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Radioelectrocomplexing: A counterelectrophoretic method for hapten—antibody interaction
Journal of lmmunological Methods 6 (1975) 209-223 © North-Holland Publishing Company
RADIOELECTROCOMPLEXING: A COUNTERELECTROPHORETIC METHOD FOR HAPTEN-ANTIBODY INTERACTION* Albert A. BENEDICT and Leonard W. POLLARD Department of Microbiology, University of Hawaii, Honolulu, Hawaii 96822, U.S.A.
Received 15 May 1974,
accepted 27 August 1974
We have described an application of a radioimmunoassay (RIA) method, known as radioelectrocomplexing (REC), which involves the anodal migration of antigen and the cathodal migration of antibody in agar electrophoresis. The agar is divided into zones of free antigen (DNp125I-HSA) and antigen bound with anti-DNP. Complete assays of anti-DNP can be performed in 2-4 hr since both immune complex formation and separation of free from bound antigen can be accomplished by electrophoresis in 60-90 min. Estimation of the weight of specifically-purified anti-DNP chicken antibodies in the nanogram range by REC is of the same order as the reported sensitivity of other RIA methods. The method was capable of demonstrating the higher avidity of the 17 S than 7 S antibody. Based on hapten inhibition the relative binding constants of DNP derivatives and anti-DNP were of the same order as reported from more definitive methods.
1. Introduction Of the many in vitro methods available for estimating the concentration of either antigen or antibody, those based on the determination of the primary interaction between antigen and antibody have certain advantages over those methods in which secondary interactions are measured. The chief advantages of these methods are (1) nonprecipitating and nonagglutinating antibodies are measured; ( 2 ) n a n o gram amounts of antigen or antibody can be measured relatively simply and rapidly if the antigen is radioactively labelled as in the case of radioimmunoassay (RIA) procedures. In addition, meaningful thermodynamic and stoichiometric characterizations can be obtained when antibody and hapten are employed as in equilibrium dialysis. A common requirement for all RIA methods is the necessity to separate free radiolabelled antigen from that bound in immune complexes, and this can be achieved by a number of methods (Hunter, 1970). Recently Simons (1973) and Simons and Benedict (1974) described an RIA method which involves counterelectrophoresis for detection of primary binding of antigens by antibody. The
* This work was supported in part by United States Public Health Service Grant AI 05660. 209
210 A.A. Benedict, L. W. Pollard, Electrophoretic method for hapten-antibody interaction method, referred to as 'radioelectrocomplexing' (REC), depends on the anodal movement of radiolabelled antigen, such as serum albumin, and the cathodal movement of antibodies. In REC both immune complex formation and separation of free from bound antigen are achieved by a single electrophoretic process; thus, complete assays can be performed in a matter of a few hours. For clinical application REC is rapid and sensitive for the estimation of alpha-fetoprotein (AFP) (Simons and Benedict, 1974), hepatitis B antibody and antigen (Simons and Benedict, 1974), and carcinoembryonic antigen (CEA) (Coller et al., 1973). Preliminary data were presented on the application of REC for measuring the binding of dinitrophenyl (DNP) conjugates and anti-DNP antibody (Simons and Benedict, 1974). In the present report quantitative data are given on the parameters governing the interaction of hapten and antibody in REC; thus, establishing firmer grounds for its clinical use.
2. Materials and methods 2.1. Antigens
2,4-Dinitrophenyl-protein conjugates were prepared according to the method of Eisen (1964). Dinitrophenyl-bovine gamma-globulin (DNP-BGG) and dinitrophenyl-human serum albumin (DNP-HSA) had approximately 60 and 40 moles of DNP per mole of protein, respectively. Radiolabelling of conjugates with either x31I or l=Sl was done by the method of Hunter and Greenwood (1962). To eliminate material which did not migrate electrophoretically as albumin, DNP-HSA was fractionated by starch block electrophoresis; the fastest moving fraction was used as antigen in REC. The concentrations of 2,4-dinitrophenyl derivatives and 2,4-dinitrophenol was determined by their extinction coefficients as given by Little and Donahue (1968). 2.2. Antisera
Adult white Leghorn chickens were given 4 intravenous injections of 2 mg DNP-BGG each over a one-year period, and were bled 7 days following the last injection. From a pool of these sera the anti-DNP antibodies were purified by affinity chromatography (Cuatrecasas et al., 1968). Briefly, antisera to which a final concentration of 0.001 M EDTA was added, were passed through SepharoseDNP-lysine columns, and the columns were washed repeatedly with borate buffer (pH 8.2, ½P = 0.16). The anti-DNP antibodies were eluted first with 0.1 M dinitrophenol, followed by elution of the remaining antibodies with 0.1 M DNP-glycine. These antibody preparations will be referred to as ' D N P - O H ' and 'DNP-glycine' antibodies, respectively. The chicken low and high molecular weight anti-DNP antibodies, referred to as 7 S and 17 S, respectively, were separated and recovered by gel filtration on Sephadex G-200.
A.A. Benedict, 1. tt/. Pollard, Electrophoretic method for hapten-antibody interaction 211
For other experiments, chickens were given either 2 intravenous injections of 2 mg DNP-BGG each 45 days apart, or 2 intramuscular injections of 2 mg DNPBGG each in Freund's complete adjuvant 30 days apart. The 7 S fractions, referred to as globulin fractions, were isolated by precipitation of the globulins with 18% Na 2SO4 followed by 2 cycles of gel filtration on Sephadex G-200 (Benedict, 1967). 2.3. Radioelectrocomplexing
Microscope slides were coated with a 3 ml of 1.2% purified agar (Difco) in Veronal buffer (pH 8.6, ½F = 0.025). Two wells, each 3 mm in diameter and 4 mm apart, were punched in the agar as four parallel pairs (fig. I); thus, duplicate or triplicate test samples and controls could be run under the same conditions. However, in many experiments duplicate test samples and a single diluent control were run per slide. Denaturation of antibody preparations were minimized by dilution in a 1 : 50 dilution of normal chicken serum (NCS), and dilutions of DNP-conjugates were made in a 1 : 100 dilution of BSA. Borate buffer was used throughout as the diluent. Five microliter amounts of each of antigen and antibody were placed in their respective wells as shown in fig. I, and electrophoresis was performed with an electric potential of 200 V (2.5 mA/slide) on meter and approximately 4 V/cm in agar for varying periods using a Veronal buffer (pH 8.6, ½I" = 0.025). The concentrations of antigen and of antibody given are those which were actually placed in the wells; that is, the'concentration per 5 ~1. After electrophoresis the agar was cut with a sharp razor blade according to the pattern given in fig. 1. When DNP HSA was complexed with antibody, radiolabel usually was found in the interwell zone (IWZ) and the zone around the antigen well (AgWZ); therefore, the AgWZ and the IWZ together were considered as the reaction zone (RZ) (fig. 1). The slabs of agar from the RZ and the zone of 'free' antigen referred to as the 'follow through zone' (FTZ) each were placed in 13 × 100 mm test tubes and were crushed to the bottom of the tube with an applicator stick. The radioactivity was determined in a gamma spectrometer. After substraction of background radioactivity from all counts, the percent of antigen bound in the RZ was calculated for each agar strip by correcting for the
Antibody
Antigen-125I
.-I~ ~---+ Follow through zone Antibody zone
well
Reaction zone
Interwell zone
Antigen zone
well
Fig. 1, Schematic diagram of radioelectrocomplexing.
