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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。
OBJECTIVE: To establish a preclinical safety evaluation of drug-induced circulating immune complex (CIC) in rats by 6months repeated administration of recombinant human interleukin 1 receptor antagonist (rhIL-1Ra) CIC test provides the basis. Methods Total IgG purified from rhIL-1Ra-immunized rabbit serum was coated on a microtiter plate as capture antibody. The anti-rhIL-1Ra positive serum of SD rats was mixed with rhIL-1Ra in different proportions to make artificial immune complexes (AIC ), The effectiveness of this method was evaluated by measuring the efficiency of AIC by double-antibody sandwich ELISA. Routinely, sera of 26 rhs subcutaneously injected with rhIL-1Ra were collected routinely and tested for CIC by the above-mentioned method. The immune complexes (ICs) in the glomeruli were also examined by immunohistochemistry to verify the method. Results The double antibody sandwich ELISA showed that the number of AIC was different. The serum CIC of rats showed the same change with the administration time, which was confirmed by the IC test results in glomeruli. Conclusion Double-antibody sandwich ELISA can detect CIC produced by long-term administration of foreign proteins in animals, and AIC can be used as a positive control in this assay.