抗HBs mAb的研制及其对野生与免疫逃逸变异HBsAg的交叉反应特征

Preparation of anti HBs monoclonal antibody and its combination characterization to both wild-type HBsAg and immune escape mutant HBsAg

目的：抗HBs G6mAb制备及其对重组野生与免疫逃逸变异HBsAg结合能力与特点的评价。方法：常规制备并纯化抗HBs mAb,以纯化抗HBs mAb IgG包被,采用ELISA对17种野生及“a”决定簇替代性变异全基因重组表达HBsAg进行检测,并与几种市售HBsAg检测试剂进行比较。结果：该杂交瘤细胞生长与分泌特性稳定,其培养上清与腹水效价分别为2048及4096×10^3;对野生HBsAg检测（ELISA）敏感性不低于0.125μg/L。该抗体可与15种变异抗原中12种发生反应（P/N≥2.5）,反应信号强度分别为低反应组（2份,与野生株A值比较,下同）平均7.55%;中反应组（1例）为59.40%;高反应组（9种）分别为野生株的92.1%-109.4%,低反应或无反应表达产物的免疫逃逸变异部位集中在HBV“a”决定簇I环的起始部即120-124序列之间。部分表达产物采用市售试剂作同步检测,平均显色强度高于或明显高于几种国内应用最为普遍的HBsAg ELISA（P〈0.05）。结论：抗HBs G6 mAb对多数免疫逃逸变异HBsAg有很强的结合能力,所针对的变异类型亦表现出某种特殊的规律。

AIM： To prepare monoclonal antibodies against HBsAg and estimate its binding capacity to both wild-type and varies of immune escape mutant-type HBsAg. METHODS： Using self-made the anti-HBs G6 mAb and HRP-labeled goat anti-HBs antibody, A sandwich ELISA for detection both wild-type and varies of immune escape mutant-type HBsAg has been developed. Applying this assay, to detect 17 species of whole gene wild-type and recombination expressed HBsAg which varied in ＂a＂ determinant, to evaluate its clinical application characteristic of this assay, we used the other commercial reagents to Comparing with it. RESULTS： One strain hybridoma cell lin secreted anti- HBs mAb （defined G6）, was obtained. It grew stably and the titers of the culture supernatant and hydroperitoneum were 2 048 and 4 096 × 10^3. The sensitivity to the wild-type HBsAg of this assay was less than 0. 125 μg/L. This anti- HBs mAb can bind with 12 in 15 species mutant HBsAg （P/N ≥ 2.5 ）, and 2 of them were low absorbent value at 450 nm（A450） group which was 7.55 percent of the wild-type, 1 of them was middle A450 value group which was 59.40 percent of the wild-type, 9 of them were high A450 value group which was 92.1 - 109.4 percent of the wild-type. The low A450 value group and the negative group were diversified in 120- 124 basyl in I loop of the HBV ＂a＂ determinant. We also used other commercial reagents, which was widely used in clinic, to detect partial species mutant HBsAg, the average A450 value was less than foregoing assay. CONCLUSION： The G6 anti-HBs mAb was able to bind most of the immune escape mutant HBsAg in the test. And there would be some special regulations between the mutant site and the recombination expressed HBsAg binding capability.