| Background and Objective: Hepatitis C virus was identified in 1989 as a major cause of the parenterally transmitted non-A,non-B hepatitis. Hepatitis C virus is transmitted by parenteral exposure to infected blood or body fluids. Chronic hepatitis C virus infection affects an estimated 170 million people worldwide and is characterized by varying degrees of inflammation and hepatic fibrosis. A proportion of patients with chronic infection will develop progressive liver damage with cirrhosis and complications of end stage liver disease over 20 to 40 years. Acute hepatitis C virus infection is usually subclinical, and there are no reliable predictive factors for chronic infection. Till now, there is no effective vaccine and therapeutic drugs for the prophylaxis and treatment of HCV infection, so blood donors scanning and early clinical diagnosis become very important in controlling the spreading of HCV. The detection of serum HCV RNA, anti-HCV antibody and HCV antigen are the major assays for the diagnosis of HCV infection. Though HCV RNA amplification by polymerase chain reaction (PCR) is the most sensitive and specific method for the detection of HCV, the complicated manipulation, easy contamination and high cost prevented PCR from application in a large scale. The low level of serum HCV antigens detected by common immunological and virological methods supports that serum HCV antigen detection can't be a common clinical method for HCV diagnosis. Anti-HCV antibody detection is a most commonly used assay in clinical practice. Enzyme-linked immunosorbent assay (ELISA) is now the routine method for HCV detection. However, this assay has many false negative in low level infections. Chemiluminescent assay has become the major assay for diagnosis of HCV infection because of its high sensitivity abroad. We have developed a new assay (Chemiluminescence enzyme immunoassay, CLEIA), anti-HCV kit, which can improve the sensitivity and meet the challenging demands of the clinical applications.Methods: 1 We put together the techniques of chemiluminescent assay and immunoassay and established a method, CLEIA kit, for anti-HCV determination using the horseradish peroxidase (HRP) labeled IgG antibody and the detection system of enhanced luminal-HRP chemiluminescence, Established the optimal experimental conditions of the CLEIA.2 The assay was evaluated its sensitivity and specificity with the pannels of National Institute for the Control of Pharmaceutial and Biological Products and BBI. The effection of common anticoagulants and the serum of particular groups was tested. The assay was also compared with ELISA kits.Results:1 A new enhanced chemiluminescence enzyme immunoassay (CLEIA) was developed. The optimal experimental conditions of the CLEIA was listed as below. Using the purified recombinant HCV proteins as coating antigen, the protein concentration for coating microtiter was 1μg/ml, and incubated overnight at 4℃, The plates were subsequently blocked with 0.01mol/L PBS (pH 7.4, 1% BSA, 5% sucrose), overnight at 4℃, and then incubated for 30 min with diluted sample (1:10) at 37℃, after washing the plates, the diluted HRP-IgG (1:4000) was added and incubated for 30 min at 37℃, washing, then the chemiluminescent substrate was added, detecting the value after 10～15 min.2 The detection limit was 0.1NCU/ml, and did not cross-react with anti-HBs,anti-ANA,anti-HIV,serum of multiple myeloma. The assay was not affected by bilirubin,cholesteron,triacylglyceral which was in normal range. The common anti-coagulants did not affected the experimental result. The sensitivity of this method was more than domestic ELISA kits, and its sensitivity and specificity were almost equal to overseas ELISA kits.Conclusions:The established CLEIA for anti-HCV is very sensitive,specific,and repeated, itwill be useful and economical as a routine test in laboratories for early diagnosis andprevention of HCV infection. |
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