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The measurement of relative antibody affinity by ELISA using thiocyanate elution
Journal of Immunological Methods, 106 (1988) 191-194
191
Elsevier JIM04614
The measurement of relative antibody affinity by ELISA using thiocyanate elution Ross A. Macdonald, Clifford S. Hosking and Colin L. Jones Department of Immunology, Royal Children~ Itospital, Parkville. Vtctoria, Australia (Received 23 June 1987. revised received 27 August 1987, accepted 7 September 1987)
A variation of the standard ELISA assay was used to determine the relative affinities of six murine monoclonal anti-dinitrophenol (DNP) antibodies for DNP-bovine serum albumin (DNP-BSA). The procedure involved exposing replicate wells containing antibody bound to antigen to increasing concentrations of the chaotropic thiocyanate ion. Resistance to thiocyanate elution was utilized as a measure of affinity and an index (affinity index) representing a 50% reduction in initial absorbance was used to rank the anti-DNP antibodies in order of increasing affinity. For comparison, the affinity constants of the six monoclonals were determined by equilibrium dialysis with 3H-~-DNP-L-lysine as antigen. A significant correlation (P < 0.02) was observed between the ranking orders obtained using the two methods, thereby demonstrating the applicability of the elution technique for the measurement of relative antibody affinity. Key words: Antibody affinity; Equilibrium dialysis; Affinity index; ELISA; Thiocyanate elution
Introduction
The affinity of an antibody for its corresponding antigen has been shown in many instances to be an important determinant of the biological efficacy of that antibody (Steward and Steensgaard, 1983). Furthermore, excessive production of low affinity antibody has been considered as an expression of immunodeficiency (Soothill and Steward, 1971), and antibody affinity is also undoubtedly involved in the immunopathology of autoimmune and immune complex diseases (Kuriyama, 1973; Steward, 1976; Shimizu et al., 1978; Steward and Steensgaard, 1983; Noble et al., 1987). Corre.~pondence to: R.A. Macdonald, Department of Immunology, Royal Children's Hospital, Flemington Rd., Parkville 3052, Victoria, Australia. Support: R.A.M. was supported in this study by the Royal Children's Hospital Research Foundation. C.L.J. was supported by an N.H. & M.R.C. Research Scholarship.
Measurement of the affinity or 'functional affinity' (Karush, 1970) of a particular immune serum, although of obvious clinical importance, is nevertheless difficult and time consuming. In a recent communication from this laboratory, a simple enzyme-linked immunosorbent assay (ELISA) procedure for the evaluation of antibody affinity was described (Pullen et al., 1986). Specifically, the relative affinities of anti-rubella virus antibodies in various sera were quantitated using an index representing the molarity of ammonium thiocyanate required to reduce the initial optical density by 50%. In the present study, we have examined in more detail the disruption of antigen-antibody binding by chaotropic ions as a means of determining relative affinity. This has been achieved by investigating the behaviour of a panel of monoclonal anti-DNP antibodies in the ELISA-based procedure.
