Tuberculin purified protein derivative (PPD) immunoassay as an in vitro alternative assay for identity and confirmation of potency.
Tuberculin 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.
Ho
Mei M
MM
Division of Bacteriology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire, UK. mho@nibsc.ac.uk
Kairo
Satnam K
SK
Corbel
Michael J
MJ
D000818:Animals; D004355:Drug Stability; D015151:Immunoblotting / Q000379:methods*; D011817:Rabbits; D014373:Tuberculin / Q000276:immunology*; D014374:Tuberculin Test / Q000379:methods / Q000592:standards*
D014373:Tuberculin