212 A.A. Benedict, L. W. Pollard, Electrophoretic method for hapten antibody interaction
presence of unbound labelled antigen in RZ as indicated by controls. This correction was made according to the following formula: corr. RZ t = RZ t -
/
(RZ t + FTZt) X (RZc + ~TZc )
Where t and c represent test and control reactions, and RZt, FTZt, RZc and FTZc are the observed cpm in the respective zones. Then, c°rr.RZ t
corr.
N antigen bound =
corr.
X 100 (RZ t + FTZ t)
2.4. Equilibrium dialysis The binding constants of the globulin preparations were determined by equilibrium dialysis according to Nisonoff and Pressman (1958). Duplicate samples of normal and of antibody-containing globulin were dialyzed for 3 days at 4°C against various concentrations of e-DNP-3,5-3 H-(N)-L-lysine (New England Nuclear). After dialysis, the protein concentration of each sample was determined by Folin analysis. Samples were counted in Aquasol (New England Nuclear) using a Packard Tricarb scintillation spectrometer until at least 20,000 counts had been registered. The amount of hapten bound at each free hapten concentration was determined for an adjusted globulin concentration of 10 mg/ml. Calculations for total binding sites and affinity constants were performed as described by Werblin and Siskind (1972) with the aid of an APL/360 computer program.
2.5. A utoradiography After electrophoresis the protein was fixed by staining the slides with Amido Black which was dissolved in methanol-acetic acid-water. Excess dye was removed by rising with methanol acetic acid-water and the slides were dried and placed on Kodak No-Screen Medical X-Ray film. Time of exposure varied from 2 - 4 days, after which time the films were processed.
3. Results
For each antigen-antibody system studied the effect of the diluents on the migration of antigen had to be determined (Simons and Benedict, 1974). Since NCS was used as the diluent to protect nanogram quantities of specifically purified
A.A. Benedict, L. W. Pollard, Electrophoretic method for hapten-antibody interaction 213 rain.
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Fig. 2. Autoradiograph of radioelectrocomplexing of DNP-HSA,! 2 sI and anti-DNP at 4 (left) and 7 (right) V/cm at various times of electrophoresis. (A) 18 ng antibody; (B) borate buffer control; (C) 7 ng antibody.
214 A.A. Benedict, L. W. Pollard, Eleetrophoretic method for hapten-antibody interaction anti-DNP from denaturation, it was necessary to establish the lowest concentration of NCS which gave nonspecific binding of DNP-HSA 12sI (DNP-*HSA). Most NCS diluted 1 : 8 bound approximately 10-25% of the antigen in the RZ. To give a margin of safety a 1 : 50 dilution of NCS was used as the diluent. Although this dilution of NCS did not result in binding the antigen in the RZ, it retarded the movement of antigen in the FTZ when compared to the migration of antigen in the control. 3.1. Conditions o f electrophoresis
In the absence of antibody and employing 4V/cm, DNP-*HSA eventually entered the anodal wick and buffer vessel only after 2.5 hr. With an increased potential of about 7 V/cm the antigen entered the anodal wick after 1.5 hr of electrophoresis. The distribution of bound antigen was studied by autoradiography at various times of electrophoresis at 4 and 7 V/cm. The amounts of anti-DNP 7 S antibody used were those which would bind 50% (7.0 ng) and 90% (18.0 ng) of antigen after 90 rain of electrophoresis at 2.5 mA/slide. As shown in fig. 2, 18 ng of antibody held the radioactivity in the RZ after 90 min at 4 V/cm; whereas at 7 V/cm radioactivity appeared in the FTZ after 50 min. Also, with the lower voltage radioactivity did not appear in the FTZ until 60 min of electrophoresis with 7 ng of antibody, but at 7 V/cm antigen was in the FTZ after 30 rain. The autoradiograms (fig. 2) showed a wide distribution of radioactivity in the FTZ, particularly when 7 V/cm was used. There was at least two major radioactive populations, one which migrated with the trailing edge of the unbound antigen (buffer control) and the other which remained near the antibody well. In between these spots was a smearing of radioactivity. The heterogenous migration in the FTZ might represent migration of immune complexes composed of excess antigen and/or the anodal migration of free antigen which had dissociated from the complexes. The problem of dissociation of complexes will be considered later. Thus, for the DNP-anti-DNP system about 4 V/cm for 75 min were chosen as standard conditions of electrophoresis. Under these conditions dissociation of complexes formed in moderate antigen excess seemed to be minimal. 3.2. Relationship o f antigen concentrations
Binding curves were determined for 4 levels of concentration of specificallypurified anti-DNP 7 S antibody. As shown in fig. 3a, the percent binding of antigen at the different antibody levels was a function of antigen concentration. If antibody is saturated, then the radioactivity in the RZ should reach a constant level for a fixed amount of antibody. As shown in fig. 3b, at different antibody levels a constant level of radioactivity in the RZ was approached as the antigen concentration was increased. The effect of varying antigen-antibody concentrations on the binding curves
A.A. Benedict, L. W. Pollard, Electrophoretic method for hapten-antibody interaction 215 a
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Fig. 3. Binding of increasing amounts of DNP-HSA 12sI at four different antibody levels. (a) . reacnon . zone (RZ); (b) radioactivity (cpm) in RZ. Percent DNP-HSA 12s I bound m also was studied by inhibiting the binding of labelled antigen at 4 antibody levels by the addition of varying concentrations of unlabelled antigen to the labelled antigen. As the antibody concentration increased (45-730 ng) the amounts of DNP-HSA required to inhibit 50% binding of labelled antigen was increased (fig. 4). A plot of the amounts of unlabelled antigen required to inhibit 50% binding of the labelled antigen at each antibody concentration yielded a straight line (fig. 5). In this experiment the concentration of the labelled antigen was negligible (< 0.1 ng); therefore, the straight line obtained could be used to determine the concentration of antibody in an unknown sample. For example, in the previous experiment (fig. 3a) the amount of labelled antigen bound (50% binding) by 50 and 100 ng of antibody was about 9 and 19 ng, respectively. From fig. 5 these amounts of antigen (9 and 19 ng) bound about 40 and 85 ng of antibody, respectively, at 50% binding; antibody values which approached those obtained in fig. 5 (50 and 100 ng, respectively). I00
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216 A.A. Benedict, L. 19. Pollard, Electrophoretic method for hapten-antibody interaction
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a
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Fig. 5. Relationship between the amount of antibody and the amount of DNP-HSA to inhibit 50 percent of binding of DNP-HSA 1251 based on the data given in fig. 4.
