The measurement of relative antibody affinity by ELISA using thiocyanate elution

  • MacDonald R
  • Hosking C
  • Jones C
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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 with3H-ε{lunate}-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. © 1988.

Author-supplied keywords

  • Affinity index
  • Antibody affinity
  • Equilibrium dialysis
  • Thiocyanate elution

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  • Ross A. MacDonald

  • Clifford S. Hosking

  • Colin L. Jones

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