0022-1759/88/$03.50 ,~.~1988 Elsevier Science Publishers B.V, (Biomedical Division)
192 Materials and methods
The ELISA procedure DNP-BSA was prepared as described by Hudson and Hay (1976). The degree of substitution, assessed spectrophotometrically, was 15:1. The standardization of the EL1SA was the result of a series of optimization experiments involving chequerboard titrations and time course reactions. Antigen was adsorbed onto 96-well microtitre plates (Nunc Immunoplate I) by incubating 100 gl/well of a 1 in 2 dilution of the dinitrophenylated-BSA (Hudson and Hay, 1976) in 0.05 M carbonate buffer, pH 9.6. at room temperature for 4 h in the dark. The plates were then washed four times with PBS containing 0.05% Tween 20 (PBSTw). Appropriate dilutions of the anti-DNP monoclonal antibodies (McAbs) were prepared in PBS-Tw containing 0.5% ( w / v ) BSA (PBS-TwBSA) and 100 ~1 aliquots were dispensed in triplicate into antigen coated wells. The plates were then incubated at 3 7 ° C for 2 h before repeating the washing step. 100 ~tl of peroxidase-conjugated sheep anti-mouse antiserum (Silenus Laboratories, Australia) diluted 1 in 1000 in PBS-Tw-BSA were added, and the plates were incubated at 37 o C for 3 h. After washing, 100 #1 of 0.04% ( w / v ) of o-phenylenediamine. 2HC1 (Abbot Laboratories, Chicago, IL) in 0.03 M citrate-phosphate buffer (pH 5.0) containing 1 in 1000 H20_~ were added to each well. The enzyme reaction was stopped between 3 and 5 min by the addition of 100/~l of 1 N H_,SO4. The optical density of the solution in each well was read in a Titertek Multiscan with an interference filter at 492 nm. A blank absorbance obtained by incubating buffer instead of a McAb dilution was subtracted from the absorbances. Ascites resulting from the implantation of a hybridoma producing antibody to an irrelevant antigen and serum from an unimmunized mouse were substituted for McAb as a negative control.
Affinity elution procedure. This procedure was similar to that described above, with the inclusion of an extra step. After the plate was washed following incubation of the dilution of McAb, ammonium thiocyanate in 0.1 M phosphate buffer, pH 6.0, was added to the appropriate wells (100 #l/well) in triplicate, in
concentrations ranging from 0 to 8 M. The plates were allowed to stand for 15 min at room temperature before thrice washing with PBS-Tw and proceeding with the assay as described above. The effect of concentrations of NH4SCN up to 8 M on the binding of DNP-BSA was investigated.
Data analysis The determination of the affinity index was performed as described previously (Pullen et al.. 1986). In this case, however, data were fitted to a graph of logl~j (% of initial absorbance) vs. molarity of N H 4 S C N by third degree polynomial regression analysis as performed by a graph generation program (Grafit/1000, Corporate Computer Systems, N J) on a Hewlett Packard 1000 computer. Initial absorbancc was taken as that obtained with no NH4SCN present.
Monoclonal antibodies Murine monoclonal anti-DNP antibodies were the kind gift of Professor M.W. Steward. Further details concerning their preparation and purification are described in Stanley et al. (1983).
Equilibrium dialysis The affinities of the McAbs for 3H-e-DNPq,lysine (3.2 C i / m m o l , New England Nuclear) were determined by equilibrium dialysis using perspex chambers, consisting of two halves separated by a dialysis membrane. A series of six hapten concentrations were used, ranging from 10 -~ to 10 ~ M. 555 /~1 of McAb. diluted (50 p,g/ml) in PBS containing 0.2% BSA and 0.05% sodium azide, were added to one side of the chamber, and 1.4 ml of appropriate concentrations of labelled hapten in the same diluent was added to the other side. "['he chambers were kept in a humidified container in a refrigerator at 4 ° C . A control chamber, prepared without antibody, was used to ascertain when equilibrium had been reached. Aliquots were then taken from both sides of the dialysis membrane and 3H counts determined in each using a Packard liquid scintillation spectrophotometer. Antibody affinity was calculated from antibodybound and free antigen concentrations as described by Stanley et al. (1983).