3.3. Sensitivity for detection o f 7 S and 17 S antibodies Binding curves of specifically-purified 7 S and 17 S anti-DNP antibodies obtained from the same serum and each having about the same Ko value (5 X 106 M-1) were determined when reacted with 3 ng of antigen (fig. 6). Approximately 0.31 and 1.25 pmoles of 17 S and 7 S antibodies, respectively, bound 50% of the antigen. About 40 times (12 pmoles) and 84 times (105 pmoles) more of normal 17 S and normal 7 S respectively, were required to bind 50% of antigen. Thus, in
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Fig. 6. Comparison of the ability of specifically-purified 17 S and 7 S anti-DNP antibodies to bind 3 ng of DNP-HSA 1251.
A.A. Benedict, L. W. Pollard, Electrophoretic method for hapten-antibody interaction 217
this experiment the 17 S antibody was 4 - 5 times more effective than the 7 S for binding antigen. In another experiment, 50% of 1 ng of antigen was bound by 4 ng or 2.3 × 10-2 pmoles of a different preparation of 7 S antibody. 3.4. Hapten inhibition o f binding
The specificities of antibodies and the relative combining constant (grel) of a hapten with antibody have been studied by the variable ability of different haptens to inhibit the reactions of antibody with conjugated haptens (Pauling et al., 1944; Landsteiner, 1962). Hapten inhibition offers an advantage over other methods for determining Kre 1 in that specifically purified antibody is not required. To determine whether REC was potentially useful for this purpose, the DNP-OH and DNP-glycine antibody preparations each were reacted with various concentrations of the following DNP derivatives: DNP-OH, DNP-glycine, e-DNP-L-lysine, and e-DNP-aminocaproate. To minimize dissociation of hapten and antibody during electrophoresis, the inhibiting hapten was added to the agar and to the buffer in the electrode vessels to the final concentration desired. The electrode vessels of the electrophoresis apparatus were partitioned with Plexiglas into five sections so that five different inhibitor concentrations could be tested under the same conditions of electrophoresis. The amounts of antibody and DNP-*HSA giving 80-85% binding were used. Figs. 7a and 7b show the inhibition curves for the DNP-OH and DNP-glycine antibody preparations, respectively. Taking the concentration of DNP-lysine which gave 50% inhibition as a standard with a Kre 1 = 1.0, the Kre 1 values for the other ligands at 50% inhibition were calculated. As shown in table 1, the Krel of the haptens for both antibodies in the order of increasing Krel of the hapten was: DNP-OH, DNP-glycine, DNP-lysine, DNP-caproate. I00
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Fig. 7. Comparison of 2,4-dinitrophenyl derivatives as inhibitors of the binding of DNPHSA12 s I and anti-DNP 7 S antibody. Antibody was eluted from an immunoadsorbent column with (a) dinitrophenol, and (b) DNP-glycine following elution of antibodies with dinitrophenol.
1.00
1.8
0.22
0.041
a Eluted from immunoadsorbent with 2,4-dinitrophenol. b Following elution with 2,4-DNP-OH, antibody eluted with DNP-glycine. c Relative binding constant.
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e-DNP-aminocaproate
110
4.5
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40
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Concentration of hapten at 50% inhibition of binding (MX 10 - 6 )
Concentration of hapten at 50% inhibition of binding (MX 10 - 6 )
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DNP-OH a
2,4-Dinitrophenol
e-DNP-lysine
Hapten
Antibody preparation
2.0
0.22
0.009
1.0
Kre 1
Table I Comparison of the relative binding constants of 2,4-DNP derivatives and two antibody preparations as determined by inhibition of binding of antiDNP and D N P - H S A - 12s I in radioelectroeomplexing.
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A.A. Benedict, L W. Pollard, Electrophoretic method for hapten-antibody interaction
219
3.5. Dissociation of complexes To study this problem further, two concentrations of anti-DNP 7 S antibody, 70 ng and 7.0 ng, which formed complexes with I0 ng of antigen in moderate A B
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®1 ~ 3 ,5 ~ 7 O Fraction Fig. 8. Autoradiogzaphs and radioactivity (percent cpm/fraction) in fractions after various times of electrophoresis at 5 mA/sfide. A. 70 ng anti-DNP ? S; B. Buffer control; C. 7.0 ng anti-DNP 7S.
220 A.A. Benedict, L. W. Pollard, Electrophoretic method for hapten-antibody interaction antibody excess and moderate antigen excess, respectively, were electrophoresed with a current of 7 V/cm. In previous experiments this amount of current seemed to accelerate apparent dissociation. Slides were removed 20, 30, 50 and 75 min after electrophoresis and one set of each was prepared for autoradiography. The agar of another set of slides was fractionated in a manner as shown in fig. 8, and the radioactivity of the fractions was determined. A comparison of the radioactivity in the fractions as determined by autoradiography and by actual counts in the fractions is presented in fig. 8. The complexes formed in antibody excess remained in the RZ (fractions 5, 6, and 7) after 75 min of electrophoresis; whereas, at this time, the complexes formed in antigen excess formed two major zones of radioactivity. One zone had the same rate of migration as the trailing half of the antigen in the buffer control, the other zone remained in the cathodal end of the FTZ (fractions 3 and 4). Although only 11% of the binding remained in the RZ for the low antibody concentration, antigen was bound as indicated by the activity in fractions 3 and 4. It is reasonable to suggest that complexes of various ratios of antibody:antigen were formed and that they were responsible for the smearing of radioactivity in the FTZ. The complexes formed in antigen excess seemed to be stable under the conditions employed in that they remained in the RZ for 75 min.
I00
• Normal globulin globulin
O Anti DNP
~
1
'," 60 O O
{ i
z ~40
20
0
~-,..A •
t
1.0
t
1.5
I
2.0
~
2.5
Hours
Fig. 9. Rate o f dissociation (migration o f D N P - H S A 12sI' out o f the reaction zone) o f anti. DNP globulin preparations with different K 0 values. Globulin-I, -2, and -3 ~have K 0 values oI 1 X l 0 s , 3 X 10 6 , and 1 X 10 8 N -1 , respectively, as determined by equilibrium dialysis.
