Immunoassay for serologic diagnosis of influenza type A using recombinant DNA produced nucleoprotein antigen and monoclonal antibody to human IgG

Abstract

Influenza type A nucleoprotein (NP) derived from the full length cloned gene expressed in E. coli was evaluated in a solid phase enzyme immunoassay (EIA) for detection of human antibody to influenza. Monoclonal antibody to human IgG was used for detection. Direct and indirect assays were developed and sera were tested in serial and single dilution formats. Preliminary results indicated that recombinant-and virion-derived NP antigens were comparable in binding ability to plastic and binding human antibody. Eighty-seven paired sera from influenza patients were tested. The most sensitive assay (indirect-serial dilution) detected 56 (64%) rises and the simplest assay (direct-single dilution) detected 43 (49%) rises, compared to 36 (41%) for complement fixation. Paired sera from 18 control patients showed no evidence of antibody rises by any of the assays. Forty-nine paired sera from influenza B infected patients were negative for antibody rises except for one borderline rise by the indirect-serial dilution assay. These results indicate that the use of recombinant DNA derived nucleoprotein for immunoassay is feasible. The sensitivity of immunoassays using NP adsorbed to the solid phase and monoclonal antibody specific for human IgG to detect bound antibody exceeded that of conventional complement fixation testing for establishing serologic evidence of influenza type A infection.

Molecular and phylogenetic analysis of human influenza virus among Iranian patients in Shiraz, Iran

“…The standard laboratory method for diagnosis of influenza is based on virus isolation in tissue culture [Harmon et al, 1989]. Several methods such as immunoflourscence assay (IFA) or enzyme linked immunosorbant assay (ELISA) have been applied for rapid detection of influenza viruses [Reina et al, 1996].…”

Expression of influenza A and B virus nucleoprotein antigens in baculovirus

“…The influenza virus-specific sequences immediately upstream from the NP initiation codons are different in type A and B viruses and could have different effects on expression levels. Optimum levels of expression have been obtained when the intervening sequences were removed and the initiation codon of the expressed < 100 100 100 Child 6 S1 < 100 < 100 < 100 $2 < 100 < 100 < 100 Normal serum Child 7 < 100 < 100 < 100 Child 8 < 100 < 100 < 100 Chimpanzee < 100 < 100 < 100 * Titres from Harmon et al (1989). t Monovalent influenza vaccine recipients.…”

“…A previous report has demonstrated that bacterially expressed NP antigen could be used in an EIA for the serological diagnosis of influenza virus infection, and the or AcBNP (right lanes) were subjected to electrophoresis and transferred to nitrocellulose and hybridized to pairs of acute phase (S1) and convalescent phase ($2) human serum specimens that had increased antibody reactivity specifically to either influenza A virus (al : results were superior to those obtained using the standard C F test (Harmon et al, 1989). The results of the analyses we present here indicated that the reactivities of the crude baculovirus-expressed antigens and highly purified bacterially expressed N P antigens were equivalent and that the titres o b t a i n e d using the baculovirusexpressed antigens in a standard E I A gave titres that correlated with titres obtained with the C F assay.…”

“…Serum samples were obtained from each group at 14 days post-infection and pooled. Adult human serum samples used in this study were from the same set as was used in a previous study with bacterially expressed influenza A virus NP (Harmon et al, 1989). These serum samples had been obtained during the acute and convalescent stages of illness of patients from whom influenza A or B viruses were isolated.…”

“…Since the NP antigen is antigenically conserved, it is an ideal candidate for expression by recombinant DNA methodology as part of the development of a new generation of diagnostic assays for influenza virus infections. We have previously reported the use of NP antigen produced in Escherichh~ coli in an experimental diagnostic assay for influenza A virus (Harmon et al, 1989). This assay was found to be superior to CF for influenza virus diagnosis.…”