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Hepatitis delta virus (HDV) is a single-stranded, ringed negative-sense small RNA virus with a genome of approximately 1700 bases (6). It replicates only in patients who are concurrently infected with hepatitis B virus (HBV) (2). Concurrent infection by HBV and HDV increases the risk of severe liver disease com-pared to infection with HBV alone; the liver cells can be seriously damaged and the incidence of chronic hepatitis, hepatocirrhosis and hepatocellular carcinoma is higher than for other types of hepatitis (16, 17). There is a high incidence of HDV within the country an it is very important to establish sensitive and specific assays for HDV diagnosis in a timely manner. Antibody to hepatitis delta virus (anti-HDV) is one of the primary diagnostic HDV markers. This assay is the usual method for diagnosis of HDV infection since viremia lasts only a few weeks, and will help to determine the stage of the disease, degree of in-fectivity, prognosis, and immune status of the patient.
Up to now, anti-HDV has been measured by com-petitive ELISA, in which HDAg and detected sample are added to an antibody-coated well (3); there are also some other methods but they generally require kits which are manufactured by foreign companies and are often too expensive for routine use in deve-loping countries (1, 12). In addition, the HDAg for preparation of anti-HDV kit mainly came from animals infected by HDV and this can be restrictive for quality control (2). In the study, we have es-tablished a double antigen sandwich Enzyme-linked Immunosorbent Assay (ELISA) protocol for detection of anti-HDV in human serum by producing high-efficient purified and high titer recombinant HDAg protein, and we have evaluated the new assay by com-parison with competitive ELISA and indirect ELISA.
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We amplified the HDV gene fragment with 340 bp by RT-PCR for efficient expression of HDAg which primarily existed in inclusion bodies. In SDS-PAGE assay, the result was a single band and showed the molecular weight of the purified HDAg to be ~16kDa; the specific identification of purified HDAg protein showed a clear positive reaction with anti-HDV HRP, and the purified HDAg protein's purity was 90% by ultraviolet scan. After reversion from denaturalization, the concentration of the purified HDAg protein was 0.5mg/mL, and its ELISA titer was 1/100 000.
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In the selection of the coated HDAg, the results showed that both 5 μg/ml and 10 μg/mL gave clear signals, so we selected a concentration of 5 μg/mL.
For the testing of four anti-HDV positive serial sera and one anti-HDV negative serum, OD values at plates coated with purified HDAg were higher than those observed with plates coated with a mixture of purified HDAg and HDAg (pD280) (data not shown), so we selected the first coating model.
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For the double antigen sandwich ELISA, the anti-HDV positive rate increased with the dilution ratio, while the anti-HDV positive rate decreased for the competitive ELISA, this results revealed a statistical association (t=2.44, P < 0.01) between the two assays. (Table 1).
Table 1. Positive numbers for 42 anti-HDV positive serial sera with two assays
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In the neutralization inhibitory test by purified HDAg, OD values of the 2 anti-HDV positive serial sera declined in an obvious manner, this showed that the activity of anti-HDV was significantly declined after combining with HDAg; the inhibitory rates were all more than 50%. In this assay, the OD values of anti-HDV positive control and anti-HDV negative control were 0.993 and 0.067 respectively (Table 2).
Table 2. The results of the neutralization assaya
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Of the 42 anti-HDV positive sera, 20 (47.6%) were HDV-RNA positive; this result suggests that there was clear correlation between anti-HDV and HDV-RNA.
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In this assay, negative controls of anti-HDV kit purchased from Noctech Ltd., Dublin, Ireland were measured and the OD values were all below 0.10, then the 2.1N value of this assay was assumed to be 0.21. Based on this data, 2 positive controls of anti-HDV positive sera were anti-HDV positive, all 60 samples infected by other hepatitis viruses and 30 normal samples were negative for anti-HDV.