Electroelution for purification of influenza A matrix protein for use in immunoassay.
A new preparative method for isolation of matrix protein from type A influenza virus was developed. Commercially available whole virus or split virus vaccines were lysed, and the soluble proteins separated by electrophoresis on polyacrylamide gel. The matrix protein was located on the gel by precipitation with KCl, and recovered by electroelution. The method was technically simple and required little direct supervision during the two-step recovery process. Yields of A matrix were consistently high, averaging 68.1% in five trials with A/Brazil/X-71. The method was also successful with other A viruses, although not with influenza B virus. Isolated A matrix had less than 0.5% contamination by hemagglutinin or nucleoprotein, as determined by immunoblotting and ELISA. Matrix protein was immunoreactive in Western blots and was detectable in concentrations as low as 1 ng/ml with ELISA. The isolated matrix provided a suitable standard for detection of matrix protein in nasal washes from patients with influenza A virus infection, and could also be used to detect anti-matrix antibodies, including monoclonal antibodies in tissue culture supernatants. The advantages of electroelution for separation of matrix protein compared to other methods were its technical simplicity, applicability to formalin-fixed influenza virus in commercially available vaccines, its consistently high yield, and its very high level of purification.
D000911:Antibodies, Monoclonal / Q000032:analysis; D000914:Antibodies, Viral / Q000032:analysis; D000956:Antigens, Viral / Q000276:immunology / Q000302:isolation & purification*; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D009980:Influenza A virus / Q000032:analysis / Q000276:immunology*; D009981:Influenza B virus / Q000276:immunology; D007252:Influenza Vaccines / Q000032:analysis*; D014763:Viral Matrix Proteins; D014764:Viral Proteins / Q000276:immunology / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral; D007252:Influenza Vaccines; D014763:Viral Matrix Proteins; D014764:Viral Proteins