Preparation And Application Of A New Naringin Monoclonal Antibody

Objective:Preparation of the naringin monoclonal antibodies, and its application study including the preliminary research on sandwich ELISA.To prepare the naringin monoclonal antibody, to establish the naringin monoclonal antibody based enzyme-linked immunosorbent assay, to prepare the naringin immunoaffinity chromatography column, and to do a preliminary study on sandwich ELISA use the existing two anti naringin antibodies, optimize its reaction condition, and try to establish a sandwich enzyme-linked immunosorbent assay.Methods:Recover the freezing hybridoma cells which can secrete anti naringin antibodies in our lab’s hybridoma cells storeroom, prepare the naringin monoclonal antibody, use the naringin immunoaffinity chromatography column to knock naringin out, and do preliminary research in sandwich ELISA of naringin.1. Recover the freezing hybridoma cells which can secrete anti naringin antibodies, prepare the naringin monoclonal antibody.2. Optimization of the coating concentration, the concentration of an anti-linear detection range of conventional indirect competitive ELISA method naringin enzyme-linked immunosorbent assay. Examine its accuracy and recoveries, and determine naringin in pharmaceutical samples.3. Activating the CNBr- activated Sepharose 4B gel, then coupling the naringin antibody, closing, washing to Miscellaneous balance, column packing and preparation the naringin immune affinity chromatography. Examine the antibody gel coupling ratio and the capacity of the column of the prepared naringin immunoaffinity chromatography column. Get evidence of its successful naringin knockout.4. Use the existing two anti naringin antibodies to do a preliminary study on sandwich ELISA, optimize the coating concentration of one naringin antibody, the working concentration of the concentration of the other naringin antibody, and the linear detection range of the sandwich ELISA, and examine its accuracy.Results:the newly prepared naringin monoclonal antibody has a various difference compare with the existing one in antibody subtypes, linear detection range and cross reactions. The newly prepared naringin monoclonal antibodies were successfully established the naringin enzyme-linked immunosorbent assay and immunoaffinity chromatography and have showed a stable performance, high precision, good recovery, repeatability to meet the requirements, and can be used at the naringin the pharmacological efficacy studies.1. The newly prepared naringin monoclonal antibody has a strong specificity, except an slight cross reaction with rutin and scutellarin(the cross-reactivity was 1.95% and 0.32%) no significant cross-reactivity (cross-reactivity ratios<0.1%), with the remaining structural analogues. Its antibody subtype is IgG2b.2. Enzyme-linked immunosorbent assay based on naringin monoclonal antibody was successfully established, the coating ratio was 1:10000, the antibody concentration was 1:64000, linear competitive range was 0.04883μg/mL-0.3125μg/mL, the linear equation:y=-0.0981n(x)+0.0078,R2=0.9919.the accuracy and repeatability are both good, hole difference between the coefficient of variation<5%, the plate difference between the coefficient of variation<10%, the average recovery was 103.1%, all these parameter suggest that it can be used for sample testing.3. The naringin immunoaffinity chromatography has been prepared, the antibody-coupled was 99.12%, the antibody gel coupling 2.938μg/mL, column capacity 51.26μg. All the data has proved that this immunoaffinity chromatography can knock naringin out.4.Have a preliminary study in sandwich ELISA, the coating ratio was 1:128000, the antibody concentration was 1:5000, linear competitive range was 200μg/mL-1200μg/mL, the linear equation:y=-0.0981n(x)+0.0078,R2=0.9919. The accuracy was good. Conclusion:The new anti-naringin monoclonal antibody was prepared successfully, this new antibody has structural and functional difference with the old one. The immunoaffinity chromatography coupled by this new anti-naringin monoclonal antibody could knock out naringin succussfuly, and the sandwich ELISA used these two antibodies was established successfully.