Titration of penicilloyl antibody by enzyme-immunoassay: method and data analysis to assign titer

Antisera to the hapten, penicilloyl, were titered in an indirect enzyme immunoassay with the marker enzyme, horseradish peroxidase. A multivalent solid-phase antigen for stable solid-phase immunocomplex adsorption and a 10 min peroxidase light-assay with photolabile ortho-phenylenediamine were features of the assay. The penicilloyl antisera titration curves formed the basis for an evaluation of 4 methods to assign titer. Titer was expressed as either the antiserum dilution-1 that produced an assigned absorbance, exhibited 50% maximal antigen-antibody binding, and equaled zero absorbance by extrapolation, or as absorbance at 10(-3) antiserum dilution. The accuracy for extrapolated titer was improved with 2 or more solid-phase antigen concentrations. Absorbance at 10(-3) antiserum dilution was most useful as a screen for relative titer and for specific titer from a standard curve generated with many test sera. The antiserum dilution may vary with a particular enzyme immunoassay.