A.A. Benedict, L. W. Pollard, Electrophoretic method for hapten-antibody interaction 221
A more rigorous examination of dissociation would be to employ antibody preparations with different known binding constants, and to form the complexes in zones other than that of antibody excess. For this purpose preliminary experiments have been performed with three anti-DNP globulin preparations with binding constants of 1 X 10 s M -1 (globulin-l), 3 X 10 6 M-1 (globulin-2), and 1 X 108 M-1 (globulin-5), respectively. In the first experiments concentrations of globulin-1 and globulin-2, which would bind about 90% of antigen after 1 hr of electrophoresis, were electrophoresed for various periods of time at 2.5 mA/slide. For both preparations, the percent of bound antigen (RZ) decreased with time (fig. 9), however, the rate of disappearance from the RZ was faster for globulin-1 than for the globulin-2. In another experiment these antibody preparations were diluted so that after 1 hr of electrophoresis they bound only 30-50% of antigen. Again there was a faster rate of disappearance of globulin-1 from the RZ (fig. 9). The faster rate of migration of globulin-1 out of the RZ is best explained on the basis of its lower binding constant rather than as a result of differences in concentrations since the preparations were equated on basis of binding of antigen after 1 hr of electrophoresis. Actually, more globulin-1 antibody was used than either of the other preparations; 50% of antigen was bound by globulin-1 and only 33% of antigen was bound by globulin-2 after 1 hr of electrophoresis.
4. Discussion
These studies have provided some quantitative data required to establish the reliablity of REC as a RIA method. This procedure was capable of furnishing relative values for some reacting parameters which were in good agreement with values obtained by more definitive methods. The order of the Kre 1 of DNP derivatives and anti-DNP was the same as that determined by fluorescence quenching and equilibrium dialysis (Eisen and Siskind, 1964). With DNP-lysine having a Kre I of 1.0, the order of increasing affinities for some antisera was: DNP-OH, DNP-glycine, DNP-lysine, DNP-caproate. Also, in accord with the findings of Eisen and Siskind (1964), the antibodies purified by elution from immunoadsorbent columns with DNP-OH bound DNP-OH better (Kre 1 = 0.041) than antibodies eluted with DNP-glycine ( K r e 1 = 0 . 0 0 9 ) although both antibody preparations bound the other ligands to the same extents. In addition, the rate of dissociation of anti-DNP and DNP-conjugate seemed to be related to the binding constants. Finally, in accordance with the valencies of 17 S and 7 S antibodies, REC revealed the greater effectiveness for antigen binding by IgM antibody. On the assumption that the molecular composition of complexes in extreme antigen excess approaches Ag2Ab, then at a constant level of antibody there will be a concentration of antigen which saturates antibody. The limiting composition of Ag2Ab seemed to have been reached; that is, when increasing concentrations of antigen were added to a limited antibody concentration no additional binding
222 A.A. Benedict, L. W. Pollard, Eleetrophoretic method for hapten-antibody interaction occurred (fig. 3). Although sufficient data have not as yet been obtained to test the assumptions which can be used to estimate the weight of antibody in an antiserum, as was done so elegantly by Osler (1971) and by Revoltella et al., (1971) with a modified Farr technique (1958), the results reported here suggest that such measurements are possible with REC. Two methods of antibody assay are available. The first involves the determination of concentration of antibody or dilution of antiserum required to give 50% binding of a standard concentration of antigen. For example, the standard concentration of antigen could be established as that amount of antigen required to give 80% binding of a reference serum. A second method of estimating antibody potency is based on reacting a constant amount of antibody with varying amounts of antigen and determining the concentration of antigen which just saturates the antibody as suggested by Revoltella et al. (1971). In this connection, estimation of the weight of anti-DNP antibodies in the low nanogram range by REC is of the same order of sensitivity as the modified Farr method. Thus, determination of standard curves and assay of antigens can be accomplished with a sensitivity similar to that which can be achieved by other RIA methods (Hunter, 1970). The immune complexes which are formed during electrophoresis may subsequently dissociate due to the electrophoretic removal of free antigen. The extent of the dissociation, in part, will depend on the antibody affinity. With relatively high electrical current and prolonged electrophoresis some dissociation occurred even with high affinity antibody. Thus, for each antigen-antibody system it is important to establish the conditions whereby association of reactants and separation of bound from free antigen is maximized, while dissociation of bound antigen is minimized. Although dissociation of immune complex is a complicating factor in REC it may be possible to use the phenomenon advantageously. Investigations are proceeding to determine whether complex dissociation can be used to measure the Kre 1. Also, dissociation might be used to detect both antigen and antibody in a serum in which they exist as complexes. Indeed, complexes composed of hepatitis B antigen and antibody have been detected by the REC method (Simons and Benedict, 1974). The smearing of radioactivity on the anodal side of the antigen well when low antibody concentrations were used indicated the migration of immune complexes of varying ratios of antibody to antigen. The FTZ (following-trough zone) does not consist only of free antigen, but rather slowly-moving complexes which are probably composed of excess antigen. Therefore, sections of the FTZ should be included in the assay for bound antigen. However, DNP-conjugates are held up somewhat in the FTZ by NCS (Simons and Benedict, 1974). Also, we have repeatedly observed nonspecific binding of DNP by some pre-immune chicken sera in radioimmunoelectrophoresis. On the other hand, some antigens are not held up in the FTZ by normal sera, and in such cases sensitivity can be increased by extending the 'bound zone' to the anodal margin of the antibody well (Simons and Benedict, 1974). Radioelectrocomplexing is practical in that a large number of samples can be
A.A. Benedict, L. I¢. Pollard, Electrophoretic method for hapten-antibody interaction
223
analyzed in a few hours. The method is relatively simple to perform; no washing or centrifugation is involved. We have extended the use of REC in our laboratory for determination of the immune responses of chickens to the synthetic copolymer of L-glutamic acid, L-alanine, and L-tyrosine (GAT ~0 ).
Acknowledgement The authors wish to thank Dr. Karen Yamaga for obtaining the equilibrium dialysis data.
References Benedict, A.A., 1967, in: Methods in immunology and immunochemistry, Vol. 1, eds. C.A. Williams and M.W. Chase (Academic Press, New York), p. 299. Coller, J.A., R.W. Crichlow and K.Y. Lo, 1973, Cancer Res. 33, 1684. Cuatrecasas, P., M. Wilcheck and C.B. Anfinsen, 1968, Proc. Natl. Acad. Sci. U.S. 61,636. Eisen, H.N., 1964, in: Methods in medical research, Vol. 10, ed. H.N. Eisen, (Y-ear Book Medical Publishers, Chicago), p. 94. Eisen, H.N. and G.W. Siskind, 1964, Biochemistry 3,996. Farr, R.S., 1958, J. Infect. Dis. 103,239. Hunter, W.M., 1970, in: Handbook of experimental immunology, ed. D.M. Weir (Blackwell Scientific Publications, Oxford), p. 608. Hunter, W.M., and F.C. Greenwood, 1962, Nature 194,495. Landsteiner, K., 1962, in: The specificity of serological reactions (Harvard Univ. Press reprinted by Dover Publications, Inc., N.Y.). Little, J.R. and H. Donahue, 1968, in: Methods in immunology and immunochemistry, Vol. 2, eds. C.A. Williams and M.W.,Chase (Academic Press, New York). Nisonoff, A. and D. Pressman, 1958, J. Immunol. 81,126. Osier, A.G., 1971, in: Methods in immunology and immunochemistry, Vol. 3, eds. C.A. Williams and M.W. Chase (Academic Press, New York), p. 73. Pauling, L., D. Pressman and A.L., Grossberg, 1944, J. Amer. Chem. Soc. 66, 330. Revoltella, R., L.T. Adler and A.G. Osier, 1971, J. Immunol. 106, 1507. Simons, M.J., 1973, Bull. Wld. Hlth. Org. 48,499. Simons, M.J. and A.A. Benedict, 1974, in: Contemporary topics in molecular immunology, Vol. 3, ed. G.L. Ada (Plenum Press, N.Y.), p. 205. Werblin, T.P. and G.W. Siskind, 1972, Immunochemistry 9,987.
