抗人生长激素单克隆抗体的制备及特点分析

Preparation and Characteristic Analysis of Monoclonal Antibodies against Human Growth Hormone

人生长激素(hGH)是垂体前叶分泌的重要激素,但有关其结构与功能的关系、抗原决定簇的数目及位置、受体结合位点以及不均一性的来源和意义等问题至今尚未解决。抗hGH单克隆抗体(McAb)不仅是研究上述问题的有用工具,而且还可用于建立更为快速、灵敏,特异和准确的GH新免疫分析法,以提高有关疾患的诊治水平。本实验用微量免疫法获得四种抗整分子hGH McAb,并对其特性进行了较详细的研究。

Four hybridomas stably secreting monoclonal antibodies (McAbs) against human growth hormone (hGH) have been obtained by fusion of immunized mice spleen cells with myeloma SP2/0 cells. After inoculation of hybridoma cells to BALB/c mice i.p., the ascitic fluids were collected and characterized, the titre of the 4 McAbs being in the range of 0.6-10.0 × 105. As determined by Ouchterlony analysis, all the 4 McAbs were IgG1. They varied in affinities, with equilibrium constants from 0.53×109 to 9.0× 109 L/mol. McAb-1 showed 52.5% cross-reactivity with human placental lactogen (hPL). McAb-2, McAb-3 and McAb-4 displayed no crossreactivity with hPL and human prolactin (hPRL). Antigenic determinants recognized by the 4 McAbs were mapped through their reactions with hGH fragment consisting of residues 1-43, cross-reaction with hPL, hPRL and antibody-antibody compe- tition test. The results showed that the antigenic determinants of the McAbs are not located in the site of hGH comprising residues 1-43 but on the three non-overlapping antigenic sites of hGH. As shown in receptor assay, all the 4 McAbs exhibited specific inhibition on the binding of pregnant rabbit liver GH receptor to 125I-hGH. It seems likly that receptor-binding site is larger than the antigenic determinant or there are more than one site on hGH surface capable of binding to the hormone receptor. The McAbs could only bind with the peak 2 (MW22000) but not the peak 1 (MW 45000) of old l25I-hGH after gel filtration. Therefore, they can be used for developing hGH RIA with such high specificity that only biolgoical hGH can be measured without offering molecular forms of nonbiolgoical hGH.