Carrier effect of concanavalin A-reactive and -non-reactive material in tuberculin PPD

Tuberculin purified protein derivative (PPD) is a very potent T-cell reactive material in tuberculin-positive individuals, but the components responsible for this reactivity have not been adequately defined. Three purified mycobacterial proteins (MPB70, the BCG 65-kDa protein, and BCG antigen 85B) with different degrees of temperature sensitivity were iodine-labelled and added to BCG culture fluid, and the mixtures were autoclaved at 120 degrees C for 30 min to simulate the initial heating procedure used to prepare PPD. SDS-PAGE followed by protein staining and autoradiography showed that the banded pattern of unheated culture fluid was completely lost after heating, and only the labelled MPB70 preparation retained most of the radioactivity in a fraction with soluble protein of the same size. Most mycobacterial proteins are extensively denatured by these procedures, which explains the previous extensive difficulties in isolating defined constituents from PPD to characterize their behaviour in B- and T-cell reactions. In assays for the carrier effect of NIP-PPD for induction of anti-NIP production in BCG-vaccinated mice, the active fractions were heterogeneous in lectin reactivity as well as in SDS-PAGE. It appears most likely that a number of Mycobacterium tuberculosis proteins give rise to core peptides which resist proteolysis and heat denaturation to possess powerful T-cell-activating ability.