RADIOELECTROCOMPLEXING: A COUNTERELECTROPHORETIC METHOD FOR HAPTEN-ANTIBODY INTERACTION* Albert A. BENEDICT and Leonard W. POLLARD Department of Microbiology, University of Hawaii, Honolulu, Hawaii 96822, U.S.A.
Received 15 May 1974,
accepted 27 August 1974
We have described an application of a radioimmunoassay (RIA) method, known as radioelectrocomplexing (REC), which involves the anodal migration of antigen and the cathodal migration of antibody in agar electrophoresis. The agar is divided into zones of free antigen (DNp125I-HSA) and antigen bound with anti-DNP. Complete assays of anti-DNP can be performed in 2-4 hr since both immune complex formation and separation of free from bound antigen can be accomplished by electrophoresis in 60-90 min. Estimation of the weight of specifically-purified anti-DNP chicken antibodies in the nanogram range by REC is of the same order as the reported sensitivity of other RIA methods. The method was capable of demonstrating the higher avidity of the 17 S than 7 S antibody. Based on hapten inhibition the relative binding constants of DNP derivatives and anti-DNP were of the same order as reported from more definitive methods.
1. Introduction Of the many in vitro methods available for estimating the concentration of either antigen or antibody, those based on the determination of the primary interaction between antigen and antibody have certain advantages over those methods in which secondary interactions are measured. The chief advantages of these methods are (1) nonprecipitating and nonagglutinating antibodies are measured; ( 2 ) n a n o gram amounts of antigen or antibody can be measured relatively simply and rapidly if the antigen is radioactively labelled as in the case of radioimmunoassay (RIA) procedures. In addition, meaningful thermodynamic and stoichiometric characterizations can be obtained when antibody and hapten are employed as in equilibrium dialysis. A common requirement for all RIA methods is the necessity to separate free radiolabelled antigen from that bound in immune complexes, and this can be achieved by a number of methods (Hunter, 1970). Recently Simons (1973) and Simons and Benedict (1974) described an RIA method which involves counterelectrophoresis for detection of primary binding of antigens by antibody. The
* This work was supported in part by United States Public Health Service Grant AI 05660. 209
210 A.A. Benedict, L. W. Pollard, Electrophoretic method for hapten-antibody interaction method, referred to as 'radioelectrocomplexing' (REC), depends on the anodal movement of radiolabelled antigen, such as serum albumin, and the cathodal movement of antibodies. In REC both immune complex formation and separation of free from bound antigen are achieved by a single electrophoretic process; thus, complete assays can be performed in a matter of a few hours. For clinical application REC is rapid and sensitive for the estimation of alpha-fetoprotein (AFP) (Simons and Benedict, 1974), hepatitis B antibody and antigen (Simons and Benedict, 1974), and carcinoembryonic antigen (CEA) (Coller et al., 1973). Preliminary data were presented on the application of REC for measuring the binding of dinitrophenyl (DNP) conjugates and anti-DNP antibody (Simons and Benedict, 1974). In the present report quantitative data are given on the parameters governing the interaction of hapten and antibody in REC; thus, establishing firmer grounds for its clinical use.
2. Materials and methods 2.1. Antigens
2,4-Dinitrophenyl-protein conjugates were prepared according to the method of Eisen (1964). Dinitrophenyl-bovine gamma-globulin (DNP-BGG) and dinitrophenyl-human serum albumin (DNP-HSA) had approximately 60 and 40 moles of DNP per mole of protein, respectively. Radiolabelling of conjugates with either x31I or l=Sl was done by the method of Hunter and Greenwood (1962). To eliminate material which did not migrate electrophoretically as albumin, DNP-HSA was fractionated by starch block electrophoresis; the fastest moving fraction was used as antigen in REC. The concentrations of 2,4-dinitrophenyl derivatives and 2,4-dinitrophenol was determined by their extinction coefficients as given by Little and Donahue (1968). 2.2. Antisera
Adult white Leghorn chickens were given 4 intravenous injections of 2 mg DNP-BGG each over a one-year period, and were bled 7 days following the last injection. From a pool of these sera the anti-DNP antibodies were purified by affinity chromatography (Cuatrecasas et al., 1968). Briefly, antisera to which a final concentration of 0.001 M EDTA was added, were passed through SepharoseDNP-lysine columns, and the columns were washed repeatedly with borate buffer (pH 8.2, ½P = 0.16). The anti-DNP antibodies were eluted first with 0.1 M dinitrophenol, followed by elution of the remaining antibodies with 0.1 M DNP-glycine. These antibody preparations will be referred to as ' D N P - O H ' and 'DNP-glycine' antibodies, respectively. The chicken low and high molecular weight anti-DNP antibodies, referred to as 7 S and 17 S, respectively, were separated and recovered by gel filtration on Sephadex G-200.
