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

Purified protein derivatives (PPDs) previously prepared by two different methods from the cultured filtrate of Mycobacterium avium subsp. paratuberculosis (MAP) ATCC strain 19698 were further characterized and compared. The traditional production method, which incorporates autoclaving prior to filtration of the culture media, was compared to a modified method that removed autoclaving during the process. PPD preparations were characterized utilizing mass spectrometry and compared for skin test responses using the guinea pig potency test. Mass spectrometry identified 131 MAP proteins among the three PPD preparations, ten of which present in all preparations. Guinea pig potency testing between PPD preparations resulted in significant difference between production lots. The overall potency of each tested lot was acceptable based upon the measured reactivity. 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 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

Survival of Brucella abortus RB51 lyophilized and liquid vaccine at different storage conditions

http://www.worldcat.org/oclc/3970802

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

Mycobacterium avium subsp. paratuberculosis (MAP) purified protein derivatives (PPDs) are immunologic reagents prepared from cultured filtrates of the type strain. Traditional pro-duction 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 preparations. Antibody bound distinct protein bands in the alternative PPDs by immunoblot analysis, 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

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

Mycobacterium avium subsp. paratuberculosis (MAP) purified protein derivatives (PPDs) are immunologic reagents prepared from cultured filtrates of the type strain. Traditional pro-duction 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 preparations. Antibody bound distinct protein bands in the alternative PPDs by immunoblot analysis, 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

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

Mycobacterium avium subsp. paratuberculosis (MAP) purified protein derivatives (PPDs) are immunologic reagents prepared from cultured filtrates of the type strain. Traditional pro-duction 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 preparations. Antibody bound distinct protein bands in the alternative PPDs by immunoblot analysis, 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

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

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 preparations. Antibody bound distinct protein bands in the alternative PPDs by immunoblot analysis, 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

Secretion of IFN-γ using whole blood from Johne’s positive animals, <i>M</i>. <i>bovis</i> sensitized animals, and negative control animals.

<p>Whole blood was incubated with selected recombinant proteins and mycobacterial PPDs for 20–22 hrs as indicated in the materials and methods. Absorbance was measured at 450 nm.</p

Cattle Sera used in this study.

<p>Cattle Sera used in this study.</p

Composition and Potency Characterization of <i>Mycobacterium avium</i> subsp. <i>paratuberculosis</i> Purified Protein Derivatives

<div><p><i>Mycobacterium avium</i> subsp. <i>paratuberculosis</i> (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 preparations. Antibody bound distinct protein bands in the alternative PPDs by immunoblot analysis, 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 <i>E</i>. <i>coli</i> 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 <i>in-vitro</i> assay reagents.</p></div

Comparative antibody responses between the PPD production methods.

<p>Immunoblots of PPD 0902 produced by an alternative production method on the left and PPD 9801 produced by the traditional method on the right. Both blots were exposed to identical bovine serum samples loaded into independent slots. Note that distinct antigenic bands appear in the alternate PPD preparation. Lanes 1–15 cattle positive for Johne’s disease, Lanes 16 & 17 cattle negative for Johne’s disease. Lane numbers correspond to cattle listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154685#pone.0154685.t001" target="_blank">Table 1</a>.</p