酶联免疫吸附试验检测人叶酸受体抗体IgM方法的建立及评价

目的 建立酶联免疫吸附试验(ELISA)检测人血浆叶酸受体抗体IgM的方法。方法 将从正常分娩健康产妇的胎盘中提取、纯化人叶酸受体蛋白稀释至5 ng/μl,作为抗原包被96孔板,以羊抗人IgM作为二抗,以健康人混合血浆作为标准品,浓度设定为1。采用ELISA检测人血浆叶酸受体抗体IgM水平,并评价该方法的灵敏度、精密度和稳定性。分别以人叶酸受体蛋白和牛源叶酸结合蛋白作为包被蛋白,检测24例健康新生儿母亲和20例神经管畸形患儿母亲的血浆标本叶酸受体抗体IgM水平,比较两种包被蛋白的检测结果。结果 ELISA方法可以检测的人血浆叶酸受体抗体IgM浓度为6.25×10-4~8.00×10-2,最低检测限为3.12×10-4。该方法检测高、中和低浓度血浆叶酸受体自身抗体IgM的批内差异分别为6.61%、3.50%和5.12%,批间差异分别为4.54%、5.49%和5.44%,此方法检测不同稀释倍数样品中叶酸受体自身抗体IgM浓度的测定值均在均数±10%范围内。人叶酸受体包被检测板测得的健康新生儿母亲(t=-11.9,P<0.001)及神经管畸形患儿母亲(t=7.35,P<0.001)的叶酸受体抗体IgM浓度均显著高于牛源叶酸结合蛋白包被检测板的测定值。结论 本研究成功建立了检测人血浆叶酸受体抗体IgM的ELISA方法,该方法以人叶酸受体蛋白作为包被蛋白,检测灵敏度高,精密度好,结果稳定。

Establishment and Evaluation of Enzyme-linked Immunosorbent Assay for Measuring Human IgM Autoantibody to Folate Receptor

Objective To establish the method of enzyme-linked immunosorbent assay (ELISA) for measuring human IgM autoantibody to folate receptor. Methods Folate receptor was extracted and purified from the healthy woman placenta. The protein was coated on 96-well plates with a concentration of 5 ng/μl. Goat monoclonal antibody was used for detecting antibody. Pooled plasma from healthy donors was used to plot the standard curve and the IgM concentration of pooled plasma was defined as 1. We set up an ELISA procedure to measure human IgM autoantibody to folate receptor. The sensitivity,precision,and stability of the method were evaluated. Further,the folate receptor and bovine folate-binding protein were used as the antigen,respectively,to determine the autoantibody levels in 24 healthy individuals and 20 individuals once gave birth to baby with neural tube defects. Results The measuring range of the method was from 6.25×10-4 to 8.00×10-2. The lowest IgM level that can be detected was 3.12×10-4. The inter-assay coefficients of variations for samples with high,medium,and low IgM levels were 6.61%,3.50%,and 5.12%,respectively. The intra-assay coefficients of variations were 4.54%,5.49%,and 5.44%,respectively. The stability test results were considered within acceptable limits. The data from folate receptor-ELISA was significantly higher than that from bovine folate binding protein-ELISA,both in the healthy group (t=-11.9,P<0.001) and in the neural tube defect group(t=7.35,P<0.001). Conclusions The folate receptor-ELISA method for measuring human IgM autoantibody to folate receptor was successfully established. The method is sensitive,repeatable,and stable.