A.A. Benedict, 1. tt/. Pollard, Electrophoretic method for hapten-antibody interaction 211
For other experiments, chickens were given either 2 intravenous injections of 2 mg DNP-BGG each 45 days apart, or 2 intramuscular injections of 2 mg DNPBGG each in Freund's complete adjuvant 30 days apart. The 7 S fractions, referred to as globulin fractions, were isolated by precipitation of the globulins with 18% Na 2SO4 followed by 2 cycles of gel filtration on Sephadex G-200 (Benedict, 1967). 2.3. Radioelectrocomplexing
Microscope slides were coated with a 3 ml of 1.2% purified agar (Difco) in Veronal buffer (pH 8.6, ½F = 0.025). Two wells, each 3 mm in diameter and 4 mm apart, were punched in the agar as four parallel pairs (fig. I); thus, duplicate or triplicate test samples and controls could be run under the same conditions. However, in many experiments duplicate test samples and a single diluent control were run per slide. Denaturation of antibody preparations were minimized by dilution in a 1 : 50 dilution of normal chicken serum (NCS), and dilutions of DNP-conjugates were made in a 1 : 100 dilution of BSA. Borate buffer was used throughout as the diluent. Five microliter amounts of each of antigen and antibody were placed in their respective wells as shown in fig. I, and electrophoresis was performed with an electric potential of 200 V (2.5 mA/slide) on meter and approximately 4 V/cm in agar for varying periods using a Veronal buffer (pH 8.6, ½I" = 0.025). The concentrations of antigen and of antibody given are those which were actually placed in the wells; that is, the'concentration per 5 ~1. After electrophoresis the agar was cut with a sharp razor blade according to the pattern given in fig. 1. When DNP HSA was complexed with antibody, radiolabel usually was found in the interwell zone (IWZ) and the zone around the antigen well (AgWZ); therefore, the AgWZ and the IWZ together were considered as the reaction zone (RZ) (fig. 1). The slabs of agar from the RZ and the zone of 'free' antigen referred to as the 'follow through zone' (FTZ) each were placed in 13 × 100 mm test tubes and were crushed to the bottom of the tube with an applicator stick. The radioactivity was determined in a gamma spectrometer. After substraction of background radioactivity from all counts, the percent of antigen bound in the RZ was calculated for each agar strip by correcting for the
Antibody
Antigen-125I
.-I~ ~---+ Follow through zone Antibody zone
well
Reaction zone
Interwell zone
Antigen zone
well
Fig. 1, Schematic diagram of radioelectrocomplexing.
212 A.A. Benedict, L. W. Pollard, Electrophoretic method for hapten antibody interaction
presence of unbound labelled antigen in RZ as indicated by controls. This correction was made according to the following formula: corr. RZ t = RZ t -
/
(RZ t + FTZt) X (RZc + ~TZc )
Where t and c represent test and control reactions, and RZt, FTZt, RZc and FTZc are the observed cpm in the respective zones. Then, c°rr.RZ t
corr.
N antigen bound =
corr.
X 100 (RZ t + FTZ t)
2.4. Equilibrium dialysis The binding constants of the globulin preparations were determined by equilibrium dialysis according to Nisonoff and Pressman (1958). Duplicate samples of normal and of antibody-containing globulin were dialyzed for 3 days at 4°C against various concentrations of e-DNP-3,5-3 H-(N)-L-lysine (New England Nuclear). After dialysis, the protein concentration of each sample was determined by Folin analysis. Samples were counted in Aquasol (New England Nuclear) using a Packard Tricarb scintillation spectrometer until at least 20,000 counts had been registered. The amount of hapten bound at each free hapten concentration was determined for an adjusted globulin concentration of 10 mg/ml. Calculations for total binding sites and affinity constants were performed as described by Werblin and Siskind (1972) with the aid of an APL/360 computer program.
2.5. A utoradiography After electrophoresis the protein was fixed by staining the slides with Amido Black which was dissolved in methanol-acetic acid-water. Excess dye was removed by rising with methanol acetic acid-water and the slides were dried and placed on Kodak No-Screen Medical X-Ray film. Time of exposure varied from 2 - 4 days, after which time the films were processed.
3. Results
For each antigen-antibody system studied the effect of the diluents on the migration of antigen had to be determined (Simons and Benedict, 1974). Since NCS was used as the diluent to protect nanogram quantities of specifically purified
A.A. Benedict, L. W. Pollard, Electrophoretic method for hapten-antibody interaction 213 rain.
O
A B
G,,30
C
O"
40
"
.......................................
°
50
,th
O
60
0, 70
Q. Q
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Q 90
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Fig. 2. Autoradiograph of radioelectrocomplexing of DNP-HSA,! 2 sI and anti-DNP at 4 (left) and 7 (right) V/cm at various times of electrophoresis. (A) 18 ng antibody; (B) borate buffer control; (C) 7 ng antibody.
214 A.A. Benedict, L. W. Pollard, Eleetrophoretic method for hapten-antibody interaction anti-DNP from denaturation, it was necessary to establish the lowest concentration of NCS which gave nonspecific binding of DNP-HSA 12sI (DNP-*HSA). Most NCS diluted 1 : 8 bound approximately 10-25% of the antigen in the RZ. To give a margin of safety a 1 : 50 dilution of NCS was used as the diluent. Although this dilution of NCS did not result in binding the antigen in the RZ, it retarded the movement of antigen in the FTZ when compared to the migration of antigen in the control. 3.1. Conditions o f electrophoresis
In the absence of antibody and employing 4V/cm, DNP-*HSA eventually entered the anodal wick and buffer vessel only after 2.5 hr. With an increased potential of about 7 V/cm the antigen entered the anodal wick after 1.5 hr of electrophoresis. The distribution of bound antigen was studied by autoradiography at various times of electrophoresis at 4 and 7 V/cm. The amounts of anti-DNP 7 S antibody used were those which would bind 50% (7.0 ng) and 90% (18.0 ng) of antigen after 90 rain of electrophoresis at 2.5 mA/slide. As shown in fig. 2, 18 ng of antibody held the radioactivity in the RZ after 90 min at 4 V/cm; whereas at 7 V/cm radioactivity appeared in the FTZ after 50 min. Also, with the lower voltage radioactivity did not appear in the FTZ until 60 min of electrophoresis with 7 ng of antibody, but at 7 V/cm antigen was in the FTZ after 30 rain. The autoradiograms (fig. 2) showed a wide distribution of radioactivity in the FTZ, particularly when 7 V/cm was used. There was at least two major radioactive populations, one which migrated with the trailing edge of the unbound antigen (buffer control) and the other which remained near the antibody well. In between these spots was a smearing of radioactivity. The heterogenous migration in the FTZ might represent migration of immune complexes composed of excess antigen and/or the anodal migration of free antigen which had dissociated from the complexes. The problem of dissociation of complexes will be considered later. Thus, for the DNP-anti-DNP system about 4 V/cm for 75 min were chosen as standard conditions of electrophoresis. Under these conditions dissociation of complexes formed in moderate antigen excess seemed to be minimal. 3.2. Relationship o f antigen concentrations
Binding curves were determined for 4 levels of concentration of specificallypurified anti-DNP 7 S antibody. As shown in fig. 3a, the percent binding of antigen at the different antibody levels was a function of antigen concentration. If antibody is saturated, then the radioactivity in the RZ should reach a constant level for a fixed amount of antibody. As shown in fig. 3b, at different antibody levels a constant level of radioactivity in the RZ was approached as the antigen concentration was increased. The effect of varying antigen-antibody concentrations on the binding curves
A.A. Benedict, L. W. Pollard, Electrophoretic method for hapten-antibody interaction 215 a
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o
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Fig. 3. Binding of increasing amounts of DNP-HSA 12sI at four different antibody levels. (a) . reacnon . zone (RZ); (b) radioactivity (cpm) in RZ. Percent DNP-HSA 12s I bound m also was studied by inhibiting the binding of labelled antigen at 4 antibody levels by the addition of varying concentrations of unlabelled antigen to the labelled antigen. As the antibody concentration increased (45-730 ng) the amounts of DNP-HSA required to inhibit 50% binding of labelled antigen was increased (fig. 4). A plot of the amounts of unlabelled antigen required to inhibit 50% binding of the labelled antigen at each antibody concentration yielded a straight line (fig. 5). In this experiment the concentration of the labelled antigen was negligible (< 0.1 ng); therefore, the straight line obtained could be used to determine the concentration of antibody in an unknown sample. For example, in the previous experiment (fig. 3a) the amount of labelled antigen bound (50% binding) by 50 and 100 ng of antibody was about 9 and 19 ng, respectively. From fig. 5 these amounts of antigen (9 and 19 ng) bound about 40 and 85 ng of antibody, respectively, at 50% binding; antibody values which approached those obtained in fig. 5 (50 and 100 ng, respectively). I00
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216 A.A. Benedict, L. 19. Pollard, Electrophoretic method for hapten-antibody interaction
-- 2 0 0 3~ ~_ I
a
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fo
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00
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Fig. 5. Relationship between the amount of antibody and the amount of DNP-HSA to inhibit 50 percent of binding of DNP-HSA 1251 based on the data given in fig. 4.
