Characterization of Bovine, Avian and Johnin Purified Protein Derivative (PPD) with FTIR Method

What is it about?

Tuberculins purified protein derivatives (PPDs) are obtained by precipitation from heat treated mycobacteria. PPDs are used in diagnosis of mycobacterial infections in humans and animals. Bovine PPD (PPDB) is obtained from Mycobacterium bovis (Mycobacterium tuberculosis complex), while Avian PPD (PPDA) and Johnin PPD (PPDJ) are extracted, respectively, from Mycobacterium avium and M. avium subsp. paratuberculosis (M. avium complex). PPDB and PPDA are used for bovine tuberculosis diagnosis, while PPDJ is experimentally used in the immunodiagnosis of paratuberculosis. Although PPDs date back to the 19th Century, limited knowledge about their composition is currently available. The goal of our study was to evaluate Fourier Transform InfraRed (FTIR) spectroscopy as a tool to dierentiate PPDB, PPDA, and three PPDJs. The results highlighted that the three PPDs have specific profiles, correlated with phylogenetic characteristics of mycobacteria used for their production. This analysis is eligible as a specific tool for dierent PPDs batches characterization and for the assessment of their composition. The entire PPD production may be eciently controlled, since the N content of each preparation is related to IR spectra, with a reference spectrum for each PPD and a standardized analysis protocol.

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Why is it important?

FTIR spectroscopy has proven to be a fast, reliable, reproducible, and inexpensive technique, which is useful for describing and characterizing extracts, as well as substrates and cells. This first application of FTIR analysis on PPDs showed that this method could be used to characterize and differentiate the PPDB, from the PPDA and from the Johnin PPD. For the future it could be interesting to check whether the analysis is able to highlight differences between the batches of the same PPD preparations. It will be necessary to analyze several lots of the same PPD preparation in order to verify the capacity of the method to evaluate the reproducibility of the PPD production process and batches. This preliminary study lay the foundations for a potential use of the FTIR analysis as a tool to characterize the different PPDs batches and to assess their composition; in fact, with a reference IR spectrum for each PPD and a standardized analysis protocol, the entire PPD production process might be controlled. In fact, on the basis of existing correlation between the IR spectra and the average N content of each PPD, it could be possible to perform a further control on production protocols and on PPD batches. Moreover, mycobacteria cultures could be characterized with FTIR analysis before the heat inactivation in order to verify how the different production steps can affect PPD composition, so that their profile could be compared with those of the final products.

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