Development Of Colloidal Gold Detection Kit For Enterovirus EV71-IgM

Hand-foot-mouth disease(HFMD)is a common disease in children.Many kinds of enteroviruses can cause HFMD,and the proportion and mortality of severe HFMD patients caused by enterovirus 71(EV71)are higher than those caused by other enteroviruses.Early diagnosis is very important to save the lives of children with severe diseases.In order to reduce the mortality of severe hand,foot and mouth disease(HFMD),it's essentiall to develop a rapid and accurate diagnosis to detect the infection of EV71.Colloidal gold immunochromatography has the advantages of fast,simple,accurate and intuitive.In this study,detection kit for IgM antibody of EV71(colloidal gold method)was successfully developed by using this method.Firstly,the EV71 IgM test reference panel of enterovirus was established with the collected serum,which was used to control the quality of reagents.The reference panel is composed of 12 positive quality control samples,12 negative quality control samples,1 repetitive quality control product and 1 series minimum detectable quality control product.Secondly,the EV71-IgM colloidal gold reagent was successfully prepared by using monoclonal antibody(MAH IgM),rabbit IgG,EV71 antigen,goat anti rabbit IgG polyclonal antibody and other raw materials.The results showed that nitrocellulose membrane(HF135)coated with 2.0 mg/mL EV71 antigen and 1.0 mg/mL goat anti rabbit IgG polyclonal antibody,the humidity of the coating is 40-60%.Large colloidal gold is used for marking,the optimum pH for marking is 8.The optimum colloidal gold labeling concentration of monoclonal antibody(MAH IgM)was 10 mg/mL,and that of rabbit IgG was 15 mg/mL;The optimum dilution ratio of monoclonal antibody(MAH IgM)was 1:10,and that of goat anti-rabbit IgG was 1:15.In the reaction system,10 ?L serum/plasma plus 2 drops of diluent was used to read the results for 15 minutes and 20 minutes.Finally,the EV71-IgM colloidal gold test paper was evaluated by the reference panel,endogenous interfering substances test,cross reaction test,plasma sample test of different anticoagulants,positive sample destruction test,stability test,comparative test of similar reagents and external clinical evaluation.The results showed that the kit had excellent performance,strong anti-interference ability and no cross-reaction with high concentration of specific antibody serum of other common pathogens with similar structure to pathogens or similar clinical symptoms.The commonly used clinical anticoagulants had no effect on the plasma test results,and the test results had a high coincidence rate with similar domestic kits.In this study,the detection kit of EV71 IgM antibody was successfully developed by using colloidal gold immunochromatography.The method is simple,quick and efficient,and can be widely used in various grassroots hospitals,community health stations and other sites.It is conducive to the early diagnosis of EV71 infection,and has important significance for the timely prevention and control of the disease.