Influence of immunogen modification of the gonadotropin releasing hormone antibody reactivity

The binding of MoAbs and PoAbs to native GnRH, azo-GnRH, GnRH-BSA and azo-GnRH-BSA conjugates was studied using a solid phase ELISA test. The use of carbonate/bicarbonate buffer (pH 9.2) significantly affected the coating of peptide to the ELISA plates. Azo-GnRH coated in PBS showed seven times less binding to MoAb than the native GnRH. Saturation analysis studies indicated comparable binding to the antibodies to the coated GnRH and GnRH-BSA but binding to azo-GnRH-BSA was higher than to azo-GnRH. The epitope recognition ability of MoAbs was therefore investigated. Blocking ELISA additivity tests were performed to analyse whether MoAbs recognised a common epitope involving a conformation or sequence of the modified peptide (azo-GnRH). The plates coated with azo-GnRH or azo-GnRH-BSA and saturated with MoAbs did not bind PoAbs. The inhibition activity was similar when GnRH or GnRH-BSA was used instead of azo-GnRH or azo-GnRH-BSA thus suggesting that the MoAbs were directed against a conformational epitope of the GnRH molecule.