药源性循环免疫复合物检测方法的建立

目的通过重组人白介素1受体拮抗剂(rhIL-1Ra)的大鼠6mon重复给药毒性试验,建立临床前安全性评价中动物体内药源性循环免疫复合物(CIC)的检测方法,为临床CIC检测提供依据。方法从rhIL-1Ra免疫后的兔血清中纯化总IgG包被于酶标板上作为捕获抗体,SD大鼠抗rhIL-1Ra阳性血清与rhIL-1Ra以不同比例混和制成人工免疫复合物(AIC),通过双抗夹心ELISA检测AIC的效率来评价该方法有效性。定期采集连续26wk皮下注射rhIL-1Ra的大鼠血清并以上述方法检测CIC,同时用免疫组化法显示肾小球中的免疫复合物(IC)来验证该方法。结果双抗夹心ELISA法可显示AIC的数量差异,大鼠血清CIC显示出与给药时间一致的变化,并与肾小球中IC检测结果相印证。结论双抗夹心ELISA法可检测出动物因长期给予外源蛋白而产生的CIC,AIC则可作为该检测体系中的阳性对照。

Establishment of the detection method for drug-derived circulation immune complex

Aim To establish a detection method for drug-derived circulation immune complex (CIC),which might emerge in animal's blood during preclinical safety evaluation,with a case of recombinant human interleukin-1 receptor antagonist (rhIL-1Ra),and to provide reference to clinical trial with this method.Methods Total IgG was purified from serum of rhIL-1Ra-immunized rabbit and coated on microplate as capture antibody.A series of artificial immune complex (AIC) was prepared by mixing the serum from rhIL-1Ra-immunized SD rat and rhIL-1Ra with different ratios,and detected by a sandwich enzyme immunosorbent assay (ELISA) for effectivity evaluation of the method.Serum samples of SD rats injected (sc) with rhIL-1Ra for 26 weeks were collected periodically and CIC in the serum was detected by the method above.Meanwhile immune complex deposited in kidney was demonstrated by immune histochemistry.Results Variant quantities of AICs could be reflected by the sandwich ELISA.CIC in the rat serum appeared variance which consisted with the time of administration and proofed by IC in glomerular capillaries of kidney.Conclusion Sandwich ELISA can detect CIC due to repeated doses of foreign protein to animals with the positive control of AIC.