Singh V
Institute of Self-Organising systems and Biophysics, North-Eastern Hill University, Meghalaya, India.
Biochem Int. 1992 Aug;27(4):579-89.
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.
使用固相酶联免疫吸附测定（ELISA）试验研究了单克隆抗体（MoAbs）和多克隆抗体（PoAbs）与天然促性腺激素释放激素（GnRH）、偶氮-GnRH、GnRH-牛血清白蛋白（BSA）及偶氮-GnRH-BSA偶联物的结合情况。使用碳酸盐/碳酸氢盐缓冲液（pH 9.2）对肽包被至ELISA板有显著影响。包被于磷酸盐缓冲盐水（PBS）中的偶氮-GnRH与MoAb的结合比天然GnRH少7倍。饱和分析研究表明，包被的GnRH和GnRH-BSA与抗体的结合相当，但与偶氮-GnRH-BSA的结合高于与偶氮-GnRH的结合。因此研究了MoAbs的表位识别能力。进行封闭ELISA加和性试验以分析MoAbs是否识别涉及修饰肽（偶氮-GnRH）构象或序列的共同表位。包被有偶氮-GnRH或偶氮-GnRH-BSA且用MoAbs饱和的板不结合PoAbs。当使用GnRH或GnRH-BSA替代偶氮-GnRH或偶氮-GnRH-BSA时，抑制活性相似，因此表明MoAbs针对的是GnRH分子的构象表位。
