Establishment and verification of a quantitative detection ELISA method for IgG antibodies against Norovirus GⅠ.1 and GⅡ.4 in human serum
10.13200/j.cnki.cjb.004504
- VernacularTitle:人血清中诺如病毒GⅠ.1和GⅡ.4型IgG抗体定量ELISA检测方法的建立及验证
- Author:
HAN Zibo
- Publication Type:Journal Article
- Keywords:
Norovirus(NoV);
Type GⅠ.1;
Type GⅡ.4;
Serum IgG antibody;
ELISA;
Four-parameter logistic model;
Receiver operating characteristic curve(ROC curve)
- From:
Chinese Journal of Biologicals
2025;38(06):691-698
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish and verify an ELISA method for quantitative detection of human serum IgG antibodies against Norovirus(NoV) GⅠ.1 and GⅡ.4,so as to provide a more accurate assay for antibody detection in the immunogenicity evaluation and seroepidemiological study of NoV vaccines.Methods High-titer human sera positive for IgG antibodies against NoV GⅠ.1 and GⅡ.4 were mixed,aliquoted,and lyophilized to prepare laboratory reference serum.The EC_(50)values of the reference serum were calibrated by fitting the results of multiple batches of ELISA tests to a four-parameter logistic model.Then a quantitative ELISA method for detection of NoV GⅠ.1 and GⅡ.4 IgG antibodies in human serum was developed with the calibrated laboratory reference serum as working standard.The linearity,determination range,precision and accuracy of the method were verified.The human sera positive and negative for GⅠ.1 and GⅡ.4 IgG antibodies were used as test samples,and based on the specificity and sensitivity of the detection results,the cut-off values used to determine the positive results were determined by using the receiver operating characteristic curve(ROC curve) analysis.Results The calibrated titers of the laboratory reference serum for GⅠ.1 and GⅡ.4 IgG antibodies were 434.2 and 456.5,respectively.A four-parameter logistic-based ELISA method was established for quantifying IgG antibodies against NoV GⅠ.1 and GⅡ.4.The method demonstrated linearity with R2 values greater than 0.99 across nine experimental batches,with the measurement ranges of GⅠ.1 IgG 2.714-0.042 and GⅡ.4 IgG 5.706-0.045.The relative standard deviation(RSDs) of reproducibility tests were not more than 10%,and the RSDs of intermediate precision tests were not more than 25%.The recovery rates from spiked tests ranged from 75% to 125%.The cut-off value of GⅠ.1 IgG was 6.705 with specificity of 96.6% and sensitivity of 96.3%,and that of GⅡ.4 IgG was 12.70 with both specificity and sensitivity of 100%.Conclusion By preparing and calibrating laboratory positive sera for NoV GⅠ.1 and GⅡ.4 IgG antibodies,a quantitative ELISA method based on a fourparameter logistic standard curve for human IgG antibodies against NoV was established,and the cut-off values for GⅠ.1 and GⅡ.4 IgG results were preliminarily determined.The method has a wide determination range with good
