Table 2.
Binding of MAb-BA5 or anti-rV10 antibodies to polymorphic LcrV proteins
| ELISA antigen | Mean IgG concn ± SEM bound to antigen |
|
|---|---|---|
| Anti-rV10a | MAb-BA5b | |
| LcrVD27 | 2.61 ± 0.22 | 61.94 ±1.04 |
| LcrVW22703 | 2.71 ± 0.09 | 63.69 ± 2.89 |
| LcrVWA-314 | 3.31 ± 0.51 | 69.73 ± 3.16 |
The average amount of anti-rV10 IgG antibody (in mg/ml) in undiluted rabbit anti-rV10 serum that bound to purified recombinant LcrV from Y. pestis KIM D27 (LcrVD27), Y. enterocolitica W22703 (LcrVW22703), or Y. enterocolitica WA-314 (LcrVWA-314) immobilized in 96-well plates was calculated from three independent experimental determinations. Samples were developed using a BD OptElA TMB substrate reagent kit (BD Biosciences), the absorbance of 450-nm light was measured, and the data were analyzed using GraphPad Prism. The absorbance values of control reactions lacking primary antibodies were subtracted from the absorbance data obtained with primary antibodies to correct for nonspecific binding. Two-tailed unpaired Student's t test was used to examine differences in antibody binding, which were judged not to be significant (LcrVD27 versus LcrVW22703, P = 0.6950; LcrVD27 versus LcrVWA-314, P = 0.2741; and LcrVW22703 versus LcrVWA-314, P = 0.3096). The experiment was repeated with three different dilutions of anti-rV10 serum (1:25,000, 1:35,000, and 1:45,000). Absorbance values were measured for each sample and antibody dilution, which generated results similar to those obtained with undiluted serum. For example, the average amounts of anti-rV10 IgG antibody (in ng/ml) in 1:35,000-diluted rabbit anti-rV10 serum were 92.95 ± 38.91 for LcrVD27, 84.01 ± 28.89 for LcrVW22703, and 97.01 ± 23.98 for LcrVWA-314 (LcrVD27 versus LcrVW22703, P = 0.7090; LcrVD27 versus LcrVWA-314, P = 0.5877; and LcrVW22703 versus LcrVWA-314, P = 0.8105).
The average amount of MAb-BA5 antibody (in ng/ml) bound to purified LcrVD27, LcrVW22703, or LcrVWA-314 immobilized in 96-well plates was calculated from three independent experimental determinations as described above. Two-tailed unpaired Student's t test was used to examine differences in antibody binding, which were judged not to be significant (LcrVD27 versus LcrVW22703, P = 0.5992; LcrVD27 versus LcrVWA-314, P = 0.0795; and LcrVW22703 versus LcrVWA-314, P = 0.2310).