preparation of agarose may limit its use (see appendix 6); it is available commercially, but it is relatively expensive.

2.4.2.2. Cellulose acetate Immunoelectrophoresis can also be performed on cellulose acetate paper (Kohn 1958,1959,1962,1968; Lomanto and Vergani 1967). The technique suffers from difficulties involved in applying the antiserum evenly on the paper. It is therefore not recommended for the beginner in the field of immunochemistry. The difficulty is not encountered in the agar technique where the application of antiserum merely consists of filling the antibody trough with antiserum. On the other hand, the cellulose acetate easily sucks up buffered solutions of antisera and may be recommended for radial immunodiffusion (see appendix 9) and electrophoresis of antigens in an antibody-containing medium (Laurel1 technique, p. 437) (Vergani et al. 1967). Cellulose acetate has also been used in combination with thin-layer gel filtration for immunochemical detection of serum proteins. The serum proteins are first separated by thin-layer gel filtration on Sephadex G 200. The cellulose acetate foil is applied onto the Sephadex surface on which the proteins have been separated. Strips of filter paper soaked with antiserum are placed parallel to either side of the cellulose membrane. By allowing immunodiffusion to develop for 48 hours, twelve different serum protein antigens have been described (Agostoni et al. 1967). 2.4.2.3. Starch gel Horizontal or vertical starch gel electrophoresis (Smithies 1955, 1959) of protein antigens may be followed by diffusion in gel by cutting the electrophoretic starch gel vertically in two halves. One half is stained Subject index p. 557

436

1MMUNOCHEMICAL IDENTIFICATION OF MACROMOLECULES

for proteins and the other half is placed in a melted agar buffer solution. After gelling, an antibody trough is made in the gel in the anodecathode direction, and an antiserum is applied in this trough. The antigen separated in the starch gel will now diffuse into the agar gel and meet the corresponding antibody and thus create multiple precipitation lines. This method is laborious, because the electrophoresis and immunodiffusion cannot be performed in one step. However, starch gel electrophoresis has a higher resolving power for the antigen mixture than agar has. Horizontal starch gel electrophoresis may produce 18 electrophoretic zones in separation of serum proteins, whereas agar gel electrophoresis results in only seven. It has been possible to separate and analyse subunits of proteins by starch gel electrophoresis (Poulik 1964a, b). By reductive alkylation followed by starch gel electrophoresis in 8 M urea (formate buffer) the L- and H-chains of immunoglobulins are separated (pp. 415-416). 2.4.2.4. Polyacrylamide gel lmmunoelectrophoresis may be performed in an acrylamide gel (Antoine 1962; Keutel 1964). Polymerized acrylamide, a linear polymer gel is an adequate inert supporting medium for electrophoresis. Although the electrophoresis can be completed within twenty minutes, immunodiffusion time is more than three times longer than in agar gel (Keutel 1964). The acrylamide immunoelectrophoresis is preferred when the samples distort agar gel during electrophoretic separation, where extreme acid or alkaline conditions are required or where antigens are to be enzymically detected. (For factors governing separation in acrylamide the companion volume by Gordon (1968) should be consulted .) 2.4.2.5. Fibrin-agar Fibrin-agar electrophoresis is mainly used in studies of proteolytic enzymes, their activators and inhibitors. Such studies involve characterization of a number of the individual steps in the catalytic processes, for example blood coagulation, by examination of the reaction products (Heimburger and Schwick 1962; Schwick 1967). The me-

IMMUNODIFFUSION IN GtLS

431

thod is based on the combination of immunoelectrophoresis and the classical test of fibrinolysis performed by means of the fibrin-plate technique (Astrup and Miillertz 1952). By mixing agar with buffer, fibrinogen and calcium ions followed by appropriate heating, a medium is obtained which possesses the electrophoretic properties of the agar and the substrate properties required for proteolytic digestion. After electrophoretic separation and incubation, every electrophoretic zone containing a proteinase will appear as a clear zone of lysis. By cutting out troughs for antisera, precipitation lines corresponding to the antigenic proteinases may appear. The troughs may also be used for application of an activator or inhibitor of proteinase function. An electrophoretic zone containing a proenzyme of a proteinase may, during diffusion towards the trough containing the activator, cause the formation of a clear zone of digested fibrin as a sign of the activation of the proenzyme. Combined with immunodiffusion on the second half of the slide, this method offers possibilities for localization of enzymes and proenzymes belonging to the group of proteinases. Similarly, it is possible to demonstrate anti-proteinase activity (Heide and Haupt 1964). 2.4.3. Electrophoresis in antibody-containing media (Laurel1 technique) In this technique, the antigen-antibody reaction occurs during the electrophoresis of an antigen mixture in an antibody containing medium (Bussard 1959; Ressler 1960a, b). Both antigen and antibody move according to their electrophoretic mobilities, and they also react together, resulting in flame-shaped precipitin zones of antigen-antibody complexes (fig. 2.6). Under the influence of the electric field, unbound antigen within the peak of the flame-shaped precipitate migrates into the precipitate which redissolves in the excess antigen. Thus, the leading edge of the flame is gradually displaced in the direction the antigen is migrating. The amount of antigen within the leading boundary edge is successively diminished because of the formation of soluble antigenantibody complexes. When the antigen is diminished to equivalence with the antibody, the antibody-antigen complex can no longer be dissolved and a stable precipitate will form at the leading edge, which Subjerf index p . 557

438

IMMUNOCHEMICAL IDENTIFICATION OF MACROMOLECULES

is thereafter stationary. The distance finally travelled by the peak depends on the relative excess of antigen over antibody and can be used as a measure of the amount of antigen present. Every precipitin band featured as a flame represents an individual antigen. By absorbing the antibody with pure antigens prior to the migration it is possible to identify the pattern (Ressler 1960a). Greater resolution can be obtained if, prior to the electrophoresis in the agar gel containing antiserum, the proteins are separated by starch gel electrophoresis; a number of precipitates of albumin and ceruloplasmin have thus been obtained (Ressler 1960a, b). Electrophoresis in an antibody-containing medium may thus be applied to studies of antigenic sites.

Fig. 2.6. Electrophoresis of serum albumin in antibody-containing agarose gel. Serum albumin dilutions run for 30, 60, 120 and 300 min in agarose gel containing 2.5% rabbit anti-albumin serum. Amount of albumin in each series from left to right: 5 , 3.75, 2.5, 2.0, 1.5, 1.0, 0.5, 0.25, and 0.125 pg. (From Laurell 1966).

The electrophoresis of an antigen mixture i n an antibody-containing agar gel has been used one-dimensionally or two-dimensionally. The techniques (see fig. 2.6, Laurell’s method) have been used for quantitative estimation of antigens in solutions containing from 500 to 12.5 pg/ml (Clarke and Freeman 1967; Laurell 1966) provided the isoelectric point of the antigen differs from that of the

IMMUNODIFFUSION IN GELS

439

antibody. Systematic studies of the migration rate in an agarose medium containing the antibody (Laurel1 1966) revealed empirically that within a certain time (2 to 10 hr) a rectilinear relationship between final migration distance of the leading edge and the amount of antigen may be established. This method is appropriate for estimation of serum proteins or other antigens in different biological fluids (vide infra) and is described in appendix 13. The migration rate of the antigens must be faster than that of the IgG globulin.

Subject index P. 557