193 TABLE I
Results
Determination of affinity index
The outcome of the ELISA affinity elution procedure, performed on the series of anti-DNP McAbs are illustrated in Fig. 1. The concentration of McAbs used in this procedure were those which, when assayed by ELISA. had optical densities near the top of the linear portion of the titre curve. The affinity indices were determined as follows. A line at y = 1.699 (1og10150% initial absorbance]) was drawn parallel to the x-axis. The point relative to the x-axis at which this line intersects the elution curve for a particular McAb was taken as the affinity index for that McAb, i.e., the molarity of NH4SCN causing a 50% reduction in initial absorbance. The affinity indices calculated for the six McAbs are shown in Table I. It will be noted that an accurate index could not be obtained for McAb no. 3 as this antibody failed to elute to an appreciable extent, even with 10 M NHaSCN. Table I also shows equilibrium constants for each of the McAbs, as determined by equilibrium dialysis. The Kendall rank correlation coefficient between the affinity indices and the equilibrium constants for each of the McAbs was 0.867 (0.01 < P < 0.02). While tho elution technique failed to correctly rank McAbs no. 2 and 6, it will be noted that their respective affinity constants are nevertheless close. Neither irrelevant antibody from mouse ascites nor serum from an unimmunized mouse showed a
AFFINITY CONSTANTS A N D AFFINITY INDICES OF SIX MONOCLONAL ANTI-DNP ANTIBODIES, AS MEAS U R E D BY EQUILIBRIUM DIALYSIS AND NHaSCN ELUTION, RESPECTIVELY Rankings are in order of increasing affinity. Kendall rank correlation 'r = 0.867, P < 0.02. Monoclonal antibody no.
1 2 3 4 5 6
Affinity Affinity index (equilibrium dialysis) (thiocyanate elution) l/tool
Rank
mol NH4SCN
Rank
2.00 x 106 1.87×107 3.80x 108 2.90 x 105 7.68x 106 1.54x 107
2 5 6 1 3 4
1.6 6.7 >8 0.2 3.9 6.9
2 4 6 1 3 5
response above the background level. DNP-BSA binding to the ELISA plate was unaffected by up to 8 M NH4SCN. and the elution procedure did not itself contribute to the optical density. Accuracy and reproducibility o f the assay
The variation in the affinity index obtained for one McAb repeatedly tested on the same plate, and on different plates on different days was determined. The interplate coefficient of variation (CV) was 10.1% (n = 7), and the intraplate CV was 5.01% (n = 4).
Discussion
- ~ .....................~ ~ - ~ , i --~...... = ................... ._1 I--
o
\
.... <>..--
~zAb
02
McAb
#4
M~b iS -4,--- ~ b #S
_o
I
0
$
I
I
I
I
I
I
I
l
1
2
3
4
5
6
7
8
9
NH4SCN CONCENTRATION (molor)
I0
I
Fig. 1. Effect upon initial absorbance by adding increasing concentrations of NH,;SCN in the anti-DNP ELISA.
A large number of techniques have been described for the determination of antibody affinity (reviewed in Sigal and Klinman, 1978). These include precipitation of complexes by various agents. separation of complexes based on size and electrophoretic mobility, changes in fluorescent properties and equilibrium dialysis. Estimates of antibody affinity have also been made using various aspects of dose-response curves in EL1SAs (Lehtonen and Eerola, 1982; Gripenberg and Gripenberg, 1983). While the reference method for affinity determination remains equilibrium dialysis, this procedure cannot be used with large, polyvalent antigens and it poses particular prob-
194
lems in the measurement of affinity of polyclonal antibodies (Steward and Steensgaard, 1983). Similarly, the precipitation technique described by Farr (1958) cannot be used for determining the quality of antibody to soluble proteins which are precipitable at 50% saturation of ammonium sulphate. By contrast, the present technique is not bound by these limitations, and may be applied to any antigen-antibody interaction for which a sandwich ELISA is appropriate, and provided binding of antigen to the ELISA plate is unaffected by NH4SCN. A number of assumptions are inherent in the elution procedure, however, and these must be taken into account. The first is that, in common with all ELISAs, the reagent concentrations arc largely unknown. While