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

Abstract

Tuberculin purified protein derivative (PPD) currently can only be standardised bydelayed hypersensitivity skin reactions in sensitised guinea pigs. An in vitro dot blotimmunoassay was developed for both identity and possibly potency estimation of PPD.Polyclonal antibodies (mainly IgG) were generated and reacted with human, bovine and, tolesser extent, avian PPD preparations in immunoblotting assays. Various PPD preparationswere analysed using SDS-PAGE coupled with Western blot and size exclusionchromatography (FPLC-SEC). The results showed that PPD preparations were mixtures ofvery heterogeneous tuberculoproteins ranging in size from very large aggregates to very smalldegraded molecules. All individual fractions of PPD separated by size were immunoreactive,although those of the largest molecular sizes appeared the most immunoreactive in this invitro dot blot immunoassay. This method is very sensitive and specific to tuberculoproteinsand can be an in vitro alternative for the in vivo intradermal skin assay which uses guinea pigsfor identity of PPD preparations. Although the capacity of PPD to elicit cell-mediatedimmune responses on intradermal testing has to be confirmed by in vivo assay, the dot blotimmunoassay offers a rapid, sensitive and animal-free alternative to in vivo testing forconfirming the identity and approximate quantification of PPD preparations. The procedurewould be particularly useful for national control laboratories for confirming the data ofmanufacturers and thus reducing the use of animals.