猪链球菌溶血素双抗体夹心酶联免疫吸附测定方法的建立

陈志萍; 陈益; 吴梦; 邓晓雨; 高雅晴; 葛良鹏; 杜华茂

猪链球菌溶血素双抗体夹心酶联免疫吸附测定方法的建立

Establishment of double-antibody sandwich enzyme-linked immunosorbent assay for suilysin detection

摘要

摘要:
目的建立猪链球菌溶血素（SLY）的双抗体夹心酶联免疫吸附测定（ELISA）方法，通过对SLY进行定量检测来评价猪链球菌产生SLY的能力与致病力。
方法用4种胆固醇依赖性溶细胞毒素的类毒素（CDC）免疫BALB/c鼠、新西兰大白兔制备高免血清，以间接ELISA方法检测高免血清与重组猪溶素（rSLY）、重组肺溶血素（rPLY）、重组李斯特菌溶血素O（rLLO）和重组产气荚膜梭菌溶血素O（rPFO）的反应性。以全人单克隆抗体1-10F（专利号CN 113957057B）为捕获抗体、高免血清为检测抗体，经正交试验优化两类抗体的工作浓度，建立双抗体夹心ELISA方法，并对猪链球菌、肺炎球菌、单增李斯特菌、产气荚膜梭菌、溶血性大肠埃希菌和金黄色葡萄球菌的培养液进行特异性试验。
结果间接ELISA方法显示兔抗rSLY^P353L血清与rPLY有交叉反应，鼠抗rPLY血清与rSLY、rLLO有交叉反应，四联血清与rPLY、rSLY、rLLO有较强反应，rPFO与3种血清的反应性较弱。经优化，双抗体夹心ELISA方法的捕获抗体包被量为100 ng，检测抗体为鼠抗rPLY血清（1∶1 000），在此条件下，检测25～800 ng/mL的rSLY呈线性关系，R²值为0.986；对猪链球菌培养液为强阳性反应，对肺炎球菌培养液为弱阳性反应，其他为阴性反应。
结论本研究建立的双抗体夹心ELISA方法能定量检测SLY，将为诊断高致病性猪链球菌感染提供新手段。

Abstract:
Objective To establish a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) assay for the quantitative detection of suilysin (SLY) and provide evidence for the evaluation of SLY producing ability and pathogenicity of Streptococcus suis.
Methods Antisera was prepared by using 4 kinds of cholesterol-dependent cytolysin (CDC) in BALB/c mice and New Zealand white rabbits. The reactivity of antisera with four recombinant CDCs, including suilysin (rSLY), pneumolysin (rPLY), listeriolysin (rLLO) and perfringolysin (rPFO), was evaluated by indirect ELISA. The concentrations of human-sourced monoclonal antibody (mAb) 1-10F (patent no. CN 113957057B), as trapping antibody, and antiserum, as testing antibody, were optimized by orthogonal test for the establishment of a double-antibody sandwich ELISA assay for SLY detection. The specificity of this assay was assessed by testing the cell-free mediums of S. suis, S. Pneumoniae, Listeria monocytogenes, Clostridium perfringens, hemolytic Escherichia coli and Staphylococcus aureus.
Results The results of indirect ELISA showed that rabbit anti-rSLY^P353L serum could react with rPLY, mouse anti-rPLY serum could react with rSLY and rLLO, and the 4-valent serum had strong reaction with rPLY, rSLY and rLLO, while rPFO had weak reaction with the three types of antisera. The optimized conditions of double antibody sandwich ELISA assay were 100 ng 1-10F IgG with mouse anti-rPLY serum dilution as 1∶1 000. In this condition, the measurement of rSLY showed a linear correlation at concentrations of 25–800 ng/mL (R²=0.986). Moreover, the specificity test of this assay showed strong positive result for cell-free medium of S. suis, weak positive result for S. pneumoniae and negative result for others.
Conclusion The established double antibody sandwich ELISA assay can detect SLY quantitatively, which can be used as a new option for the diagnosis of highly pathogenic S. suis infection.

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