this may be so, the present assay is performed in antigen and substrate excess. Therefore, it is not unreasonable to assume that the optical density is wholly and linearly dependent upon the amount of McAb bound to the ELISA plate. This latter point has been demonstrated in this laboratory by Dr. C.L. Jones (manuscript in press). Secondly, it is assumed that immunologic equilibrium has occurred before the elution procedure. In this assay, the incubation time chosen was that at which little further binding of McAb was detected, Finally, it is assumed that antibodies of a specific affinity are not preferentially detected. While this point does not apply to the current study, it does bear relevance to analyses of polyclonal sera. In this context, it is likely that the chaotropic ion SCN affects the binding of all antibodies of a particular class to a similar extent, such that the immunoglobulin molecule itself is not irreversibly denatured (Eddington. 1971 ), and that whether a particular antibody molecule is eluted or not is solely dependent upon its affinity. In this respect, the elution procedure adequately evaluates binding of low affinity antibodies, which are difficult to measure by other methods (Stanley et al., 1983). In summary, a comparison of the chaotropic elution and equilibrium dialysis techniques for determining the quality of monoclonal antibodies for a monovalent hapten showed that the results from the two methods gave nearly identical rank orders. Provided certain constraints arc observed,
the present procedure represents a simple, reproducible and rapid means of ascertaining antibody affinity. Whether the results of this assay in polyclonal systems (Pullen et al., 1986) represent a measurement of 'average' avidity (Steward and Steensgaard, 1983), or a point at some other loci on the affinity distribution function, is not known. References Eddington. T.S. (1971) Dissociation of antibexJv from erythrocyte surfaces by chaotropic ions. J. lmmunol. 106, 673. Farr, R.S. (1958) A quantitative immunochemical measure of the primary interaction between I-BSA and antibody,. J. Infect. Dis. 103, 239. Gripenberg, M. and (~'ripenberg, O. (1983) Expression of ,mtibody activity measured by' ELISA. Anti-ssDNA antibody activity characterized by the shape of the dose-response Curve.
Hudson, L. and Hay. F.C. (1976) Practical Immunology. Blackwcll Scientific, Oxford, p. 5. Karush, F. (197(/) Affinity and the immune response. Ann. N.Y. Acad..'~i. 169, 56. Kuriyama, T. (1973) Chronic glomerulonephritis induced b~, prolonged immunization in the rabbit. Lab. Invest. 28. 224. l,ehtonen, O.P. and t?.erola, E. (1982) The effect of different antibody affinities on ELISA absorbance and titrc. J. lmmunol. Methods 62, 315. Noble, B.. Steward. M.W., Vladutiu, A. and Brentjens, J.R. (1987) Relationship of the quality of circulating anti-BSA antibodies to the severity of glomerulonephritis in rats with chronic serum sickness. Clin. Exp. Immunol. 67, 277. Pullen, GR., Fitzgerald, M.G. and Hosking, C.S. (1986) Antibody avidity determination by EI,ISA using thiocyanatc elution. J. immunol. Methtxts 86. 83. Shimizu. F., Mossmann, H., Takayima, H. and Vogt. A. 11978) F,ffect of antibody avidity of the induction of renal injur), in anti-glomerular basement membrane nephritis. Br. I Exp. Pathol. 59, 624. Sigal, N.H. and Klinman, N.R. (1978) The B-cell clonotype repertoire. Adv. hnmunol. 26, 255. S~thill. J.F. and Steward, M.W. (1971) The immunological significance of the heterogeneity of antibody affinity. Clin. Exp. Immunol. 9, 193. Stanley. C.. Lew. A.M. and Steward, M.W. ( 1 9 8 3 ) l h c measurement of antibody affinity: a comparison of five techniques utilizing a panel of monoclonal anti-DNP antibodies and the effect of high affinity antibody on the measurement of low affinity antibody. J. lmmunol. Methods 64, 119. Steward. M.W. (1976) In: D.C. Dumonde (Ed.), Infection and Immunology in the Rheumatic Diseases. Blackwell Scientific. Oxford, p. 439. Steward, M.W. and Steensgaard, J. (1983) Antibody Affinity: Thermodynamic Aspects and Biological Significance. CRC Press, B~ca Raton, FL.
