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. 2017 Dec 6;6:e30051. doi: 10.7554/eLife.30051

Figure 4. Anti-LigB mAb binding characterization.

(A) A representative example of domain-level epitope ELISA-based mapping for anti-LigB mAbs (full dataset Figure 4—figure supplement 1). The mean ±S.D. for three trials is displayed. (B) Flow cytometry was used to measure the ability of anti-LigB mAbs to bind directly to live Leptospira (Figure 4—figure supplement 2B). The representative fluorescence emission displays the relative binding of fluorescently-labelled secondary anti-mouse IgG antibodies to PBS-treated cells (negative control, right panel) and to mAb C5-treated cells (left panel).

Figure 4.

Figure 4—figure supplement 1. Domain-level epitope mapping of anti-LigB mAb library determined by ELISA.

Figure 4—figure supplement 1.

An array of double and single Ig-like domains from (A) LigB1-7 (1 μg/well) or (B) LigB7-12 (1 μg/well) and BSA (negative control) were coated on the microtiter plates for epitope mapping of mAbs. A fixed concentration (1 μM) of individual mAbs was applied to the LigB-coated wells. The binding levels of the mAbs were measured by rabbit anti-mouse IgG antibody conjugated with HRP (1:5000). Colors correspond to relative domain position. All experiments were conducted in three trials, the mean ±S.D. of which were shown in bar charts. Positive binding domains were identified by comparing with BSA and statistically significant (t-test; p<0.05) differences were marked by an asterisk and indicated in Table 1.
Figure 4—figure supplement 2. Cell surface binding of anti-LigB mAbs.

Figure 4—figure supplement 2.

(A) Flow cytometry was used to generate light scatter plots to obtain the mean fluorescence index (MFI) of FITC-positive cells from two negative control experiments (positive experiment displayed in Figure 4B). The mAb C22 (no binding to LigB1-7) binds poorly to live Leptospira interrogans Pomona (upper panel). The anti-LigB1-7 mAb C5 binds poorly to live non-pathogenic Leptospira biflexa, which does not express LigA or LigB proteins (lower panel). (B) The mean FITC-positive (MFI) count was obtained for a set of mAbs from libraries C and V. Each value represents the mean ± S.D. from two individual trials of two replicates. Statistically significant (t-test; p<0.05) differences were calculated from the comparisons between bacteria bound mAbs and corresponding weakly bound mAbs (*; C22, upper panel; V33, lower panel) or PBS control (#; both panels).