The CMG2 ELISA for evaluating inhibitors of the binding of anthrax toxin protective antigen to its receptor.
BACKGROUND
Anthrax 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, PA(83) is enzymatically cleaved to a 63 kDa product (PA(63)), followed by binding of LF or EF, receptor-mediated internalisation of these factors, and production of their toxic effects. The high-affinity binding of PA(83) 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.


METHODS
One of the two known anthrax toxin receptors (capillary morphogenesis factor 2; CMG2) was adsorbed onto wells of a 96-well plate. Either PA(83) or PA(63) 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.


RESULTS
Best results were obtained by overnight incubation of CMG2 in PBS at 4 degrees C. CMG2 was used at 1 microg/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 microg/ml. PA(83) was also used at 1 microg/ml, but the assay values reached a plateau at approx. 10 microg/ml. Binding was divalent cation-dependent, almost irreversible, and free CMG2 was a potent inhibitor of binding (I(50) in the nM range). Binding of PA(63) was similar to that of PA(83).


CONCLUSIONS
The 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.
Dawson
Raymond M
RM
DSTO Melbourne, Defence Science and Technology Organisation, Melbourne, VIC, Australia. ray.dawson@dsto.defence.gov.au
D000818:Animals; D000942:Antigens, Bacterial / Q000276:immunology / Q000494:pharmacology*; D001427:Bacterial Toxins / Q000276:immunology / Q000494:pharmacology*; D001665:Binding Sites / Q000187:drug effects; D002021:Buffers; D002118:Calcium / Q000737:chemistry / Q000494:pharmacology; D002412:Cations / Q000737:chemistry / Q000494:pharmacology; D002413:Cations, Divalent / Q000737:chemistry / Q000494:pharmacology; D002457:Cell Extracts / Q000737:chemistry; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000737:chemistry / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000737:chemistry* / Q000187:drug effects / Q000276:immunology; D011485:Protein Binding; D018000:Receptors, Peptide / Q000187:drug effects* / Q000276:immunology; D013696:Temperature; D013997:Time Factors; D014841:von Willebrand Factor / Q000737:chemistry / Q000378:metabolism
C447150:ANTXR2 protein, human; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D002021:Buffers; D002412:Cations; D002413:Cations, Divalent; D002457:Cell Extracts; D008565:Membrane Proteins; D018000:Receptors, Peptide; C030325:anthrax toxin; C070309:anthrax toxin receptors; D014841:von Willebrand Factor; D004492:Edetic Acid; D002118:Calcium