191
Elsevier JIM04614
The measurement of relative antibody affinity by ELISA using thiocyanate elution Ross A. Macdonald, Clifford S. Hosking and Colin L. Jones Department of Immunology, Royal Children~ Itospital, Parkville. Vtctoria, Australia (Received 23 June 1987. revised received 27 August 1987, accepted 7 September 1987)
A variation of the standard ELISA assay was used to determine the relative affinities of six murine monoclonal anti-dinitrophenol (DNP) antibodies for DNP-bovine serum albumin (DNP-BSA). The procedure involved exposing replicate wells containing antibody bound to antigen to increasing concentrations of the chaotropic thiocyanate ion. Resistance to thiocyanate elution was utilized as a measure of affinity and an index (affinity index) representing a 50% reduction in initial absorbance was used to rank the anti-DNP antibodies in order of increasing affinity. For comparison, the affinity constants of the six monoclonals were determined by equilibrium dialysis with 3H-~-DNP-L-lysine as antigen. A significant correlation (P < 0.02) was observed between the ranking orders obtained using the two methods, thereby demonstrating the applicability of the elution technique for the measurement of relative antibody affinity. Key words: Antibody affinity; Equilibrium dialysis; Affinity index; ELISA; Thiocyanate elution
Introduction
The affinity of an antibody for its corresponding antigen has been shown in many instances to be an important determinant of the biological efficacy of that antibody (Steward and Steensgaard, 1983). Furthermore, excessive production of low affinity antibody has been considered as an expression of immunodeficiency (Soothill and Steward, 1971), and antibody affinity is also undoubtedly involved in the immunopathology of autoimmune and immune complex diseases (Kuriyama, 1973; Steward, 1976; Shimizu et al., 1978; Steward and Steensgaard, 1983; Noble et al., 1987). Corre.~pondence to: R.A. Macdonald, Department of Immunology, Royal Children's Hospital, Flemington Rd., Parkville 3052, Victoria, Australia. Support: R.A.M. was supported in this study by the Royal Children's Hospital Research Foundation. C.L.J. was supported by an N.H. & M.R.C. Research Scholarship.
Measurement of the affinity or 'functional affinity' (Karush, 1970) of a particular immune serum, although of obvious clinical importance, is nevertheless difficult and time consuming. In a recent communication from this laboratory, a simple enzyme-linked immunosorbent assay (ELISA) procedure for the evaluation of antibody affinity was described (Pullen et al., 1986). Specifically, the relative affinities of anti-rubella virus antibodies in various sera were quantitated using an index representing the molarity of ammonium thiocyanate required to reduce the initial optical density by 50%. In the present study, we have examined in more detail the disruption of antigen-antibody binding by chaotropic ions as a means of determining relative affinity. This has been achieved by investigating the behaviour of a panel of monoclonal anti-DNP antibodies in the ELISA-based procedure.
