COMPOSITION AND IMMUNOREACTIVITY OF THE A60 COMPLEX AND OTHER CELL-FRACTIONS FROM MYCOBACTERIUM-BOVIS BCG

Surface static cultures of Mycobacterium bovis BCG contained cells emb edded in an extracellular matrix, whose mechanical removal yielded fre e cells that were pressure disrupted and fractionated into cytoplasm a nd walls. Cell envelopes were either mechanically disrupted or extract ed with detergents. Intracellular and extracellular fractions were ana lysed for proteins, polysaccharides, and antigen 60 (A60), a major com plex immunodominant in tuberculosis. A60 was present in extracellular matrix, cytoplasm and walls: it represented a substantial portion of t he proteins and polysaccharides of these fractions. While the protein/ polysaccharide ratio varied according to the origin of A60 preparation s, the electrophoretic patterns of A60 proteins (which accounted for t he immunogenicity of the complex) remained unchanged. Western blots po inted to the proteins present within the 29-45 kDa range as the A60 co mponents endowed with the highest immunogenicity level. Since the most heavily stained protein bands in SDS-PAGE patterns were located outsi de the region best recognized by antisera, a striking discordance was found between concentration and immunogenicity patterns of A60 protein s. The electrophoretic patterns of A60- and non-A60-proteins from cyto plasm were also different. A60 complexes in dot blots and some electro phoresed A60 proteins reacted with monoclonal antibodies directed agai nst lipoarabinomannan (LAM), a highly immunogenic polymer of cell enve lope. This contaminating compound was removed from A60 with organic so lvents and detergents. SDS-PAGE and Western blot patterns of proteins from delipidated A60 were similar to those of native A60 proteins.