Abstract

Canine distemper virus (CDV) infection causes mass mortality in diverse carnivore species. For effective virus surveillance, rapid and sensitive assays are needed to detect CDV in field samples. In this study, after BABL/c mice were immunized with recombinant CDV-fusion (F) protein, monoclonal antibodies (mAbs) against recombinant CDV-F protein (designated 1A5, 1A6, and 7D5) were produced using traditional hybridoma cell technology. Next, capture antibody (1A6, 800 ng/well) and horseradish peroxidase (HRP)–conjugated detection antibody (HRP-7D5, 1:100, 500 ng/well) were used in a double monoclonal antibody–based sandwich enzyme-linked immunosorbent assay (ELISA) for CDV detection after optimization of both mAb amounts per well using a checkerboard titration test. Based on sandwich ELISA test results for 120 known CDV-negative samples, the cutoff value for a positive result was set to an OD_450 nm value ≥ 0.196. As compared with test results obtained from commercial immune colloidal gold test strips, the low limits of detection for the two assays were revealed to be 100 TCID₅₀ per 100 μL. In addition, the sandwich ELISA agreed 100% and 96.4% with commercial immune colloidal gold test strips when testing serum and stool samples. The sandwich ELISA assay provided statistically similar CDV detection. Thus, the sandwich ELISA developed here to detect CDV in fecal and serum samples provided good sensitivity, high specificity, and good reproducibility and should serve as an ideal method for large-scale surveillance of CDV infections in carnivores.

Key points

• Three CDV mAbs that recognized different epitopes and bound to virion were generated.

• The sandwich ELISA based mAbs to detect CDV in fecal and serum samples was developed.

• The sandwich ELISA is an ideal method for detecting CDV infections in the field.

中文翻译：

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基于双重单克隆抗体的夹心酶联免疫吸附测定技术的开发，用于检测犬瘟热病毒

摘要

犬瘟热病毒（CDV）感染导致各种食肉动物物种大量死亡。为了进行有效的病毒监视，需要快速灵敏的检测方法来检测现场样本中的CDV。在这项研究中，用重组CDV融合（F）蛋白免疫BABL / c小鼠后，使用传统的杂交瘤细胞技术生产了针对重组CDV-F蛋白（命名为1A5、1A6和7D5）的单克隆抗体（mAb）。接下来，将捕获抗体（1A6，800 ng /孔）和辣根过氧化物酶（HRP）结合的检测抗体（HRP-7D5，1：100，500 ng /孔）用于基于双单克隆抗体的三明治酶联免疫吸附剂使用棋盘滴定测试优化每孔两个mAb量后进行CDV检测的ELISA（ELISA）检测。根据120种已知CDV阴性样品的三明治ELISA测试结果，_{450 nm}值≥0.196。与从商业免疫胶体金测试条获得的测试结果相比，这两种测定的最低检测限为每100μL100 TCID ₅₀。此外，在检测血清和粪便样品时，夹心ELISA与商业免疫胶体金试纸的一致性为100％和96.4％。夹心ELISA分析提供了统计上相似的CDV检测。因此，这里开发的夹心ELISA检测粪便和血清样品中的CDV具有良好的敏感性，高特异性和良好的重现性，应该成为大规模监测食肉动物CDV感染的理想方法。

关键点

•产生了识别不同表位并结合病毒粒子的三个CDV mAb。

•开发了基于夹心ELISA的mAb，可检测粪便和血清样品中的CDV。

•夹心ELISA是在现场检测CDV感染的理想方法。