| Vesicular stomatitis,also known as Pseudoaphthosis,is a high contagious diseaseof bovine,horse,pig,sheep and humans caused by vesicular stomatitis virus.Thedisease is characterized by the formation of vesicles that progress into oral cavitymucosa,tongue,lip,nipple and the coronary band.The disease has been reported inSouth America, Europe, African,and provinces of Xinjiang,Jilin since1821outbreakin the USA. The disease has a worldwide distribution. The VSV epidemic in manycountries caused the world’s meat market confusion and caused severe economiclosses.At present,classical detection methods have been describde for the detecetionof VSV,including virus isolation, complement fixation and neutralization tests,ELISA, semi-nested PCR and real-time PCR.However,the application of thesemethods is limited by time-consuming, require laboratory operations or specialfacilities,which makes these methods unsuitable for clinical samples detection. Assuch,the current methods would not be useful for managing an outbreak of thedisease.Therefore,it is necessary to develop a rapid,specific and easily performedassay for the rapid detection and surveillance of VSV infection in animal.In recent years,the method using hybridoma technique for preparing monoclonalantibody(McAb) has become increasingly mature through continuous improvementand have been used widely in modern life science reserch.Such as disease diagnosisand diagnosis methods were established with the McAb.In this study,Balb/c micewere immunized subcutaneously with VSV purified by sucrose density gradientcentrifugation, their splenic mononuclear cells were isolated and fused with murinemyeloma cells(SP/20). The hybridoma cell were screened using Enzyme-linkedimmunosorbent assay(ELISA) and access to two single-secreting anti-VSVMcAb,named1A2and4C3. ELISA titers of2McAbs between1:25600and1:51200. Identification of antibody subclasses showed that the two monoclonal antibodies1A2and4C3were IgG1.Western blot analysis showed that1A2recognized VSV-Gprotein. By indirect immunofluorescence identification,2McAbs can be combinedwith VSV.Monoclonal antibody only reacted specifieally with the vesicular stomatitisvirus and don’t reacted with other common virus antigen.Monoclonal antibodies playimportant role in slip of indicator paper.Gold immunochromtographic assay is a new detecting technology that bases onthe antigen-antibody reaction and combines colloidal gold labeling techniques andchromatography analysis technique,using colloidal gold as a tracer.The technique ischaracterized with simple and convenient operation,result judgment intuitive,suitablefor clinical diagnosis.In this study,an immunochromtographic strip was successfullydeveloped for the detection of VSV,combining McAb and GICA. Transmissionelectronic microscope images show that colloidal gold particles were showing nodifference in size,well-dispersed,and that the average diameter of particles was about30nm,colloidal gold by OD value method to determine the best combinationmonoclonal antibodies pH8.5,the best combination of a concentration of35μg/mL.The colloidal gold-labeled MAb1A2solution was dispensed onto glass fiberpaper and the polyclonal antibody or the goat anti-mouse antibody was dispensed at thetest or the control line on the NC membrane.The sample pad,pretreated conjugatepad,Ncmembrane and absorbent pads were glued together on a support board andassembled into a test strip. The immunochromatographic strip was capable ofspecifically detecting VSV with5μg/mL. At the same time,sandwich ELISA methodis also established to detect VSV using the McAb and Polyelonal antibody withgood linear range between3.75μg/mL-500μg/mL in its standard curve,its sensitivity is3.75μg/mL.Compared with a sandwich ELISA method,VSV gold immunoehro-温表matography assay strip has the advantages of high sensitivity,better stability andspecificity, good repeatability and handling convenience of the detectingproeess.Therefore, it is playing an important role in the early diagnosis of VSV. |
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