Establishment And Application Of A Double Antibody Sandwich ELISA For Detecting M Protein Of Vesicular Stomatitis Virus

Vesicular stomatitis(VS)is a type of zoonotic infectious disease caused by the infection of vesicular stomatitis virus(VSV),which is a highly contagious disease.The disease as an insect-borne diseases,sick animals,invisible poison,culex mosquitoes are the main disseminators and carriers,mainly through saliva and blisters to the outside environment.It can also occur in the body passing through the egg.In clinical practice,it can infect a variety of mammals,it is characterized with vesicular stomatitis lesions in mouth,lips,tongue,nipple and mucosa or skin of infected animals.In addition,it also can cause the occurrence of infected meningoencephalitis,and there are obvious neurological symptoms.People can occasionally infection with flu-like symptoms,and sometimes even cause viral encephalitis.Thus,vesicular stomatitis has important public health significance,it will not only cause some loss for the aquaculture industry,but also have a certain threat for human health.The symptoms of vesicular stomatitis are very similar to those of some diseases,such as FMD,VES,SVD.et al.And they are very easy to be misdiagnosed.It is easy to spread to other place.Therefore,the differential diagnosis for these diseases is of great significance.At present,there are many ways to detect VSV,but some of the traditional methods have some disadvantages such as complex operation,long detection period,and not conducive to clinical testing.Although some molecular biology methods have some characteristics such as short detection cycle and more sensitive.But they are need professional laboratory equipment and professional testing staff,and some methods are easy to make false positive results and have misdiagnosed.The double antibody sandwich ELISA overcome some of the drawbacks of traditional detection methods,it has the characteristics of simple operation and stable detection period.At the same time,the method has good sensitivity,high specificity,and it can achieve the level of molecular biology detection.The method does not require professional experimental equipment and high experimental conditions.It is more suitable for large-scale clinical testing than the molecular biology detection technology,Therefore the double antibody sandwich ELISA is widely used in veterinary clinical.In this experiment,a double antibody sandwich ELISA for detecting M protein of vesicular stomatitis virus was developing using VSV monoclonal antibody(MAb)as capture antibody and the rabbit polyclonal antibodies against VSV-M protein as detecting antibody.And we optimized the conditions of the detection method.We evaluated the sensitivity,specificity and repeatability of the method.The results showed: this method had high sensitivity,the lowest possible detection of VSV was 2.5 ?g / m L.and the method had well specificity.In the detection of VSV,SVDV,FMDV,ORFV,Only the result of VSV showed positive,the result of other three virus were negative.Repetitive test results showed that the repetitive coefficient of variation of the method was less than 10%.The above results showed that the double antibody sandwich ELISA for detecting M protein of vesicular stomatitis virus had high sensitivity,good specificity and good reproducibility.After that,we used this method to detect VSV positive samples and negative samples stored in the laboratory.At the same time,we used RT-PCR to verify the method.The coincidence rate of RT-PCR detection method and the double antibody sandwich ELISA for detecting M protein of vesicular stomatitis virus was 97%.Then we used this method to detect 200 beef mouth swabs from Nong an and Song yuan of Jilin Province.And we used RT-PCR to verify the method.The results showed that: the results of 200 samples of cattle oral swab samples were negative,and it was consistent with RT-PCR results.Therefore,the establishment and application of a double antibody sandwich ELISA for detecting M protein of vesicular stomatitis virus will provide technical support for the warning,forecast,prevention and control of VSV.And it will generate potential economic and social benefits.