3.3. Sensitivity for detection o f 7 S and 17 S antibodies Binding curves of specifically-purified 7 S and 17 S anti-DNP antibodies obtained from the same serum and each having about the same Ko value (5 X 106 M-1) were determined when reacted with 3 ng of antigen (fig. 6). Approximately 0.31 and 1.25 pmoles of 17 S and 7 S antibodies, respectively, bound 50% of the antigen. About 40 times (12 pmoles) and 84 times (105 pmoles) more of normal 17 S and normal 7 S respectively, were required to bind 50% of antigen. Thus, in
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Fig. 6. Comparison of the ability of specifically-purified 17 S and 7 S anti-DNP antibodies to bind 3 ng of DNP-HSA 1251.
A.A. Benedict, L. W. Pollard, Electrophoretic method for hapten-antibody interaction 217
this experiment the 17 S antibody was 4 - 5 times more effective than the 7 S for binding antigen. In another experiment, 50% of 1 ng of antigen was bound by 4 ng or 2.3 × 10-2 pmoles of a different preparation of 7 S antibody. 3.4. Hapten inhibition o f binding
The specificities of antibodies and the relative combining constant (grel) of a hapten with antibody have been studied by the variable ability of different haptens to inhibit the reactions of antibody with conjugated haptens (Pauling et al., 1944; Landsteiner, 1962). Hapten inhibition offers an advantage over other methods for determining Kre 1 in that specifically purified antibody is not required. To determine whether REC was potentially useful for this purpose, the DNP-OH and DNP-glycine antibody preparations each were reacted with various concentrations of the following DNP derivatives: DNP-OH, DNP-glycine, e-DNP-L-lysine, and e-DNP-aminocaproate. To minimize dissociation of hapten and antibody during electrophoresis, the inhibiting hapten was added to the agar and to the buffer in the electrode vessels to the final concentration desired. The electrode vessels of the electrophoresis apparatus were partitioned with Plexiglas into five sections so that five different inhibitor concentrations could be tested under the same conditions of electrophoresis. The amounts of antibody and DNP-*HSA giving 80-85% binding were used. Figs. 7a and 7b show the inhibition curves for the DNP-OH and DNP-glycine antibody preparations, respectively. Taking the concentration of DNP-lysine which gave 50% inhibition as a standard with a Kre 1 = 1.0, the Kre 1 values for the other ligands at 50% inhibition were calculated. As shown in table 1, the Krel of the haptens for both antibodies in the order of increasing Krel of the hapten was: DNP-OH, DNP-glycine, DNP-lysine, DNP-caproate. I00
/e / .~/~
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I 7
I 6
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Fig. 7. Comparison of 2,4-dinitrophenyl derivatives as inhibitors of the binding of DNPHSA12 s I and anti-DNP 7 S antibody. Antibody was eluted from an immunoadsorbent column with (a) dinitrophenol, and (b) DNP-glycine following elution of antibodies with dinitrophenol.
1.00
1.8
0.22
0.041
a Eluted from immunoadsorbent with 2,4-dinitrophenol. b Following elution with 2,4-DNP-OH, antibody eluted with DNP-glycine. c Relative binding constant.
2.5
20
DNP-glycine
e-DNP-aminocaproate
110
4.5
4.5
40
1000
9.0
Concentration of hapten at 50% inhibition of binding (MX 10 - 6 )
Concentration of hapten at 50% inhibition of binding (MX 10 - 6 )
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DNP-OH a
2,4-Dinitrophenol
e-DNP-lysine
Hapten
Antibody preparation
2.0
0.22
0.009
1.0
Kre 1
Table I Comparison of the relative binding constants of 2,4-DNP derivatives and two antibody preparations as determined by inhibition of binding of antiDNP and D N P - H S A - 12s I in radioelectroeomplexing.
t~
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E
~
~
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to 0o .~
A.A. Benedict, L W. Pollard, Electrophoretic method for hapten-antibody interaction
219
3.5. Dissociation of complexes To study this problem further, two concentrations of anti-DNP 7 S antibody, 70 ng and 7.0 ng, which formed complexes with I0 ng of antigen in moderate A B
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®1 ~ 3 ,5 ~ 7 O Fraction Fig. 8. Autoradiogzaphs and radioactivity (percent cpm/fraction) in fractions after various times of electrophoresis at 5 mA/sfide. A. 70 ng anti-DNP ? S; B. Buffer control; C. 7.0 ng anti-DNP 7S.
220 A.A. Benedict, L. W. Pollard, Electrophoretic method for hapten-antibody interaction antibody excess and moderate antigen excess, respectively, were electrophoresed with a current of 7 V/cm. In previous experiments this amount of current seemed to accelerate apparent dissociation. Slides were removed 20, 30, 50 and 75 min after electrophoresis and one set of each was prepared for autoradiography. The agar of another set of slides was fractionated in a manner as shown in fig. 8, and the radioactivity of the fractions was determined. A comparison of the radioactivity in the fractions as determined by autoradiography and by actual counts in the fractions is presented in fig. 8. The complexes formed in antibody excess remained in the RZ (fractions 5, 6, and 7) after 75 min of electrophoresis; whereas, at this time, the complexes formed in antigen excess formed two major zones of radioactivity. One zone had the same rate of migration as the trailing half of the antigen in the buffer control, the other zone remained in the cathodal end of the FTZ (fractions 3 and 4). Although only 11% of the binding remained in the RZ for the low antibody concentration, antigen was bound as indicated by the activity in fractions 3 and 4. It is reasonable to suggest that complexes of various ratios of antibody:antigen were formed and that they were responsible for the smearing of radioactivity in the FTZ. The complexes formed in antigen excess seemed to be stable under the conditions employed in that they remained in the RZ for 75 min.
