ABSTRACT
Anti-hepatitis A virus IgM capture ELISA was developed by using the reagents produced in the NIV laboratory. The major reagents of the assay were anti-human IgM antibody, hepatitis A virus (HAV) and anti-HAV IgG-horse radish peroxidase (HRP) conjugate. Of these, anti-human IgM antibodies were generated in rabbit against IgM secreted by human hybridoma clone(G3). HAV was derived from buffalo green money kidney cell line infected with HM-175 strain. Virus purified from the cell lysates was used for immunization of rabbits and guinea-pigs. There was very low anti-HAV response. A seropositive rhesus monkey was inoculated with monkey adapted strain of HAV to boost the anti-HAV antibody titre. Anti-HAV IgGs derived from hyperimmune sera of monkey and hepatitis A patient were conjugated with HRP. The preparations of conjugate--particularly human antibody--HRP conjugate yielded highly satisfactory results in anti-HAV capture ELISA. The assay appears to be specific, sensitive and quick and is useful in differentiating acute HAV infection from other acute infections caused by B, E and non-A non-B hepatitis viruses.
Subject(s)
Animals , Chlorocebus aethiops , Enzyme-Linked Immunosorbent Assay , Guinea Pigs , Hepatitis A/diagnosis , Hepatitis A Antibodies , Hepatitis Antibodies/blood , Hepatovirus/immunology , Humans , Immunoglobulin M/blood , RabbitsABSTRACT
Studies were carried out to determine the effect of prolongation of incubation periods, cocultivation with normal buffalo green monkey kidney (BGMK) cells and different concentrations of foetal calf serum (FCS) on the production of hepatitis A virus (HAV) by BGMK cell line persistently infected with HAV strain HM175. HAV could be detected from week 1 onwards. However, maintenance of cultures beyond this period was found to yield substantially higher quantities of virus. Cocultivation of persistently infected cells with normal BGMK cells also improved the antigen yields. Different concentrations of FCS did not show any effect on the amount of virus produced. The cell line was maintained up to 46 passages during which there was continuous production of HAV in the cells and release of small amounts of virus in the culture supernatants. Cell associated and cell free viral particles were found to be infectious. Supernatant derived virus was a highly suitable inoculum for infecting other susceptible cell lines. Persistently infected BGMK cell line appears to be a reliable and economical source to derive HAV in adequate amounts for diagnostic and research purposes.