0022-1759/88/$03.50 ,~.~1988 Elsevier Science Publishers B.V, (Biomedical Division)
192 Materials and methods
The ELISA procedure DNP-BSA was prepared as described by Hudson and Hay (1976). The degree of substitution, assessed spectrophotometrically, was 15:1. The standardization of the EL1SA was the result of a series of optimization experiments involving chequerboard titrations and time course reactions. Antigen was adsorbed onto 96-well microtitre plates (Nunc Immunoplate I) by incubating 100 gl/well of a 1 in 2 dilution of the dinitrophenylated-BSA (Hudson and Hay, 1976) in 0.05 M carbonate buffer, pH 9.6. at room temperature for 4 h in the dark. The plates were then washed four times with PBS containing 0.05% Tween 20 (PBSTw). Appropriate dilutions of the anti-DNP monoclonal antibodies (McAbs) were prepared in PBS-Tw containing 0.5% ( w / v ) BSA (PBS-TwBSA) and 100 ~1 aliquots were dispensed in triplicate into antigen coated wells. The plates were then incubated at 3 7 ° C for 2 h before repeating the washing step. 100 ~tl of peroxidase-conjugated sheep anti-mouse antiserum (Silenus Laboratories, Australia) diluted 1 in 1000 in PBS-Tw-BSA were added, and the plates were incubated at 37 o C for 3 h. After washing, 100 #1 of 0.04% ( w / v ) of o-phenylenediamine. 2HC1 (Abbot Laboratories, Chicago, IL) in 0.03 M citrate-phosphate buffer (pH 5.0) containing 1 in 1000 H20_~ were added to each well. The enzyme reaction was stopped between 3 and 5 min by the addition of 100/~l of 1 N H_,SO4. The optical density of the solution in each well was read in a Titertek Multiscan with an interference filter at 492 nm. A blank absorbance obtained by incubating buffer instead of a McAb dilution was subtracted from the absorbances. Ascites resulting from the implantation of a hybridoma producing antibody to an irrelevant antigen and serum from an unimmunized mouse were substituted for McAb as a negative control.
Affinity elution procedure. This procedure was similar to that described above, with the inclusion of an extra step. After the plate was washed following incubation of the dilution of McAb, ammonium thiocyanate in 0.1 M phosphate buffer, pH 6.0, was added to the appropriate wells (100 #l/well) in triplicate, in
concentrations ranging from 0 to 8 M. The plates were allowed to stand for 15 min at room temperature before thrice washing with PBS-Tw and proceeding with the assay as described above. The effect of concentrations of NH4SCN up to 8 M on the binding of DNP-BSA was investigated.
Data analysis The determination of the affinity index was performed as described previously (Pullen et al.. 1986). In this case, however, data were fitted to a graph of logl~j (% of initial absorbance) vs. molarity of N H 4 S C N by third degree polynomial regression analysis as performed by a graph generation program (Grafit/1000, Corporate Computer Systems, N J) on a Hewlett Packard 1000 computer. Initial absorbancc was taken as that obtained with no NH4SCN present.
Monoclonal antibodies Murine monoclonal anti-DNP antibodies were the kind gift of Professor M.W. Steward. Further details concerning their preparation and purification are described in Stanley et al. (1983).
Equilibrium dialysis The affinities of the McAbs for 3H-e-DNPq,lysine (3.2 C i / m m o l , New England Nuclear) were determined by equilibrium dialysis using perspex chambers, consisting of two halves separated by a dialysis membrane. A series of six hapten concentrations were used, ranging from 10 -~ to 10 ~ M. 555 /~1 of McAb. diluted (50 p,g/ml) in PBS containing 0.2% BSA and 0.05% sodium azide, were added to one side of the chamber, and 1.4 ml of appropriate concentrations of labelled hapten in the same diluent was added to the other side. "['he chambers were kept in a humidified container in a refrigerator at 4 ° C . A control chamber, prepared without antibody, was used to ascertain when equilibrium had been reached. Aliquots were then taken from both sides of the dialysis membrane and 3H counts determined in each using a Packard liquid scintillation spectrophotometer. Antibody affinity was calculated from antibodybound and free antigen concentrations as described by Stanley et al. (1983).
