Unusual discrimination against carrier protein antibodies during partial purification of hapten-protein polyclonal antibodies to plant stress hormones.

ABSTRACT

Polyclonal antibodies (PcAb) were raised against t-zeatin riboside (t-ZR) and abscisic acid (ABA). t-ZR-BSA and ABA-BSA antibodies had high titre and specificity to haptens but also contained BSA specific antibodies as observed in double immuno diffusion and quantitative precipitation tests. Partial purification of antiserum by precipitation, desalting, and ion-exchange chromatography almost completely eliminated interference from BSA specific antibodies. Consequently, very little or no reaction was observed in dot-immunoblot assays even when high concentrations of BSA were probed with partially purified t-ZR-BSA IgG. Further studies with ABA antiserum showed that discrimination against BSA occurred during chromatography and not during salt fractionation. Because antibodies to both hapten and carrier protein were predominantly of IgG class, this unusual discrimination against carrier protein Ab was possibly influenced by two approaches followed for DEAE chromatography, viz. (i) adsorption of IgG at pH 8 followed by elution; or (ii) adsorption of contaminating proteins at neutral pH while specific IgG comes off as unbound fraction.