A STUDY OF THE ADVANTAGES AND LIMITATIONS OF IMMUNOBLOTTING PROCEDURES FOR THE DETECTION OF ANTIBODIES AGAINST INFLUENZA-VIRUS

An immunoblotting procedure was used to determine the specificity and examine some of the properties of antibodies produced following infect ion of mice with influenza virus or inoculation with noninfectious mat erial 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, hemagglut inin (HA) and neuraminidase (NA). When influenza viral proteins were s eparated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions, each of the anti-viral antise ra 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 dir ected to HA also bound well to viral HA separated by SDS-PAGE under no nreducing conditions but failed to recognize epitopes on HA1 separated under reducing conditions. These results suggest that when HA is redu ced and immobilized on a solid support, it does not display the confor mational features essential for the integrity of all epitopes. The imm unoblotting procedure was also used to determine the isotype of anti-v iral antibody directed against individual viral proteins and to detect matrix protein 2 (M2) in purified influenza virions and influenza-inf ected cells using antisera raised to a synthetic peptide representing a sequence within the M2 protein.