193 TABLE I
Results
Determination of affinity index
The outcome of the ELISA affinity elution procedure, performed on the series of anti-DNP McAbs are illustrated in Fig. 1. The concentration of McAbs used in this procedure were those which, when assayed by ELISA. had optical densities near the top of the linear portion of the titre curve. The affinity indices were determined as follows. A line at y = 1.699 (1og10150% initial absorbance]) was drawn parallel to the x-axis. The point relative to the x-axis at which this line intersects the elution curve for a particular McAb was taken as the affinity index for that McAb, i.e., the molarity of NH4SCN causing a 50% reduction in initial absorbance. The affinity indices calculated for the six McAbs are shown in Table I. It will be noted that an accurate index could not be obtained for McAb no. 3 as this antibody failed to elute to an appreciable extent, even with 10 M NHaSCN. Table I also shows equilibrium constants for each of the McAbs, as determined by equilibrium dialysis. The Kendall rank correlation coefficient between the affinity indices and the equilibrium constants for each of the McAbs was 0.867 (0.01 < P < 0.02). While tho elution technique failed to correctly rank McAbs no. 2 and 6, it will be noted that their respective affinity constants are nevertheless close. Neither irrelevant antibody from mouse ascites nor serum from an unimmunized mouse showed a
AFFINITY CONSTANTS A N D AFFINITY INDICES OF SIX MONOCLONAL ANTI-DNP ANTIBODIES, AS MEAS U R E D BY EQUILIBRIUM DIALYSIS AND NHaSCN ELUTION, RESPECTIVELY Rankings are in order of increasing affinity. Kendall rank correlation 'r = 0.867, P < 0.02. Monoclonal antibody no.
1 2 3 4 5 6
Affinity Affinity index (equilibrium dialysis) (thiocyanate elution) l/tool
Rank
mol NH4SCN
Rank
2.00 x 106 1.87×107 3.80x 108 2.90 x 105 7.68x 106 1.54x 107
2 5 6 1 3 4
1.6 6.7 >8 0.2 3.9 6.9
2 4 6 1 3 5
response above the background level. DNP-BSA binding to the ELISA plate was unaffected by up to 8 M NH4SCN. and the elution procedure did not itself contribute to the optical density. Accuracy and reproducibility o f the assay
The variation in the affinity index obtained for one McAb repeatedly tested on the same plate, and on different plates on different days was determined. The interplate coefficient of variation (CV) was 10.1% (n = 7), and the intraplate CV was 5.01% (n = 4).
Discussion
- ~ .....................~ ~ - ~ , i --~...... = ................... ._1 I--
o
\
.... <>..--
~zAb
02
McAb
#4
M~b iS -4,--- ~ b #S
_o
I
0
$
I
I
I
I
I
I
I
l
1
2
3
4
5
6
7
8
9
NH4SCN CONCENTRATION (molor)
I0
I
Fig. 1. Effect upon initial absorbance by adding increasing concentrations of NH,;SCN in the anti-DNP ELISA.
A large number of techniques have been described for the determination of antibody affinity (reviewed in Sigal and Klinman, 1978). These include precipitation of complexes by various agents. separation of complexes based on size and electrophoretic mobility, changes in fluorescent properties and equilibrium dialysis. Estimates of antibody affinity have also been made using various aspects of dose-response curves in EL1SAs (Lehtonen and Eerola, 1982; Gripenberg and Gripenberg, 1983). While the reference method for affinity determination remains equilibrium dialysis, this procedure cannot be used with large, polyvalent antigens and it poses particular prob-
194
lems in the measurement of affinity of polyclonal antibodies (Steward and Steensgaard, 1983). Similarly, the precipitation technique described by Farr (1958) cannot be used for determining the quality of antibody to soluble proteins which are precipitable at 50% saturation of ammonium sulphate. By contrast, the present technique is not bound by these limitations, and may be applied to any antigen-antibody interaction for which a sandwich ELISA is appropriate, and provided binding of antigen to the ELISA plate is unaffected by NH4SCN. A number of assumptions are inherent in the elution procedure, however, and these must be taken into account. The first is that, in common with all ELISAs, the reagent concentrations arc largely unknown. While