A Simple Purification Method of <i>Vibrio cholerae</i> Non-O1 Hemagglutinin/Protease by Immunoaffinity Column Chromatography Using a Monoclonal Antibody

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A new simple purification method (I) for Vibrio cholerae non-O1 hemagglutinin/protease (NAG-HA/P) was developed. The method (I) requires only an immunoaffinity column chromatography using a monoclonal antibody against NAG-HA/P. The method (I) is much simpler than previously reported purification method (II) (Honda, T. et al, Infection and Immunity 57: 2799-2803, 1989) which required four or more complicated chromatographic procedures. Method (I) also gave an improved recovery rate (about 27%) compared with (II). The molecular weight of NAG-HA/P purified by method (I) was mainly 34 kilodaltons (kDa) with a little of 32kDa, whereas that of NAG-HA/P purified by (II) was usually 32kDa. Immunological analysis by the Ouchterlony double gel diffusion test and Western blotting test using polyclonal antibody against 32kDa protein revealed that the 34 and 32kDa proteins are immunologically indistinguishable and thus it is supposed that 34K protein is an isoform or a preform of the 32K protein.

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