用兔抗人分化抑制因子3抗体建立双抗体夹心ELISA法及初步临床应用

Development of sandwich ELISA for detecting inhibitor of differentiation 3 and preliminary clinical application

目的制备兔抗人分化抑制因子3(Id3)多克隆抗体,建立检测血清Id3的双抗体夹心ELISA技术。方法用纯化的Id3重组蛋白免疫新西兰大白兔,制备兔抗Id3多克隆抗体,通过多肽亲和层析柱去除非特异性成分,免疫细胞化学染色、免疫双向扩散及间接ELISA法检测抗体特异性和效价。制备辣根过氧化物酶(HRP)标记抗Id3,以纯化Id3重组蛋白为标准抗原制备标准曲线,建立定量检测人Id3的双抗体夹心ELISA法。结果免疫细胞化学染色显示,制备的兔抗血清能与人Id3蛋白特异性结合,所得兔抗血清效价分别为1∶8(琼脂双向扩散)和1∶8000(ELISA)。ELISA法的包被抗体和酶标记抗体的工作浓度分别为5μg/ml和1∶4000,标准曲线为Y=0.0793+0.0188X,r=0.997。检测线性范围为2～64U/ml,平均回收率为98.2%,批内平均变异系数(CV)为4.4%,批间平均CV为7.2%。该方法可用于人血清Id3含量的检测。结论兔抗人Id3多克隆抗体的制备及夹心ELISA法的建立为今后研究Id3蛋白的功能及其在临床和基础医学中的应用奠定了基础。

Objective To develop a sandwich ELISA for detecting human inhibitor of differentiation 3 （Id3） protein in sera by preparing rabbit polyclonal antibody against human Id3.Methods New Zealand rabbits were immunized with purified recombinant Id3 protein to produce the polyclonal antibody against Id3.The rabbit antisera were collected and purified with polypeptide affinity column.The titer and specificity of rabbit antisera were determined by double immunodiffusion in agar-gel,ELISA and immunocytochemistry staining technique.The purified antibodies were labeled with horseradish peroxidase （HRP）,and the quantitative sandwich ELISA was developed by using standard curve with a serially diluted Id3 antigen standard.Results Immunocytochemistry staining showed that the prepared antisera specifically reacted with Id3.The titer of the rabbit polyclonal antibodies against Id3 was 1∶ 8 determined by double immunodiffusion and 1∶ 8 000 for ELISA.The working concentrations of coating antibody and enzyme labeled antibody in the sandwich ELISA were 5 μg/ml and 1∶ 4 000 respectively.The regression equation of standard curve for the sandwich ELISA was Y = 0.079 3 ＋ 0.018 8X （r = 0.997） .The linear range of ELISA was from 2-64 U/ml.The average recovery rate was 98.2% and the intra and interassay coefficients of variation （CV） were 4.38% and 7.19% respectively.Conclusions Polyclonal antibodies against human Id3 was prepared and sandwich ELISA for the quantitative analysis of Id3 protein was developed.The developed method can be a useful tool for quantitative analysis of Id3 protein in human serum.