Development of an improved production method, determination of protein composition, and potency characterization of Mycobacterium avium subsp. paratuberculosis purified protein derivative

Mycobacterium avium subsp. paratuberculosis (MAP) purified protein derivatives(PPDs) are an immunologic reagent prepared from the cultured filtrate of MAP ATCC strain19698. PPD was produced using MAP ATCC Strain 19698 in Povitsky bottles and Erlenmeyerflasks. Traditional production consisted of floating culture incubation at 37C, live organisminactivation by autoclaving 30 minutes at 121C, and coarse filtration to remove cellulardebris. An alternative production method was developed involving removal of the autoclaving step during the production process. Proteins in both production methods were subsequentlyprecipitated by adding trichloroacetic acid to a final 4% concentration. Culture production inErlenmeyer flasks was superior to that of traditionally used Povitsky bottles as determined byfloating culture mat density and time required for growth. Defined protein bands on SDS-PAGEgels were masked by smearing of the gel tracks containing PPD produced by the traditionalmethod whereas protein bands were evident in the alternate PPD preparations. Results indicate that the current production procedure and an alternate production method produced PPDequivalent to the reference NVSL PPD Lot 9801. PPD preparations will be used for furtheranalysis to define protein components and identification of possible antigenic components.