Figure 3. The L2 and L5 IgGs React Selectively with TEM8 and Block Binding and Toxicity of Anthrax Toxin Proteins.
(A) L2 and L5 antibodies were used for flow cytometry staining of 293 cells stably transfected with mouse Tem8 (293-mTEM8) or a FLAG-tagged human TEM8 (293-Flag-hTEM8).
(B) L2 and L5 were used for flow cytometry staining of CHO/PR230 (CHO) cells (an anthrax toxin receptor-deficient cell line) that had been stably transfected with human TEM8 (CHO-TEM8) or human CMG2 (CHO-CMG2). FITC-labeled protective antigen (PA-FITC), which binds both TEM8 and CMG2, was used as a positive control.
(C) Western blot analysis was used to evaluate the expression of TEM8 and CMG2 in stably transfected CHO-TEM8 and CHO-CMG2 cells.
(D) L2 antibodies were used for cell-surface Immunofluorescence labeling of CHO and CHO-TEM8 cells.
(E) The ability of L2 and L5 antibodies to block binding of PA-FITC to CHO-TEM8 cells was measured by flow cytometry.
(F) The viability of CHO and CHO-TEM8 cells was evaluated 48 hours post-treatment with lethal toxin.
(G) The ability of L2 and L5 antibodies to protect cells from toxicity following treatment with 1µg of lethal toxin was evaluated. In this assay the EC50 for L2 and L5 were 1.9nM and 16.6nM respectively. Values in (F) and (G) represent mean ± SE. See also Figure S2.