Development of an enzyme immunoassay using recombinant expressed antigen to detect hepatitis delta virus antibodies

Abstract

Two generic enzyme immunoassays (EIAs) were developed for detection of anti-hepatitis delta virus antibodies (anti-HD) and compared with a commercially available radioimmunoassay. Both generic assays were configured as blocking assays and used hepatitis delta antigen (HDAg) derived from infected chimpanzee liver (EIA-1) or from Escherichia coli transformed with a plasmid containing an insert from within an open reading frame encoding HDAg (EIA-2). Absolute sensitivity was ascertained by endpoint titration, which demonstrated essentially identical endpoints for EIA-1 and EIA-2. The absolute sensitivities of the EIAs were approximately four times greater than that of the radioimmunoassay. Specificity and sensitivity were ascertained by testing a panel of 176 serum specimens by each assay. The specimens were selected to represent a panel composed of sera from individuals with or without markers of viral hepatitis as follows: (i) serologically confirmed by exclusion as posttransfusion non-A, non-B hepatitis; (ii) acute or chronic hepatitis B virus infection, positive for hepatitis B surface antigen; (iii) resolved hepatitis B virus infection, positive for anti-hepatitis B surface antigen; (iv) acute hepatitis A virus infection, positive for anti-hepatitis A virus immunoglobulin M; and (v) normal human sera. All three assays for anti-HD gave similar specificity and sensitivity values. In conclusion, the recombinant expressed HDAg can replace antigen derived from infected liver tissue as a diagnostic reagent used to configure an EIA for detection of anti-HD. Furthermore, the results suggest that the expressed antigen contains the important immunodominant epitope(s).

Improving Access to Anti-HDV Testing: Development and Validation of an Affordable In-House ELISA Assay

“…19 Previous HDAg production has been difficult for diagnostic purposes because of the possible biohazards involved in using human serum, biopsy samples, or animal HDAg. 20 The extraction and purification of HDAg from the bacterial expression system ensure the total and harmless production of antigens, which could be utilized for anti-HDV antibody detection. 21 Purified, recombinant HDAg shows a good antigenic response and high affinity toward anti-HDV in HDV-positive serum.…”

“…There has to be a lot of particular and minimally antigenic viral protein available for the creation of enzyme-linked immunosorbent tests and radioimmuno assays . Previous HDAg production has been difficult for diagnostic purposes because of the possible biohazards involved in using human serum, biopsy samples, or animal HDAg …”