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Thiocyanate elution estimation of relative antibody affinity
Journal of Immunological Methods, 115 (1988) 153-154 Elsevier
153
JIM 05033
Letter to the editors
Thiocyanate elution estimation of relative antibody affinity T o n y J. Hall a n d C o r i n n e Heckel The Merrell Dow Research Institute, 16, rue d'Ankara, 67084 Strasbourg Cedex, France (Letters JIM 05033 and JIM 05038 received and accepted between 1 August 1988 and 9 September 1988)
Dear Editors, Recently, MacDonald et al. (1988) have described a simple ELISA-based assay for determining relative antibody affinity using thiocyanate elution. In their paper six anti-dinitrophenol monoclonal antibodies (mAbs) of known affinity were studied and a good correlation was found between known antibody affinity (determined by equilibrium dialysis) and the thiocyanate concentration required to reduce antibody binding in ELISA by 50% (50% inhibitory concentration: ICs0). We have used this assay on a number of antiphosphocholine mAbs whose relative affinity in terms of ICs0 values for nitrophenylphosphocholine (NPPC) has been measured by hapten inhibition in ELISA (Hall, 1988). However, using thioeyanate elution in the same ELISA system we found a poor correlation between the ICs0 values for NPPC inhibition and thioeyanate elution (see Table I). It is well established that relative antibody affinity can be measured by hapten inhibition in ELISA (Stanley et al., 1983; Nieto et al., 1984) and we have used mAbs with widely differing affinities based on NPPC inhibition values (IC~0 0.007-6.6 mM), but these were not well correlated with thioeyanate elution ICs0 values ( r = 0.457, P > 0.05). We have only used IgG mAbs so that avidity effects due to antibody valency, which should be considered when antibody
Correspondence to: T.J. Hall, The MerreU Dow Research Institute, 16, rue d'Ankara, 67084 Strasbourg Cedex, France.
affinity is being estimated by ELISA (Stevens, 1987), are unlikely to be a factor. Since mAb isotypes were not disclosed in the Macdonald et al. paper it is not clear whether this was a factor in their experiments, in which mAbs of widely differing affinity were also studied. The thiocyanate elution ELISA could be considered as a useful and simple method for determining relative antibody affinity of mAbs to complex antigens such as proteins or cell surface antigens. However, we would like to draw attention to the fact that this methodology may not be applicable to all mAbs a n d / o r antigen systems.
TABLE I mAb
Isotype
ICs0 for NPPC (mM)
IC for thiocyanate (M)
PCG3-2 PCG1-4 PCG1-6 PCGI-10 PCG3-3 PCGl-13
IgG3,~. 2 IgGl,x IgGl,r IgG1,A 1 IgG3,hl IgGl,r
0.007 (1) 0.01 (2) 0.02 (3) 0.057 (4) 0.44 (5) 6.6 (6)
1,84 (2) 0,15 (1) 2.96 (6) 2.02 (4) 1.85 (3) 2.35 (5)
Key: Anti-PC mAbs were isotyped and NPPC ICs0 values were determined by ELISA on PC-histone-coated plates using subclass specific alkaline phosphatase-labelled rabbit anti-mouse IgG1 and IgG3 antibodies (Zymed, California, U.S.A.) as previously described (Hall, 1988). Sodium thiocyanate ehition was assessed as described by Macdonald et al. (1988), using thiocyanate concentrations of 0.1-3.0 M. ICs0 values were calculated from standard binding curves of each mAb which were included on the ELISA plates in each experiment. Pearson's rank correlation coefficient between the ranked ICso values for NPPC and thiocyanate for each of the mAbs (shown in brackets) was 0.457 (P > 0.05).
0022-1759/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
154
References Hall, T.J. (1988) A newly characterized group of antiphosphocholine antibodies: Group III antibodies. Mol. Immunol. 25, 447. Macdonald, R.A., Hosking, C.S. and Jones, C.L. (1988) The measurement of relative antibody affinity by ELISA using thiocyanate elution. J. Immunol. Methods 106, 191. Nieto, A., Gaya, A., Jansa, M., Moreno, C. and Vires, J. (1984) Direct measurement of antibody affinity distribution by hapten-inhibition enzyme immunoassay. Mol. lmmunol. 21, 537.
Stanley, C., Lew, A.M. and Steward, M.W. (1983) The measurement of antibody affinity: a comparison of five techniques utilizing a panel of monoclonal antiDNP antibodies and the effect of high affinity antibody on the measurement of low affinity antibody. J. Immunol. Methods 64. 119. Stevens, F.J. (1987) Modification of an ELISA-based procedure for affinity determination: correlation necessary for use with bivalent antibody. Mol. lmmunol. 24, 1055.
