Akinwolere O A, Kumararatne D S, Bartlett R, Goodall D M, Catty D
Department of Immunology, Medical School, University of Birmingham.
J Clin Pathol. 1994 May;47(5):405-10. doi: 10.1136/jcp.47.5.405.
To evaluate two of the recent methods of coating microtitre plates in the enzyme linked immunosorbent assay (ELISA) for detecting human antibodies against meningococcal capsular polysaccharides A and C with a view to validating a specific meningococcal antibody assay for routine clinical use.
Two four-layer ELISA protocols were standardised: one method utilised meningococcal polysaccharides conjugated to poly-L-lysine polypeptide for coating the microtitre plates; another used polysaccharides mixed with methylated human serum albumin (mHSA). Titration curves were plotted for the ELISAs and the squared Pearson correlation coefficient (R2) was used to determine the degree of accuracy of fit of the curves. Specificity tests were performed by inhibition and adsorption studies.
Both methods gave good titration curves with a high R2 of > 0.98, indicating a high degree of accuracy in forming the curves. The titration end point after vaccination, obtained by the mHSA method, was 20 times higher, however, than that obtained by the poly-L-lysine method. Specificity tests showed that in the ELISA using polysaccharide/poly-L-lysine, antibody activity of a pre-vaccination serum sample was inhibited by 37%, and of post-vaccination serum by 50% with 1000-fold excess antigen. Antibody activity (post-vaccination) was reduced by 51% and 59%, respectively, by adsorption with antigen-coated Sepharose beads or adsorption with suspensions of killed meningococci. In contrast, antibody activity of a pre-vaccination serum was inhibited by 60% and a post-vaccination serum by 90% in ELISA employing polysaccharides mixed with mHSA. Reproducibility was better with the use of methylated human serum albumin than with poly-L-lysine; the former showed intrabatch and interbatch coefficients of variation of 4% and 2%, respectively, compared with 43% (intrabatch) and 16% (interbatch) obtained with the poly-L-lysine.
It is concluded that the antibody assay using meningococcal polysaccharides groups A and C mixed with mHSA is much better than that using polysaccharides coupled with poly-L-lysine.
评估酶联免疫吸附测定（ELISA）中两种最新的微孔板包被方法，用于检测人类抗A和C群脑膜炎球菌荚膜多糖抗体，以验证一种用于常规临床的特异性脑膜炎球菌抗体检测方法。
标准化了两种四层ELISA方案：一种方法利用与聚-L-赖氨酸多肽偶联的脑膜炎球菌多糖包被微孔板；另一种方法使用与甲基化人血清白蛋白（mHSA）混合的多糖。绘制ELISA的滴定曲线，并使用平方Pearson相关系数（R2）来确定曲线拟合的准确程度。通过抑制和吸附研究进行特异性测试。
两种方法都给出了良好的滴定曲线，R2均大于0.98，表明在形成曲线方面具有高度准确性。然而，通过mHSA方法获得的接种疫苗后的滴定终点比通过聚-L-赖氨酸方法获得的高20倍。特异性测试表明，在使用多糖/聚-L-赖氨酸的ELISA中，接种疫苗前血清样本的抗体活性被1000倍过量抗原抑制了37%，接种疫苗后血清的抗体活性被抑制了50%。通过用包被抗原的琼脂糖珠吸附或用灭活脑膜炎球菌悬液吸附后，接种疫苗后血清的抗体活性分别降低了51%和59%。相比之下，在使用与mHSA混合的多糖的ELISA中，接种疫苗前血清的抗体活性被抑制了60%，接种疫苗后血清的抗体活性被抑制了90%。使用甲基化人血清白蛋白的重现性比使用聚-L-赖氨酸更好；前者的批内和批间变异系数分别为4%和2%，而聚-L-赖氨酸的批内变异系数为43%，批间变异系数为16%。
得出结论，使用A和C群脑膜炎球菌多糖与mHSA混合的抗体检测方法比使用与聚-L-赖氨酸偶联的多糖的方法要好得多。
