A study of the advantages and limitations of immunoblotting procedures for the detection of antibodies against influenza virus

Abstract

An immunoblotting procedure was used to determine the specificity and examine some of the properties of antibodies produced following infection of mice with influenza virus or inoculation with noninfectious material with Alhydrogel or complete Freund's adjuvant. The noninfectious material used was beta-propiolactone-inactivated influenza virus and a preparation (HANA) enriched for the surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). When influenza viral proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions, each of the anti-viral antisera tested exhibited strong binding. Under reducing conditions, however, much weaker binding was observed especially towards the HA1 subunit of HA. This was particularly apparent with antisera raised to virus or HANA in the absence of adjuvant. A panel of monoclonal antibodies directed to HA also bound well to viral HA separated by SDS-PAGE under nonreducing conditions but failed to recognize epitopes on HA1 separated under reducing conditions. These results suggest that when HA is reduced and immobilized on a solid support, it does not display the conformational features essential for the integrity of all epitopes. The immunoblotting procedure was also used to determine the isotype of anti-viral antibody directed against individual viral proteins and to detect matrix protein 2 (M2) in purified influenza virions and influenza-infected cells using antisera raised to a synthetic peptide representing a sequence within the M2 protein.

Myelitis associated with influenza a virus infection

“…The antibody index (AI) was 9.9 (normal 51.9; AI=[Influenza A antibody level in CSF : Influenza A antibody level in serum]6[total IgG in serum: total IgG in CSF]) (Ukkonen et al, 1981). In immunoblot analysis with an IgG concentration of 8.8 mg/L for CSF and 9.7 mg/L for serum against a recent reference strain, A Beijing/353/89 (H3N2), the antibodies appeared reactive (Kapaklis-Deliyannis et al, 1993). In nonreducing conditions, both serum and CSF gave distinct bands to HA2 and M proteins and a weaker band to NP.…”