鹅星状病毒1型抗体间接ELISA检测方法的建立及临床应用

Establishment and clinical application of an indirect ELISA method for detection of antibodies aganist goose astrovirus

鹅星状病毒1型(GAstV-1)是雏鹅内脏和关节痛风的重要病原,严重危害我国养鹅业的健康发展。建立血清学方法可以快速、准确地检测出GAstV-1的感染,有利于对该病的防控。本研究通过大肠杆菌表达GAstV-1的衣壳蛋白VP70,以纯化的重组GAstV-1衣壳蛋白rVP70包被酶标板、通过棋盘滴定法对抗原包被浓度、血清稀释度、酶标抗体稀释度以及各反应时间进行优化,建立检测GAstV-1抗体的间接ELISA。应用该方法对临床鹅血清样品进行了检测和分析。ELISA优化的条件是,抗原包被浓度2μg/mL、4℃包被12 h,以10%脱脂乳为封闭液,一抗血清最佳稀释度为1∶100,酶标抗体最佳稀释度为1∶6 000,一抗和二抗反应时间均为0.5 h;待检样本的样本OD_(450)值/阳性对照OD_(450)值(S/P)≥0.28判定为阳性。应用该间接ELISA对2021年江苏省各地区的180份临床鹅血清样本进行GAstV-1抗体检测,阳性率约65.5%。本研究应用rVP70蛋白为包被抗原建立的抗体检测方法可特异敏感地检出鹅感染GAstV-1,可为该病的临床防控提供技术支撑。

Goose astrovirus type 1(GAstV-1) is an important pathogen causing visceral and joint gout in goslings, which seriously endangers the development of goose industry in China. The establishment of a serological method mayquickly and accurately detect the infection of GAstV-1, which is helpful for the prevention and control of the disease. In this study, the capsid protein of GAstV-1 was expressed in E. coli, and the purified recombinant goose astrovirus capsid protein was coated by means of anenzyme-labeled plate. Through the chessboard titration against the original coating concentration, serum dilution, enzyme-labeled antibody dilution and optimization of reaction time, an indirect ELISA was established to detect the GAstV-1 antibody. Goose serum samples collected inmany regions of Jiangsu Province in 2021 were detected and analyzed usingthis method. The optimized conditions of the ELISA method wereas follows: the antigen coating concentration was 2 μg/mL;the coating was at 4 ℃ for 12 h, 10% skim milk was used as sealing solution, the optimum dilution of the sera was 1∶100, the best dilution of the second antibody was 1∶6 000, the reaction time of the first antibody and the second antibody was 0.5 h, and the sample OD_(450)/positiv OD_(450)(S/P) value≥ 0.28 of the sample to be tested was judged to be positive.The indirect ELISA detection method was initially used to detect GAstV-1 antibodies in the 180 clinical goose serum samples collected in various regions of Jiangsu Province in 2021, withapositive rate of about 65.5%. The antibody detection method established hereusing the rVP70 protein as an coating antigen specifically and sensitively detected GAstV-1 infection of goose, which provided technical support for prevention and control of the disease.