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Enzyme-linked immunosorbent assays which are based on species- or type-specific glycolipids antigens and in which rabbit antisera are prepared with homologous strains are capable of distinguishing among serological variants of the Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum complex, Mycobacterium chelonei subspecies chelonei and abscessus, Mycobacterium simiae I and II, Mycobacterium kansasii, Mycobacterium szulgai, Mycobacterium xenopi, and Mycobacterium fortuitum biovariant peregrinum. The immunoreactive glycolipids can be divided into two classes. Those resistant to alkali, the C-mycoside glycopeptidolipids, are present in the M. avium-M. intracellulare-M. scrofulaceum, the M. chelonei subspecies chelonei and abscessus, and the M. simiae I and II complexes and in M. fortuitum biovariant peregrinum. The alkali-labile glycolipid antigens, the lipooligosaccharides, are present in M. kansasii, M. szulgai, and M. xenopi. In one study, the combination of enzyme-linked immunosorbent assay and alkaline susceptibility was compared with seroagglutination in the identification of 60 clinical isolates of nontuberculous mycobacteria: 45 showed perfect concordance, 9 could be narrowed to one, two, or three possibilities, and the rest did not correspond. In a second study involving 43 clinical isolates that were untypable by seroagglutination or were autoagglutinable, the results of enzyme-linked immunosorbent assay and thin-layer chromatography of glycolipid antigens were compared: 21 showed clear concordance. The results demonstrate that enzyme-linked immunosorbent assay is particularly useful in assessing the antigenicity of lipids, and sensitivity, ease, and rapidity recommend it as an adjunct to seroagglutination and thin-layer chromatography for the identification of nontuberculous mycobacteria.