The Study Based On Establish A Method To Evaluate IgG Anti-Vi In Human Sera

Salmonella typhi is the pathogen of typhoid fever,which causes an estimated 21 million new cases of typoid fever and 216,000 death every year.Typoid fever is an invasive enteric infection that is spread through ingestion of contaminated water and food,it is found most commonly in developing countries where infrastructural facilities are inadequate.Currently,the mainly preparations to prevent typhoid fever are oral immune Ty21 a attenuated live vaccine and muscle injected typhoid Vi polysaccharide vaccine.Ty21 a attenuated live vaccine has some defects such as genetic background is not clear and poor immune effect and so on,all the clinical trials in Nepal,South Africa and China showed that typhoid Vi polysaccharide vaccine can provide 70% protection,along with typhoid Vi polysaccharide vaccine is widely used in the world,its good security,and about 70% of the protection to prevent the spread of typhoid disease made great contributions,but due to the Vi polysaccharide is T cells independent antigen,so it is generally believed that the children under 5 years of age have poor immune effect,especially for children under the age of 2 who does not produce the immune response.In recent years,with the development of the conjugate vaccine technology,several scientific research institutions in international have carried out the typhoid Vi conjugate vaccine clinical trial work,National Institutes of Health(NIH)has completed phase ? clinical trial in Vietnam,the results showed that typhoid Vi conjugate vaccine has good safety and effect,it can produce 91.1% protection in 2-5 years old children in high-risk region of Vietnam,and the protection can sustain 4 years.Meanwhile,phase ? clinical research in Vietnam determined that 3.52 ELISA unit(EU)is the minimum protective antibody of typhoid Vi,and it was recognised by the WHO.Clinical trial of Typhoid Vi polysaccharide protein conjugate vaccine developed by Lanzhou institute of biological products co.,LTD in China also concluded that it has optimal effect than typhoid Vi polysaccharide vaccine.The international scientific research institutions focus on development of typhoid Vi conjugate vaccine,heralding that the effective tools to prevent this old disease will be created.Accordingly,the establishment of the detection method to assessment of typhoid vaccine's protection effect has become an important link in the vaccine development.The Objective of this study is to establish an accurate and fast method to evaluate IgG anti-Vi in human vaccinating Typhiod Vi conjugate vaccine,and meanwhile,the method should be easy to operate.Usually Enzyme-linked immunosorbent assay(ELISA)is the most commonly used methods to test IgG antibody levels of typhoid Vi.Base on published date of Enzyme-linked immunosorbent assay(ELISA),by the ELISA method of National Institutes of Health(NIH)as the main reference,we studied on the coating antigen(Vi polysaccharide from S.typhi and Vi polysaccharide from Citrobacter freundii),plates with different binding characteristics,the coating concentration,reaction times of enzyme marked IgG,reaction conditions of samples and required time to chromogenic,placement time after termination,meanwhile,we did optimizing to those items.Sumed up those factors,we established a method to evaluate IgG anti-Vi in human sera called ELIS A procedure,under the guide of methodology verification requirements,then we did a verification to this ELIS A procedure,it contained consistency of standard cirve,specificity,linear ity,accuracy,precision and lowest limitation of quantitation.The results showed that the standard curve has a good consistency;The inhibitor(PS)and the concentration of specific antibody have a typical dose-dependent pattern when quality control(QC)sera was absorbed by PS at different concentration,the maximum inhibition ratio was approximately 92%,indicating that this ELISA procedure has a good specificity;Sera dilution and specific antibody concentration showed linear relationship with R2=0.999,indicating that this ELISA procedure has a good linear ity;Antibody concentration of QC sera is 76.80 EU/mL,CV=8.55%,it's no signif icant difference with those published by NIH(75 EU/mL,CV<15%),so we can say that this ELIS A procedure has a good accuracy;Both repeatability(CV?15%)and intermediate precision(CV?20%)are up to the detection standard,indicating that this ELIS A procedure has a good precision;Meanwhile,we determined 0.00078EU/mL is the lowest limitation of quantitation.