An assay to quantitate the binding of Leishmania amastigotes to macrophages

A leishmania amastigote radiobinding assay has been developed using organisms labeled with tritiated uracil. These labeled amastigotes resemble freshly isolated unlabeled amastigotes in metabolic activity, buoyant density, morphology, viability and their ability to transform into promastigotes. Organisms routinely incorporate between 5 x 10(-3) and 3 x 10(-2) cpm per amastigote, which allows the detection of as little as 1 x 10(4) amastigotes per assay well. This radiolabeling technique has been used to quantitate the attachment of amastigotes to macrophages adherent to either 13 mm coverslips or to 96 well plates. It can also be used to screen monoclonal antibodies to macrophage surface proteins involved in amastigote binding. Once incorporated, the label remains amastigote associated, even after intact organisms have been internalized by macrophages. It remains parasite associated until the organisms have been degraded by macrophages, at which time label is released into the supernatant. Thus, a small adaptation of the binding assay can be used to compare the intracellular survival of amastigotes in macrophages following various experimental manipulations. This amastigote radiolabeling assay, therefore, represents an important step toward determining the receptors on macrophages involved in amastigote recognition and can also be used to study the degradation of intracellular pathogens by macrophages.