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Composition and Potency Characterization of Mycobacterium avium subsp. paratuberculosis Purified Protein Derivatives

Randal T. Capsel, Charles O. Thoen, Timothy A. Reinhardt, John D. Lippolis, Renee Olsen, Judith R. Stabel, John P. Bannantine
Plos One 2016 v.11 no.5 pp. e0154685
Escherichia coli, Mycobacterium avium subsp. paratuberculosis, antibodies, antigens, autoclaving, blood, erythema, filtrates, filtration, guinea pigs, immunoblotting, interferon-gamma, mass spectrometry, polyacrylamide gel electrophoresis, recombinant proteins
Mycobacterium avium subsp. paratuberculosis (MAP) purified protein derivatives (PPDs) are immunologic reagents prepared from cultured filtrates of the type strain. Traditional production consists of floating culture incubation at 37°C, organism inactivation by autoclaving, coarse filtration, and protein precipitation. Three traditional production PPDs were used in this study including lot 9801, which served as a reference and has been used in the field for decades. Alternative production PPDs (0902A and 0902B), in which the autoclaving step was removed, were also analyzed in this study. SDS-PAGE analysis revealed protein smearing in traditional PPDs, but distinct bands were observed in the alternative PPD prep- arations. Antibody bound distinct protein bands in the alternative PPDs by immunoblot anal- ysis, whereas an immunoreactive smear was observed with the traditional PPDs. Mass spectrometry identified 194 proteins among three PPD lots representing the two different production methods, ten of which were present in all PPDs examined. Selected proteins identified by mass spectrometry were recombinantly expressed and purified from E. coli and evaluated by the guinea pig potency test. Seven recombinant proteins showed greater erythema as compared to the reference PPD lot 9801 in paired guinea pigs and were able to stimulate interferon-gamma production in blood from Johne’s positive animals. These results suggest that autoclaving culture suspensions is not a necessary step in PPD production and specific proteins could supplant the PPD antigen for intradermal skin testing procedures and for use as in-vitro assay reagents.