| Labeled immunoassay with high sensitivity and specificity has been widely used in basic research and clinical examinations.In recent years,the development of new theories and new techniques has enormously improved the techniques in labeled immunoassay.The most widely used immunolabeling techniques are radioimmunoassay(RIA) and enzymatic immunoassay(EIA).Although sensitive and specific,both of these assays have their inherent limitations in the labeling agent used. In RIA,the radioactive agents for labeling cause inevitable radiation hazards with also short-life of the iodinated labels.In EIA,the enzymes used are often easily inactivated to lower the sensitivity of the assay,and enzyme labeling of large molecules often results in alterations in the spatial structure of the labeled molecules to affect the sensitivity.The commonly used fluorescent molecules as the labeling agents also gives rise to unstable assay sensitivity due to the possible interferences by various factors including the scattered light,background fluorescence of the sample and fluorescence quenching,and therefore can hardly meet the needs in detection of trace levels of antigens or antibodies.The emergence of time-resolved fluoroimmunoassay(TRFIA) developed in the early 1980s provides a solution to the above technical obstacles.TRFIA,which has overcome the limitations in RIA and EIA without using radioactive isotopes,is characterized by rapidity,high sensitivity and stability and has been widely used in biomedical studies and clinical examinations.Hepatitis C virus(HCV) antibody(anti-HCV) is a nonprotective antibody generated after HCV infection.Currently the clinical examination of anti-HCV commonly utilizes EIA,which easily result in missed diagnosis because of the relatively low sensitivity of the assay.The IgM antibody to hepatitis B core antigen (HBc-IgM) is generated by host immune response against HBV infection,and HBc-IgM positivity often suggests a recent HBV infection.As the earliest specific HBV antibody present in the serum of HBV-infected individuals,HBc-IgM is a marker of HBV replication with infectious potentials.Currently clinical HBc-IgM detection relies rhostly on microparticle enzyme immunoassay(MEIA), enzyme-linked immunosorbent assay(ELISA) and solid-phase radioimmunoassay (SPRIA).Compared to MEIA and SPRIA,ELISA has better clinical applicability without the use of radioisotope,but its sensitivity still remains insufficient and the enzyme markers can be unstable.Hepatitis B virus pre-S1 antigen(HBV-preS1Ag) is encoded by pre-S1 gene in HBV S gene region,and can be detected in acute hepatitis B prior to the elevation of transaminase in early stage of the HBV infection,serving also as an early diagnosis indicators for HBV infection.Conversion of preS1 positivity earlier than that of HBV-DNA positivity is the earliest sign of HBV clearance and suggests favorable prognosis,whereas sustained preS1 positivity often indicates high likeliness of the development of chronic hepatitis.Additional detection of anti-HBc-IgM and HBV-preSlAg can reinforce the diagnosis of acute hepatitis B, and therefore has become routine examinations in addition to clinical examination of the common 5 indices for hepatitis B.In this study,the author developed TRFIA kits for detecting Hepatitis C virus (HCV) antibody,anti-HCV HBc-IgM and Hepatitis B virus pre-S1 antigen separately using the indirect method,immunocapture method and antibody-sandwich method. The performance of the detection kit was evaluated in terms of the analytical sensitivity,determination of cutoff value,specificity,precision,and stability,with also comparison with other diagnostic ELISA kit manufactured in China to evaluate the feasibility of our kits for clinical application.The results showed that the accuracy and sensitivity of the TRFIA reagent kit for HCV antibody detection met the requirements of the National reference standards,with cutoff value of 2.1 folds of the negative control fluorescence.No cross reactivity was found with HIV,TP,anti-HBs, anti-HBe,or anti-HBc.The intra-assay coefficients of variation were 3.2%-9.3%and the inter-assay coefficients of variation were 4.6%-8.9%.No significant decline in the value of fluorescence was detected after preservation of the reagents at 37℃for seven days or at 4℃for one year,and the intra-assay coefficients of variation remains below 10%and the inter-assay coefficients of variation remains below 15%. The testing samples were free of interferences by triglycerides,bilirubin,and hemoglobin,and comparison of the results of the TRFIA kit and ELISA demonstrated greater sensitivity of the kit than ELISA in detecting the HCV antibodies in 508 serum samples.For HBc-IgM detection,the cutoff value of the TRFIA kit was 2.5 folds of the negative control fluorescence value,and no cross-reactivity with anti-HBs, anti-HBc-IgG or anti-HBe was found.The intra- and inter-assay coefficients of variation were all below 10%;the intra- and inter-assay coefficients of variation remains below 10%after preservation of the reagents at 37℃for 7 days or at 4℃for one year.The testing results were not subjected to interference by triglycerides, bilirubin,or hemoglobin,and the TRFIA kit demonstrated greater specificity than ELISA in detecting anti-HBc IgM in 584 serum samples.For HBV pre-S1 antigen detection,the cutoff value of the TRFIA kit was 2.5 folds of the negative control fluorescence value without cross reactivity with HBsAg or HBeAg.The intra-assay coefficients of variation were 3.2%-8.3%,and the inter-assay coefficients of variation were 5.1%-10.9%;the intra-assay coefficients of variation remains below 10%,the inter-assay coefficients of variation remains below 15%after preservation of the reagents at 37℃for 7 days or at 4℃for one year.The testing results were not influenced by triglycerides,bilirubin,or hemoglobin.The TRFIA kit showed greater sensitivity than ELISA in detecting the HBV pre-S1 antigen in 280 serum samples. The above results demonstrate that the TRFIA reagent kit for HBV and HCV we developed meet the demand for clinical application,have the prospects for clinical determination. |
