关键词: 乙型肝炎表面抗原, 乙型肝炎病毒, 化学发光微粒子免疫分析法, 性能评价

Abstract:

Objective To establish a chemiluminescent microparticle immunoassay（CMIA） for the quantitative determination of hepatitis B surface antigen（HBsAg） and conduct performance evaluation. Methods Mouse-specific anti-HBsAg antibody was coated on the surface of magnetic particle to bind the corresponding HBsAg. The specific anti-HBsAg antibody labeled with acridinium ester was added to form a double antibody sandwich immune complex on the surface of magnetic particle. Pre-trigger solution and trigger solution were added. The acridinium ester on the complex was excited to emit photons,and the relative light unit was proportional to HBsAg concentration. The best reaction conditions were screened,and the performance of self-established CMIA for determining HBsAg（linear range,determination limit,precision,interference,cross-reaction,stability,correlation and consistency with electrochemiluminescence method） was evaluated. Results The optimal concentration of magnetic particles was 0.4 mg/mL,and the optimal dilution ratio of acridinium ester-labeled antibody was 1:200（a concentration of 250 ng/mL）. The amounts of reaction diluent,sample and reagent were 100,20 and 50 μL,respectively. The linear range of self-established CMIA to determine HBsAg was 20-100 000 IU/mL,the minimum determination limit was 9.9 IU/mL,and the within-run and between-run coefficents of variation（CV） were <10.0%. The self-established CMIA was not affected by the interference factors,such as hemoglobin<400 mg/L,bilirubin<4 mg/L,rheumatoid factor<800 IU/mL,triglyceride<2 mg/L,sodium citrate<22 mmol/L and ethylenediaminetetraacetic acid（EDTA）<8 mmol/L. Hepatitis A virus（HAV）,hepatitis C virus（HCV）,hepatitis E virus（HEV）,human immunodeficiency virus（HIV）,human T-lymphotropic virus（HTLV）,cytomegalovirus（CMV）and Epstein-Barr virus（EBV）positive samples did not cross-react with this assay. The prepared reagent could be stable for 6 d at 37 ℃,it could be stable for 4 weeks after opening lid at 2-8 ℃,and it could be stable for 6 months when stored in a sealed container at 2-8 ℃. The correlation and consistency between self-established CMIA and electrochemiluminescence method were good（r=0.978,P<0.001）. Conclusions The self-established CMIA has a wide linear range for determining HBsAg,high concentration samples can be directly determined without dilution,and the determination performance is good,which has certain clinical application prospects.

Key words: Hepatitis B surface antigen, Hepatitis B virus, Chemiluminescent microparticle immunoassay, Performance evaluation