Development And Performance Evaluation Of Hepatitis B Virus Surface Antigen Immunoassay Reagents

Objectives:Hepatitis B surface antigen(HBsAg),as a serological marker of hepatitis B virus infection,has been widely used in clinical detection to judge hepatitis B infection.The early HBsAg laboratory detection is mainly qualitative detection,but it can only be used as a basis for the diagnosis of hepatitis B infection.However,the classification of chronic hepatitis B(CHB)disease stage,the evaluation of covalent closed circular DNA(cccDNA)level and the response of hepatitis B virus(HBV)infection to antiviral therapy depend on the quantitative detection of HBsAg developed in recent years.Quantitative detection of HBsAg is the most important means for the diagnosis and treatment of hepatitis B.In foreign countries,American Abbott microparticle chemiluminescence immunoassay and Roche electrochemiluminescence immunoassay are commonly used.The domestic HBsAg quantitative detection market is mostly monopolized by multinational corporations.Although the imported reagent has high sensitivity and specificity,simple operation and accurate and reliable results,it is difficult to be widely used in small and medium-sized hospitals because of its high cost.At present,there are many domestic chemiluminescence immunoassay kits,but there is still a certain gap in sensitivity and specificity compared with imported reagents.there is an urgent need to develop a new domestic chemiluminescence immunoassay kit to shorten this gap.The development of an economical,rapid,accurate and practical domestic chemiluminescence immunoassay kit can not only provide convenience for clinical treatment,but also save a lot of foreign exchange,reduce the cost of detection reagents and reduce the economic burden of patients.At present,chemiluminescence immunoassay reagents are more and more used in clinical testing field because of their good specificity,high sensitivity and simple operation.Methods:1.Preparation of alkaline phosphatase(ALP)labeled antibody:the HBsAg sheep polyantibody solution was digested with pepsin,and its Fc end was cut off,and the antibody F(ab’)2HBsAg sheep polyantibody fragment was purified.F(ab’)2HBsAg sheep polyclonal antibody fragment was coupled with ALP to obtain F(ab’)2HBsAg sheep polyantibody labeled with ALP.2.Preparation of magnetic particle-mouse anti-HBsAg monoclonal antibody:the magnetic particle 80mg coupled with amino or carboxyl group was added to 100 mL 0.1 M hydroxyethyl piperazine ethyl thiosulfonic acid(HEPES)buffer solution,and 10 mg mouse anti-HBsAg monoclonal antibody was added after stirring 40 min at room temperature,then 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide(EDC)with 8 mg/mL concentration was added and reacted at 2℃ for 1 h.The magnetic particle-mouse anti-HBsAg monoclonal antibody can be obtained by washing it in 0.01 M PBS buffer for 3 times and finally dissolving it to 1 L with 0.01 MPBS.3.Establishment of detection method:using labeled ALP-F(ab’)2HBsAg sheep polyclonal antibody and magnetic particle-mouse anti-HBsAg monoclonal antibody as raw materials,a magnetic particle chemiluminescence method for quantitative determination of HBsAg was established.After optimizing the conditions,the performance evaluation and clinical comparison were carried out systematically.Results:1.In this study,the Fc end of the antibody was cut off by gastrin enzyme to obtain the antibody fragment F(ab’)2 with small molecular weight,which retained all the biological activities,and the magnetic particles were directly coated with monoclonal antibody to improve the reaction efficiency.These two methods improved the sensitivity of the kit.2.After condition optimization and performance evaluation,the lowest detection limit of this method is 0.01 IU/ml;in fact,the relative deviation between measured concentration and theoretical concentration is within±20.0%;it is not disturbed by rheumatoid factor RF)and other diseases,and the coefficient of variation(CV)of precision is 5.018,less than 7.5%.The linear range is 0.05～200.00 IU/mL.Some studies have shown that when the HBsAg concentration is more than 25 IU/mL,the ELISA appears the posterior band phenomenon,and the absorbance A value no longer increases with the increase of HBsAg concentration.Compared with the test results of the Abbott kit,the results show that the sensitivity of the newly developed kit is 94.12%,the specificity is 99.11%,and the total coincidence rate is 96.80%.The kappa value was 0.938 and the area under the ROC curve(AUC)was 0.989.In terms of quantitative detection,the newly developed HBsAg detection system had a high correlation with Architect HBsAg(R2=0.913,P<0.001).Conclusion:The microparticle chemiluminescence immunoassay(MCLIA)kit developed in this study can meet the requirements of clinical detection kit in various indexes(accuracy,sensitivity,specificity,etc.),and compared with the American Abbott microparticle chemiluminescence immunoassay kit.The results show that the detection performance of the developed kit is close to that of Abbott,and has the same detection ability as the expensive imported kit,which can be further optimized.And then popularize and apply it in clinic.