153
JIM 05033
Letter to the editors
Thiocyanate elution estimation of relative antibody affinity T o n y J. Hall a n d C o r i n n e Heckel The Merrell Dow Research Institute, 16, rue d'Ankara, 67084 Strasbourg Cedex, France (Letters JIM 05033 and JIM 05038 received and accepted between 1 August 1988 and 9 September 1988)
Dear Editors, Recently, MacDonald et al. (1988) have described a simple ELISA-based assay for determining relative antibody affinity using thiocyanate elution. In their paper six anti-dinitrophenol monoclonal antibodies (mAbs) of known affinity were studied and a good correlation was found between known antibody affinity (determined by equilibrium dialysis) and the thiocyanate concentration required to reduce antibody binding in ELISA by 50% (50% inhibitory concentration: ICs0). We have used this assay on a number of antiphosphocholine mAbs whose relative affinity in terms of ICs0 values for nitrophenylphosphocholine (NPPC) has been measured by hapten inhibition in ELISA (Hall, 1988). However, using thioeyanate elution in the same ELISA system we found a poor correlation between the ICs0 values for NPPC inhibition and thioeyanate elution (see Table I). It is well established that relative antibody affinity can be measured by hapten inhibition in ELISA (Stanley et al., 1983; Nieto et al., 1984) and we have used mAbs with widely differing affinities based on NPPC inhibition values (IC~0 0.007-6.6 mM), but these were not well correlated with thioeyanate elution ICs0 values ( r = 0.457, P > 0.05). We have only used IgG mAbs so that avidity effects due to antibody valency, which should be considered when antibody
Correspondence to: T.J. Hall, The MerreU Dow Research Institute, 16, rue d'Ankara, 67084 Strasbourg Cedex, France.
affinity is being estimated by ELISA (Stevens, 1987), are unlikely to be a factor. Since mAb isotypes were not disclosed in the Macdonald et al. paper it is not clear whether this was a factor in their experiments, in which mAbs of widely differing affinity were also studied. The thiocyanate elution ELISA could be considered as a useful and simple method for determining relative antibody affinity of mAbs to complex antigens such as proteins or cell surface antigens. However, we would like to draw attention to the fact that this methodology may not be applicable to all mAbs a n d / o r antigen systems.
TABLE I mAb
Isotype
ICs0 for NPPC (mM)
IC for thiocyanate (M)
PCG3-2 PCG1-4 PCG1-6 PCGI-10 PCG3-3 PCGl-13
IgG3,~. 2 IgGl,x IgGl,r IgG1,A 1 IgG3,hl IgGl,r
0.007 (1) 0.01 (2) 0.02 (3) 0.057 (4) 0.44 (5) 6.6 (6)
1,84 (2) 0,15 (1) 2.96 (6) 2.02 (4) 1.85 (3) 2.35 (5)
Key: Anti-PC mAbs were isotyped and NPPC ICs0 values were determined by ELISA on PC-histone-coated plates using subclass specific alkaline phosphatase-labelled rabbit anti-mouse IgG1 and IgG3 antibodies (Zymed, California, U.S.A.) as previously described (Hall, 1988). Sodium thiocyanate ehition was assessed as described by Macdonald et al. (1988), using thiocyanate concentrations of 0.1-3.0 M. ICs0 values were calculated from standard binding curves of each mAb which were included on the ELISA plates in each experiment. Pearson's rank correlation coefficient between the ranked ICso values for NPPC and thiocyanate for each of the mAbs (shown in brackets) was 0.457 (P > 0.05).
0022-1759/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
154
References Hall, T.J. (1988) A newly characterized group of antiphosphocholine antibodies: Group III antibodies. Mol. Immunol. 25, 447. Macdonald, R.A., Hosking, C.S. and Jones, C.L. (1988) The measurement of relative antibody affinity by ELISA using thiocyanate elution. J. Immunol. Methods 106, 191. Nieto, A., Gaya, A., Jansa, M., Moreno, C. and Vires, J. (1984) Direct measurement of antibody affinity distribution by hapten-inhibition enzyme immunoassay. Mol. lmmunol. 21, 537.
Stanley, C., Lew, A.M. and Steward, M.W. (1983) The measurement of antibody affinity: a comparison of five techniques utilizing a panel of monoclonal antiDNP antibodies and the effect of high affinity antibody on the measurement of low affinity antibody. J. Immunol. Methods 64. 119. Stevens, F.J. (1987) Modification of an ELISA-based procedure for affinity determination: correlation necessary for use with bivalent antibody. Mol. lmmunol. 24, 1055.