Tuberculin Purified Protein Derivative (PPD) Immunoassay as an In Vitro Alternative Assay for Identity and Confirmation of Potency

Abstract

KEY WORDS ABSTRACTTuberculin purified protein derivative (PPD) currently can only be standardised by delayed hypersensitivity skin reactions in sensitised guinea pigs. An in vitro dot blot immunoassay was developed for both identity and confirmation of potency estimation of PPD. Polyclonal antibodies (mainly IgG) were generated and immunoreacted with human, bovine and, to lesser extent, avian PPD preparations. Combining size exclusion chromatography (FPLC-SEC) and dot blot immunoassay, the results showed that PPD preparations were mixtures of very heterogeneous tuberculoproteins ranging in size from very large aggregates to very small degraded molecules. All individual fractions of PPD separated by size were immunoreactive, although those of the largest molecular sizes appeared the most immunoreactive in this in vitro dot blot immunoassay. This method is very sensitive and specific to tuberculoproteins and can be an in vitro alternative for the in vivo intradermal skin assay which uses guinea pigs for identity of PPD preparations. Although the capacity of PPD to elicit cell-mediated immune responses on intradermal testing has to be confirmed by in vivo assay, the dot blot immunoassay offers a rapid, sensitive and animal-free alternative to in vivo testing for confirming the identity of PPD preparations with appropriate potencies. This alternative assay would be particularly useful for national regulatory laboratories for confirming the data of manufacturers and thus reducing the use of animals.

Purified protein derivatives of tuberculin — past, present, and future

“…The prolonged heating of crude tuberculin to prepare PPD contributed to the denaturation, par-tial degradation, and aggregation of many of the protein components. Numerous studies identified PPD as mixtures of very heterogeneous proteins ranging in size from very large aggregates to very small degraded molecules (Klausen et al, 1994;Rowland et al, 1999;Ho et al, 2006). Similarly, little was known regarding which of these components in PPD was responsible for the DTH response.…”

Spectroscopic Characterization of Bovine, Avian and Johnin Purified Protein Derivative (PPD) with High-Throughput Fourier Transform InfraRed-Based Method

“…The proteomic characterization of human and veterinary PPDs has been described by other authors [27,30], showing that PPD contains hundreds of different proteins. In fact, several studies defined PPDs as very heterogeneous compositions of proteins that range in size from very large aggregations to very small degraded segments [25,31,32,33]. In the study of Borsuk et al [27], two bovine PPDs (Brazilian and English) and two avian PPDs (Brazilian and English) were characterized by LC-MS/MS to effectively study their protein profile; a total of 171 different proteins were identified, 77.9% of which was represented by cytoplasmic proteins, while 22.4% was represented by membrane or secreted proteins.…”