The CMG2 ELISA for evaluating inhibitors of the binding of anthrax toxin protective antigen to its receptor

IntroductionAnthrax toxin comprises a protective antigen (PA) of MW 83 kDa, a lethal factor (LF) and an edema factor (EF). Upon binding to its receptor on cell surfaces, PA83 is enzymatically cleaved to a 63 kDa product (PA63), followed by binding of LF or EF, receptor-mediated internalisation of these factors, and production of their toxic effects. The high-affinity binding of PA83 to its receptor is essential for the intoxication process. To study the interaction between the PA and its receptor, and inhibition of the binding, an enzyme-linked immunosorbent assay (ELISA) was developed.MethodsOne of the two known anthrax toxin receptors (capillary morphogenesis factor 2; CMG2) was adsorbed onto wells of a 96-well plate. Either PA83 or PA63 was then added to the receptor-coated wells, followed by sequential addition of anti-PA antibody, anti-species antibody-enzyme conjugate, and enzyme substrate at appropriate time intervals.ResultsBest results were obtained by overnight incubation of CMG2 in PBS at 4 °C. CMG2 was used at 1 μg/ml because of the cost of the commercial product. The rate of change of absorbance was low, and was measured over 3 h to obtain accurate results. The assay results increased almost linearly with CMG2 concentration to 10 μg/ml. PA83 was also used at 1 μg/ml, but the assay values reached a plateau at approx. 10 μg/ml. Binding was divalent cation-dependent, almost irreversible, and free CMG2 was a potent inhibitor of binding (I50 in the nM range). Binding of PA63 was similar to that of PA83.DiscussionThe high-affinity binding and divalent cation dependence confirm the validity of the assay as a model for toxin-receptor binding in vivo and as a means of evaluating toxin-receptor binding and inhibitors of the binding. Attempts to use crude lysates of J774A.1 cells or von Willebrand factor as an alternative source of anthrax toxin receptor were not successful.