One-incubation time-resolved fluoroimmunoassay based on monoclonal antibodies in detection of influenza A and B viruses directly in clinical

Abstract

A new modified enzyme immunoassay screening method was developed for testing hybridoma cultures, so as to select antibodies useful for solid phase assays. Samples of hybridoma cultures were incubated for 1h with purified nucleoprotein preparation in microtiter wells precoated with rabbit anti-influenza A or B immunoglobulin, followed by washing and addition of anti-mouse HRPO-conjugate. The monoclonal antibodies were then used in one-incubation time-resolved fluoroimmunoassay (TR-FIA) for detecting influenza viral proteins in nasopharyngeal aspirate specimens. The sample and europium (Eu)-labelled monoclonal detector antibody (100 μl of each) were added simultaneously to microtiter wells precoated with anti-virus monoclonal antibody, and incubated for 1 h. After washing and addition of the enhancement solution the strips were shaken for 10 min before measurement of the fluorescence using a photon counting fluorometer. All of the monoclonal antibodies screened by our modified method and Eu-labelled worked as detector antibodies. Many of these monoclones also functioned as capture antibodies on solid phase. A total number of 309 (influenza A) and 104 (influenza B) nasopharyngeal aspirate specimens taken mainly from hospitalized children with acute respiratory disease were tested with the TR-FIA, using monoclonal antibodies produced by our modified screening method in comparison with monoclonal antibodies previously reported elsewhere (Walls et al., 1986). Results were similar, and superior to those obtained with routinely used indirect enzyme immunoassay based on polyclonal antibodies. The results of this study indicate that the one-incubation TR-FIA using monoclonal antibodies selected by the modified screening method is a highly sensitive and rapid method for detecting influenza A and influenza B virus in clinical specimens.