上转发光免疫层析技术快速检测猪抗旋毛虫IgG抗体方法的建立

Establishment of an up-converting phosphor - lateral flow (UPT-LF) method for rapid detection of anti-Trichinella IgG antibodies in swine

基于上转发光免疫层析(UPT-LF)技术,旨在建立UPT-LF快速检测猪抗旋毛虫IgG抗体的方法。采用间接法制备UPT-LF试纸条,通过共价偶联使二抗山羊抗猪IgG和上转发光纳米颗粒(UCP)结合,并加入样品垫和被检血清结合,以旋毛虫肌幼虫丝氨酸蛋白酶抑制因子基因(WM5)表达的蛋白WM5为猪抗旋毛虫IgG特异性结合抗原,WM5(2 g/L)与兔抗山羊IgG(0.5 g/L)喷点于分析膜上作为UPT-LF试纸条检测带(T)与质控带(C),该试纸条命名为Tsp-UPT-LF。Tsp-UPT-LF通过对95份阴性血清检测,确定Tsp-UPT-LF的Cutoff值为0.099。Tsp-UPT-LF和ELISA分别对273份旋毛虫感染猪血清样本进行检测,总符合率为92.31%,一致性系数Kappa(K)值为0.837,表明两种方法具有高度一致性。Tsp-UPT-LF检测旋毛虫、猪囊虫、华支睾吸虫感染的猪血清Tsp-UPT-LF呈现对旋毛虫良好的特异性。Tsp-UPT-LF检测感染50 000条旋毛虫肌幼虫28 d猪血清为阳性,T/C值0.109,122 d阳性最强,T/C值为0.207,至425 d时阳性值有所下降,抗旋毛虫IgG阳性率为71.4%。本研究建立了基于UPT-LF技术快速检测猪抗旋毛虫IgG抗体的方法,为猪旋毛虫即时感染检测、确保食品安全提供了一种可参考的检测方法。

The study is to develop an up-converting phosphor-lateral flow(UPT-LF)assay for rapid detection of anti-Trichinella IgG antibodies on site.The UPT-LF strip was prepared by indirect method.The second antibodies of the goat anti-pig IgG was covalent coupled with up-converting phosphor(UCP)particles and then bound with serum samples in sample pad.The protein WM5 expressed by the serine protease inhibitor gene(WM5)of Trichinella spiralis(T.spiralis)larvae was used as binding antigen in the strip.WM5(2 g/L)and rabbit anti-goat IgG(0.5 g/L)were fixed on the test line(T)and control line(C)of the nitrocellulose membrane,respectively.The test strip was named as Tsp-UPT-LF.The cutoff value of Tsp-UPT-LF was defined as 0.099 by detection of 95 negative serum samples.Tsp-UPT-LF also showed excellent specificity in detection of pig serum infected with T.spiralis,Cysticercus or Clonorchis sinensis.273 pig serum samples were detected by Tsp-UPT-LF and ELISA,respectively.The total consistent rate of TspUPT-LF and ELISA was 92.31%,while Kappa value is 0.837,with the excellent consistency.TspUPT-LF positive result was 0.109 T/C for pig serum infected with 50 000 T.spiralis on the day28,while the max detection value was 0.207 T/C on the day 122.T/C descended on the day 425.The rate of positive results was 71.4%for anti-TrichinellaIgG of pig serum infected with 50 000 T.spiralis.The established UPT-LF assay for rapid detection of anti-Trichinella IgG antibody in swine,provides a reference method for the point-of-care testing(POCT)of T.spiralis and food safety.