Luminex Liquid Chip Technology To Detect 6 Kinds Of Arboviruses Method Research

Arbovirus is also called arthropod-borne virus, which can infect and reproduce (?) arthropods like ticks, mosquitoes, midges, sandflies but not pathogenic to (?) arthoropods. However, once Arbovirus spread into human or animals by blood-sucking insect bites, severe disease may appear. There are many kinds of arboviruses.537kinds of arboviruses have been discorvered so far, of which more than130were confirmed pathogenic to human or animals. China has vast territory and complicated geographical environment, the vectors are numerous and many arboviruses existed in our country. In addition, with the increase of international exchanges, the possibility of spread of foreign arbovirus disease into China is increaseing significantly. Hence, the development of, high throughput, rapid, accurate detection methods of arbovirus is importantAs a high-throughput detection technology, Liquid phase chip technology was developed in1990’s by Luminex Company in America.This flow cytometric technique, combine of fluorescence, laser coding, digital signal processing and traditional biochemical technology, is a multiple-founctional analyse platform, which can qualitatively and quantitatively analize multiple targets simultaneously. So it has the advantage of high throughput, high sensitivity, good repeatability, saving sample and time. Presently, the application of liquid phase chip technology in virus detection mainly focused on nucleic acids or antibodies, no report has been found on detecting viruses directly. In this study, six flavivirus arbovirus were used as detecting targets, including tick-borne encephalitis virus, Japanese encephalitis virus, West Nile virus, eastern equine encephalitis virus, Sindbis virus and dengue virus By conjugating specific monoclonal antibodies to different fluorescent microspheres, and followin principle of’double antibody sandwich" method, the liquid phase chip technique was established for detection and identification of the six arboviruses.Firstly, the specific antibodies were prepared. The procedure was as following:the mice was inoculated with hybridoma cells, ascites was harvested and the antibodies were purified by using Protein A/G-plus Agarose antibody purification column.. Secondly, the antibodies were screened and paired by using sandwich ELISA and the capture antibody and detecting antibody were selected. Then, the luminex liquid chip for viral antigen detection was established. After repeated experiments, the optimal capture antibody was selected as20μg. The optimized incubating time for antigens, detecting antibody and SA-PE was1.5h、45min and15～30min respectively. The suitable quality for SA-PE was1:100. The inactivated WNV, JEV, SINV, EEEV and DENV were serially diluted for evaluate the sensitivity of the method. The results showed that the sensitivity of TBEV could reach25PFU, while the sensitivity for WNV and JEV were both195.31PFU. Various unrelated antigens and pathogens (BSA, enterovirus71, yellow fever virus, influenza A virus, chikungunya virus, respiratory syncytial virus, eastern equine encephalitis virus, influenza virus type B) were detected to evaluate the specificity of the method. The results showed that no positive signals were detected with the unrelated pathogens, which indicated that the established method had good specificity. The coefficient of variation (CV) of mean fluorescence intensity values was less than6%for three repeating experiments, which indicated that the method had good stability. The established Liquid phase chip technology was also compared with traditional ELISA for detection, the results showed that the Liquid phase chip had better sensitivity and the same specificity compared with ELISA.In order to verify the accuracy of the method for detecting of multiple pathogens, the six viruses were mixed together as the detecting targets, the working solution of biotinylated detection antibodies contained the mixture of six corresponding antibodies. The results showed that all the six microspheres conjucted with the relative viruses had strong positive fluorescence signals, and the other microspheres had no signals. This proved that the method could be used for detection of multiple pathogens simutanously. The interassay coefficient of variation was less than9%for repeated tests, which indicated that the method had good stability.Arbovirus is a kind of widely spread and serious viral pathogens. So far, no research of detecting of arboviruses directly by liquid chip method has been reported. In this study, we established a liquid chip method for detecting of six arboviruses by employing specific monoclonal antibodies and the sandwich format. This method provides a new and high-throughput technique for detection of arboviruses, which can be used for early rapid identification of pathogens. This may have important significance on clinical treatment and epidemiological survillence.