基于鹅T细胞表面CD4分子胞外区的双抗体夹心ELISA检测方法的建立

Development and optimization of a double antibody sandwich ELISA based on the extracellular region of goose T cell surface CD4 molecule

为建立鹅CD4分子(goCD4)双抗体夹心ELISA(DAS-ELISA)检测方法,本研究以鼠抗goCD4-exc多克隆抗体为包被抗体,兔抗goCD4-exc多克隆抗体为检测抗体,确立判定标准,优化反应条件,建立了goCD4DAS-ELISA的检测方法。试验结果显示该方法最低检出量达5 pg/mL,具有较好的灵敏度。批内批间变异系数均低于2%,具有较好的重复性。将建立的双抗体ELISA方法初步应用于检测小鹅瘟病毒(GPV)感染的鹅脾单核细胞中CD4分子表达水平变化情况,结果显示GPV感染鹅脾单核细胞中goCD4分子表达水平极其显著高于未感染组。本研究建立的检测goCD4的DAS-ELISA方法有利于进一步研究禽类细胞免疫水平变化,并对实际生产中禽病检测及防控提供技术参考。

In this paper, we aimed at developing and optimizing a double antibody sandwich ELISA （DAS-ELISA） for the detection of goose T cell surface CD4 molecule based on the purified goose CD4 molecule （goCD4） as antigen, the mouse anti-goCD4-exc PAb as coated antibody and rabbit anti-goCD4-exc PAb as detected antibody. As shown in the result, the OD450nm≥0.416 was considered as positive results, on the contrary, OD450nm〈0.416 was negative results. The coefficients of variation in intra- and inter-assay were both less than 2%. And the minimum detectable dose of the developed DAS-ELISA was 5 pg/mL. Additionally, the developed DAS-ELISA was applied to detect the goCD4 expression level of goose PBMCs post GPV infected. The expression of goCD4 mRNA level in the infected birds was significantly higher than that in uninfected birds. These data shown the established DAS-ELISA was sensitivity and specificity, and the assay had the potentially to be used the detection of the cell-mediated immune responses in waterfowl.