I00
• Normal globulin globulin
O Anti DNP
~
1
'," 60 O O
{ i
z ~40
20
0
~-,..A •
t
1.0
t
1.5
I
2.0
~
2.5
Hours
Fig. 9. Rate o f dissociation (migration o f D N P - H S A 12sI' out o f the reaction zone) o f anti. DNP globulin preparations with different K 0 values. Globulin-I, -2, and -3 ~have K 0 values oI 1 X l 0 s , 3 X 10 6 , and 1 X 10 8 N -1 , respectively, as determined by equilibrium dialysis.
A.A. Benedict, L. W. Pollard, Electrophoretic method for hapten-antibody interaction 221
A more rigorous examination of dissociation would be to employ antibody preparations with different known binding constants, and to form the complexes in zones other than that of antibody excess. For this purpose preliminary experiments have been performed with three anti-DNP globulin preparations with binding constants of 1 X 10 s M -1 (globulin-l), 3 X 10 6 M-1 (globulin-2), and 1 X 108 M-1 (globulin-5), respectively. In the first experiments concentrations of globulin-1 and globulin-2, which would bind about 90% of antigen after 1 hr of electrophoresis, were electrophoresed for various periods of time at 2.5 mA/slide. For both preparations, the percent of bound antigen (RZ) decreased with time (fig. 9), however, the rate of disappearance from the RZ was faster for globulin-1 than for the globulin-2. In another experiment these antibody preparations were diluted so that after 1 hr of electrophoresis they bound only 30-50% of antigen. Again there was a faster rate of disappearance of globulin-1 from the RZ (fig. 9). The faster rate of migration of globulin-1 out of the RZ is best explained on the basis of its lower binding constant rather than as a result of differences in concentrations since the preparations were equated on basis of binding of antigen after 1 hr of electrophoresis. Actually, more globulin-1 antibody was used than either of the other preparations; 50% of antigen was bound by globulin-1 and only 33% of antigen was bound by globulin-2 after 1 hr of electrophoresis.
4. Discussion
These studies have provided some quantitative data required to establish the reliablity of REC as a RIA method. This procedure was capable of furnishing relative values for some reacting parameters which were in good agreement with values obtained by more definitive methods. The order of the Kre 1 of DNP derivatives and anti-DNP was the same as that determined by fluorescence quenching and equilibrium dialysis (Eisen and Siskind, 1964). With DNP-lysine having a Kre I of 1.0, the order of increasing affinities for some antisera was: DNP-OH, DNP-glycine, DNP-lysine, DNP-caproate. Also, in accord with the findings of Eisen and Siskind (1964), the antibodies purified by elution from immunoadsorbent columns with DNP-OH bound DNP-OH better (Kre 1 = 0.041) than antibodies eluted with DNP-glycine ( K r e 1 = 0 . 0 0 9 ) although both antibody preparations bound the other ligands to the same extents. In addition, the rate of dissociation of anti-DNP and DNP-conjugate seemed to be related to the binding constants. Finally, in accordance with the valencies of 17 S and 7 S antibodies, REC revealed the greater effectiveness for antigen binding by IgM antibody. On the assumption that the molecular composition of complexes in extreme antigen excess approaches Ag2Ab, then at a constant level of antibody there will be a concentration of antigen which saturates antibody. The limiting composition of Ag2Ab seemed to have been reached; that is, when increasing concentrations of antigen were added to a limited antibody concentration no additional binding
222 A.A. Benedict, L. W. Pollard, Eleetrophoretic method for hapten-antibody interaction occurred (fig. 3). Although sufficient data have not as yet been obtained to test the assumptions which can be used to estimate the weight of antibody in an antiserum, as was done so elegantly by Osler (1971) and by Revoltella et al., (1971) with a modified Farr technique (1958), the results reported here suggest that such measurements are possible with REC. Two methods of antibody assay are available. The first involves the determination of concentration of antibody or dilution of antiserum required to give 50% binding of a standard concentration of antigen. For example, the standard concentration of antigen could be established as that amount of antigen required to give 80% binding of a reference serum. A second method of estimating antibody potency is based on reacting a constant amount of antibody with varying amounts of antigen and determining the concentration of antigen which just saturates the antibody as suggested by Revoltella et al. (1971). In this connection, estimation of the weight of anti-DNP antibodies in the low nanogram range by REC is of the same order of sensitivity as the modified Farr method. Thus, determination of standard curves and assay of antigens can be accomplished with a sensitivity similar to that which can be achieved by other RIA methods (Hunter, 1970). The immune complexes which are formed during electrophoresis may subsequently dissociate due to the electrophoretic removal of free antigen. The extent of the dissociation, in part, will depend on the antibody affinity. With relatively high electrical current and prolonged electrophoresis some dissociation occurred even with high affinity antibody. Thus, for each antigen-antibody system it is important to establish the conditions whereby association of reactants and separation of bound from free antigen is maximized, while dissociation of bound antigen is minimized. Although dissociation of immune complex is a complicating factor in REC it may be possible to use the phenomenon advantageously. Investigations are proceeding to determine whether complex dissociation can be used to measure the Kre 1. Also, dissociation might be used to detect both antigen and antibody in a serum in which they exist as complexes. Indeed, complexes composed of hepatitis B antigen and antibody have been detected by the REC method (Simons and Benedict, 1974). The smearing of radioactivity on the anodal side of the antigen well when low antibody concentrations were used indicated the migration of immune complexes of varying ratios of antibody to antigen. The FTZ (following-trough zone) does not consist only of free antigen, but rather slowly-moving complexes which are probably composed of excess antigen. Therefore, sections of the FTZ should be included in the assay for bound antigen. However, DNP-conjugates are held up somewhat in the FTZ by NCS (Simons and Benedict, 1974). Also, we have repeatedly observed nonspecific binding of DNP by some pre-immune chicken sera in radioimmunoelectrophoresis. On the other hand, some antigens are not held up in the FTZ by normal sera, and in such cases sensitivity can be increased by extending the 'bound zone' to the anodal margin of the antibody well (Simons and Benedict, 1974). Radioelectrocomplexing is practical in that a large number of samples can be
A.A. Benedict, L. I¢. Pollard, Electrophoretic method for hapten-antibody interaction
223
analyzed in a few hours. The method is relatively simple to perform; no washing or centrifugation is involved. We have extended the use of REC in our laboratory for determination of the immune responses of chickens to the synthetic copolymer of L-glutamic acid, L-alanine, and L-tyrosine (GAT ~0 ).
Acknowledgement The authors wish to thank Dr. Karen Yamaga for obtaining the equilibrium dialysis data.
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