Na Yang1, Lin-lin Wang1, Yue Yuan1, Rong-wei Ye1, Ai-guo Ren1. 1. Peking University Institute of Reproductive and Child Health,the Key Laboratory on Reproductive Health of Ministry of Health,Department of Epidemiology and Health Statistics,School of Public Health,Peking University,Beijing 100191,China.
Abstract
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.
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.
Authors: Na Yang; Linlin Wang; Richard H Finnell; Zhiwen Li; Lei Jin; Le Zhang; Robert M Cabrera; Rongwei Ye; Aiguo Ren Journal: Birth Defects Res A Clin Mol Teratol Date: 2016-05-11