this may be so, the present assay is performed in antigen and substrate excess. Therefore, it is not unreasonable to assume that the optical density is wholly and linearly dependent upon the amount of McAb bound to the ELISA plate. This latter point has been demonstrated in this laboratory by Dr. C.L. Jones (manuscript in press). Secondly, it is assumed that immunologic equilibrium has occurred before the elution procedure. In this assay, the incubation time chosen was that at which little further binding of McAb was detected, Finally, it is assumed that antibodies of a specific affinity are not preferentially detected. While this point does not apply to the current study, it does bear relevance to analyses of polyclonal sera. In this context, it is likely that the chaotropic ion SCN affects the binding of all antibodies of a particular class to a similar extent, such that the immunoglobulin molecule itself is not irreversibly denatured (Eddington. 1971 ), and that whether a particular antibody molecule is eluted or not is solely dependent upon its affinity. In this respect, the elution procedure adequately evaluates binding of low affinity antibodies, which are difficult to measure by other methods (Stanley et al., 1983). In summary, a comparison of the chaotropic elution and equilibrium dialysis techniques for determining the quality of monoclonal antibodies for a monovalent hapten showed that the results from the two methods gave nearly identical rank orders. Provided certain constraints arc observed,
the present procedure represents a simple, reproducible and rapid means of ascertaining antibody affinity. Whether the results of this assay in polyclonal systems (Pullen et al., 1986) represent a measurement of 'average' avidity (Steward and Steensgaard, 1983), or a point at some other loci on the affinity distribution function, is not known. References Eddington. T.S. (1971) Dissociation of antibexJv from erythrocyte surfaces by chaotropic ions. J. lmmunol. 106, 673. Farr, R.S. (1958) A quantitative immunochemical measure of the primary interaction between I-BSA and antibody,. J. Infect. Dis. 103, 239. Gripenberg, M. and (~'ripenberg, O. (1983) Expression of ,mtibody activity measured by' ELISA. Anti-ssDNA antibody activity characterized by the shape of the dose-response Curve.
Hudson, L. and Hay. F.C. (1976) Practical Immunology. Blackwcll Scientific, Oxford, p. 5. Karush, F. (197(/) Affinity and the immune response. Ann. N.Y. Acad..'~i. 169, 56. Kuriyama, T. (1973) Chronic glomerulonephritis induced b~, prolonged immunization in the rabbit. Lab. Invest. 28. 224. l,ehtonen, O.P. and t?.erola, E. (1982) The effect of different antibody affinities on ELISA absorbance and titrc. J. lmmunol. Methods 62, 315. Noble, B.. Steward. M.W., Vladutiu, A. and Brentjens, J.R. (1987) Relationship of the quality of circulating anti-BSA antibodies to the severity of glomerulonephritis in rats with chronic serum sickness. Clin. Exp. Immunol. 67, 277. Pullen, GR., Fitzgerald, M.G. and Hosking, C.S. (1986) Antibody avidity determination by EI,ISA using thiocyanatc elution. J. immunol. Methtxts 86. 83. Shimizu. F., Mossmann, H., Takayima, H. and Vogt. A. 11978) F,ffect of antibody avidity of the induction of renal injur), in anti-glomerular basement membrane nephritis. Br. I Exp. Pathol. 59, 624. Sigal, N.H. and Klinman, N.R. (1978) The B-cell clonotype repertoire. Adv. hnmunol. 26, 255. S~thill. J.F. and Steward, M.W. (1971) The immunological significance of the heterogeneity of antibody affinity. Clin. Exp. Immunol. 9, 193. Stanley. C.. Lew. A.M. and Steward, M.W. ( 1 9 8 3 ) l h c measurement of antibody affinity: a comparison of five techniques utilizing a panel of monoclonal anti-DNP antibodies and the effect of high affinity antibody on the measurement of low affinity antibody. J. lmmunol. Methods 64, 119. Steward. M.W. (1976) In: D.C. Dumonde (Ed.), Infection and Immunology in the Rheumatic Diseases. Blackwell Scientific. Oxford, p. 439. Steward, M.W. and Steensgaard, J. (1983) Antibody Affinity: Thermodynamic Aspects and Biological Significance. CRC Press, B~ca Raton, FL.