Abstract
BackgroundIn assays for anti-hepatitis E virus (HEV) immunoglobulin M (IgM), large volumes of the patient's sera cannot be easily obtained for use as a positive control. In this study, we investigated an alternative chemical method in which rabbit anti-HEV IgG was conjugated with human IgM and was used as a positive control in the anti-HEV IgM assay. Rabbit anti-HEV IgG was isolated from immune sera by chromatography on protein A-Sepharose and was conjugated with human IgM by using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) as a crosslinker.ResultsThe specific anti-HEV IgG antibody titer was 100,000 times that of the negative control, i.e., prebleed rabbit serum. The results of anti-HEV IgM enzyme-linked immunosobent assay showed that the antibody conjugate was similar to anti-HEV IgM antibodies produced in humans. The results of a stability experiment showed that the antibody conjugate was stable for use in external quality assessment or internal quality control trials.ConclusionsWe concluded that the chemically conjugated rabbit-human antibody could be used instead of the traditional serum control as a positive control in the anti-HEV IgM assay.
Highlights
In assays for anti-hepatitis E virus (HEV) immunoglobulin M (IgM), large volumes of the patient's sera cannot be obtained for use as a positive control
We investigated the feasibility of replacing the conventional positive controls with an antibody conjugate in which rabbit anti-HEV IgG was conjugated with human IgM by using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) as a crosslinker [20]
Because anti-HEV assays based on open reading frame 2 (ORF2) were shown to be more sensitive than those based on ORF3 [21], we used recombinant protein NE2 with an ORF2 immunodominant epitope as the antigen
Summary
In assays for anti-hepatitis E virus (HEV) immunoglobulin M (IgM), large volumes of the patient's sera cannot be obtained for use as a positive control. We investigated an alternative chemical method in which rabbit anti-HEV IgG was conjugated with human IgM and was used as a positive control in the anti-HEV IgM assay. Rabbit anti-HEV IgG was isolated from immune sera by chromatography on protein A-Sepharose and was conjugated with human IgM by using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) as a crosslinker. The immunoglobulin M (IgM) antibody-capture enzymelinked immunosorbent assay (ELISA) was designed to detect IgM antibodies and is a valuable marker for rapid diagnosis of acute viral infection [6,9,10]. In some cases, the anti-HEV IgM assay had relatively low sensitivity.
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