The Establishment Of A Double-antibody Sandwich ELISA Kit For Helicobacter Pylori CagL Antigen

Purpose:Helicobacter pylori(H.pylori)is an important pathogen of diseases and has high infectivity,with a global infection rate of 50%.CagL protein is a chaperone protein of HP,it is a surface protein of the HPIV-type secretory system,encoded by the hp0539 gene sequence and plays an important role in initiating abnormal cell pathway to cause pathological clinical changes.In this experiment,CagL monoclonal antibody and polyclonal antibody were used to establish a double antibody sandwich ELISA method for detecting CagL antigen in human stool samples,made and assembled the kit to provide an effective tool for detecting HP infection.Method:In this experiment,a rabbit-derived CagL polyclonal antibody was prepared,and the CagL monoclonal antibody obtained in the laboratory was used as a primary monoclonal antibody plate,and then sealed.The rabbit-derived CagL polyclonal antibody was used as a secondary antibody,and then a horseradish peroxidase-labeled goat anti-rabbit IgG antibody was added to react with the secondary antibody,the color is showed by adding a substrate to detect whether the sample contains a CagL antigen.The checkerboard square array titration method was used to determine the optimal coating concentration of the primary antibody and the optimal dilution of the secondary antibody and theenzyme-labeled antibody.Thereby,the antigen antibody was more fully reacted.The sensitivity,specificity,repeatability,and stability test were performed on the initially prepared kit,and observe whether the kit was successfully developed.Result:The optimal coating concentration of the primary antibody was determined by checkerboard titration to be 0.048 ?g/mL,and the optimal dilutions of the secondary antibody and the enzyme-labeled antibody were 1:32000 and 1:4000,respectively.The sensitivity test of the kit showed that the minimum protein concentration for this method was 9.75?g/ml.Specificity test: It shows that the method has good specificity and does not cross-react with other flora in the feces.Repeatability test:Repeated test on 10 test samples showed that the in-batch and inter-batch repeatability were both good.The in-batch coefficient of variation is between 1.65% and 10.5%,and the inter-batch coefficient of variation is between 1.3% and 9.35%,which basically satisfies the coefficient of variation of less than 10%,indicating that the kit has good repeatability.Stability test: The coated ELISA plate was stored at 37 ° C and 4 ° C respectively for 105 d,and the same sample was taken out every 15 d to observe the change of absorbance value.The results showed that the P/N value of the kit was greater than 2.1 in 105 d,which satisfied the judgment standard of the kit.The above results indicate that the CagL double antibody sandwich ELISA antigen detection method established inthis experiment basically meets the advantages of high sensitivity,strong specificity and good stability,and can be further applied in clinical research.