Characterization and Quantitation of Membrane Antigens of New World Leishmania Species by Using Monoclonal Antibodies in Western Blot and Flow Microfluorometric Assays1

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Abstract
Membrane‐specific monoclonal antibodies generated against promastigotes of New World Leishmania species were used in Western blot, ELISA, and flow microfluorometric assays to characterize their antigen specificity and to determine the external surface distribution of the reactive epitopes. Three major membrane antigens of molecular weight 72 KD, 55 KD, and 42 KD were identified as well as a dominant antigen that migrated as a broad band on SDS‐PAGE, corresponding to a molecular weight of 10–15 KD. By dot‐ELISA this antigen was also found to be excreted by promastigotes into their culture medium. One minor membrane antigen of 25 KD and a triplet component of 66, 58, and 56 KD were also identified. While assays performed on air‐dried promastigotes revealed the almost ubiquitous presence of the 72 KD and 55 KD antigens, indirect immunofluorescent staining of live promastigotes followed by flow cytometric analysis revealed that these antigens had no external exposure. Antibodies binding the 55 KD component were also reactive toward purified mammalian tubulin. The remaining antigens had a variable distribution on the eight isolates utilized, and these quantitative differences could be used to distinguish isolates of the Leishmania mexicana complex from those belonging to the Leishmania braziliensis complex.