FIG 4.

Antibody-mediated ligand-blocking assays for second-generation loop-specific antibodies. Whole, iron-stressed gonococci or E. coli cells expressing recombinant TbpA were applied to a nitrocellulose membrane and allowed to dry. (A) To determine loop antibody-blocking capability, blots were blocked with antibodies and then HRP-Tf was applied. For both blots, HRP was detected with Opti-4CN. Lane – contained no antibody and therefore represents the maximal amount of hTf bound. (B) A similar assay was performed with whole cells in a microtiter dish for an ELISA. 3,3′,5,5′-Tetramethylbenzidine (Thermo) was used as the peroxidase substrate, and the OD₄₂₀ was measured. The positive control is the condition without antibody (No Ab), and the negative control is incubation with unlabeled hTf (Tf). The data represent specific binding obtained by subtracting the values obtained with strains without Tf receptors from those obtained with the experimental strains. Significant differences are noted (*, P < 0.01; #, P < 0.05). Statistics were calculated with the Student t test.