新型重组人促红细胞生成素抗体中和活性检测方法的建立及其在毒理研究中的应用

Development of a method for detection of neutralizing activity of antibody against novel recombinant human erythropoietin

目的建立新型重组人促红细胞生成素(Novel recombinant human erythropoietin,HuEPO)抗体中和活性的检测方法,并应用于毒理研究中样品的分析。方法通过考察新型rHuEPO对人UT7/EPO细胞增殖的促进作用及抗新型rHuEPO抗体(兔抗阳性血清)对该细胞增殖的抑制作用,建立抗新型rHuEPO抗体中和活性检测方法,并应用于毒理实验中对相关抗体血清中和活性的检测。结果新型rHuEPO对人UT7/EPO细胞的增殖具有促进作用,在0.125～128 ng/ml浓度范围内作用显著;抗新型rHuEPO抗体在10-1～10-4范围内对人UT7/EPO细胞的增殖具有明显的抑制作用,抑制曲线呈中和抗体性质。毒理实验中,抗新型rHuEPO抗体(SD大鼠血清)显著抑制人UT7/EPO细胞的增殖,且具有特异性;抗新型rHuEPO抗体(Beagle犬血清)对人UT7/EPO细胞的增殖无影响。结论成功建立了一种新型rHuEPO抗体中和活性检测方法,并成功应用于毒理实验样品分析,为新型rHuEPO在长期毒性实验中的免疫原性评价提供了一种有效的检测手段。

Objective To develop a method for detection of neutralizing activity of antibody against novel recombinant human erythropoietin （rHuEPO） and apply to analysis of samples in toxicological test. Methods The promoting effect of novel rHuEPO on proliferation of human UT7/EPO cells as well as the inhibitory effect of antibody（rabbit antiserum） against the novel rHuEPO on the proliferation were evaluated, based on which a method for detection of neutralizing activity of antibody against novel rHuEPO was de- veloped and used for relevant antibodies in toxicological test. Results The novel rHuEPO at a concentration of 0. 125 ~ 128 ng/ml showed significantly promoting effect on the proliferation of UT7/EPO cells. However, the antibody against novel rHuEPO, at a con- eentration of 10^-1 -10^-4, showed significantly inhibitory effect on the proliferation, and the inhibition curve was of characteristics of neutralizing antibody. In toxicological test, SD rat serum against novel rHuEPO showed significantly specific inhibitory effect, while Beagle canine serum against novel rHuEPO showed no significant effect, on the proliferation of human UT7 / EPO cells. Conclusion A method for detection of neutralizing activity against novel rHuEPO was successfully developed and used for analysis of samples in toxicological test, which provided an effective tool for evaluation of immunogenicity of novel rHuEPO in long-term toxicity test.