Fig 1.
Comparison of LPS binding in indirect ELISA with different buffers and blocking agents.
FITC-LPS from E. coli serotype O111:B4 was incubated at 1.25 μg/ml in PBS-EDTA (PBS), with or without 0.05% Tween 20 (PBS-TW2) or Triton X-100 (PBS-TTX), in wells coated with polymyxin B (PMX) or lysozyme (LSZ) (1 μg/well), or empty wells (control), which were previously blocked with BSA (A) or OVA (B). Each assay was performed in triplicate. Mean values ± SD are shown in the graph.
Fig 2.
Influence of detergents on LPS binding to casein hydrolysate (hCAS)-blocked or empty plates.
FITC-LPS isolated from E. coli serotype O111:B4 was incubated at 5 μg/ml in PBS-EDTA (control, red line) or in PBS-EDTA supplemented with twofold dilutions of Tween 20 (TW2, A) or Triton X-100 (TTX, B), starting at concentrations of 0.05 and 0.1% (w/v) respectively. Samples were tested in triplicate (mean values ± SD). CMC: critical micelle concentration (0.0074 and 0.0155% w/v, for TW2 and TTX, respectively).
Fig 3.
LPS binding in indirect ELISA.
FITC-LPSs from E. coli serotypes O111:B4 (1.25 μg/ml) and O55:B5 (2.5 μg/ml), diluted in PBS-EDTA with 0.05% Triton X-100, were added to plates coated with several proteins and peptides (1 μg/well) and blocked with BSA. Data represent the mean values ± SD of duplicate samples. HB, hemoglobin; HF1, histone f1 fraction; LF, lactoferrin; LSZ, lysozyme; MEL, melittin; MF6p, synthetic FhHDM-1/MF6p; MYO, myoglobin; P3L, A. simplex peptide: MCQCVQKYGTEFCKKRLA; PMX, polymyxin B; BSA, bovine serum albumin.
Fig 4.
Influence of pH and ionic strength on LPS binding to casein hydrolysate (hCAS)-coated plates.
FITC-LPSs from E. coli serotypes O111:B4 and O55:B5, diluted at 5 μg/ml in PBS-EDTA (pH 7.2) and acetate buffer-EDTA (pH 5.6) prepared 0.15 M, or the same buffers containing 1M NaCl, were added to hCAS-coated plates.
Fig 5.
Competitive hCAS-ELISA performed under neutral conditions.
FITC-LPSs (5 μg/ml) from E. coli serotypes O111:B4 (A) and O55:B5 (B) were preincubated with different concentrations of proteins and peptides (40, 10, 1 and 0.25 μg/ml) in PBS-EDTA (pH 7.2) and added to the wells coated with casein hydrolysate (hCAS). Data are expressed as percentage inhibition of LPS binding to hCAS by the target molecule and are the mean values ±SD of duplicate wells. The average values of the optical density (492 nm) of control wells (without inhibitor) were 0.311 (±0.019, red dashed line) and 0.300 (±0.025, red dashed line) for serotypes O111:B4 and O55:B5, respectively. Differences were considered significant at p <0.05. NS: not significant. BSA, bovine serum albumin; HB, hemoglobin; HF1, histone f1 fraction; LF, lactoferrin; LSZ, lysozyme; MEL, melittin; MF6p, synthetic FhHDM-1/MF6p; MYO, myoglobin; P3L, A. simplex peptide: MCQCVQKYGTEFCKKRLA; PMX, polymyxin B.
Fig 6.
Competitive hCAS-ELISA performed under acidic conditions.
FITC-LPSs (5 μg/ml) from E. coli serotypes O111:B4 (A) and O55:B5 (B) were preincubated with several concentrations of proteins and peptides (40, 10, 1 and 0.25 μg/ml) in acetate buffer-EDTA (pH 5.6) and added to casein hydrolysate (hCAS)-coated wells. Data are expressed as percentage inhibition of LPS binding to hCAS by the target molecule. Results are the means ±SD of duplicate wells. The average values of the optical density (492 nm) of control wells (without inhibitor) were 1.009 (±0.030, red dashed line) and 0.746 (±0.046, red dashed line) for serotypes O111:B4 and O55:B5, respectively. Differences were considered significant at p <0.05. NS: not significant. BSA, bovine serum albumin; HB, hemoglobin; HF1, histone f1 fraction; LF, lactoferrin; LSZ, lysozyme; MEL, melittin; MF6p, synthetic FhHDM-1/MF6p; MYO, myoglobin; P3L, A. simplex peptide: MCQCVQKYGTEFCKKRLA; PMX, polymyxin B.
Fig 7.
FITC-LPSs from E. coli serotypes O111:B4 (A) and O55:B5 (B) were preincubated, at 1.25 and 2.5 μg/ml, respectively, with three different concentrations of proteins and peptides (10, 1 and 0.25 μg/ml) in PBS-EDTA with 0.1% BSA and added to wells containing biotinylated polymyxin B captured by deglycosylated avidin. Results are expressed as percentage inhibition of LPS binding to captured polymyxin B by the target protein/peptide, and are the mean values ±SD for duplicate wells. The average optical densities (492 nm) of control wells (without inhibitor) were 0.904 (±0.042, red dashed line) and 0.842 (±0.036, red dashed line) for serotypes O111:B4 and O55:B5, respectively. Differences were considered significant at p <0.05. NS: not significant. HB, hemoglobin; HF1, histone f1 fraction; LF, lactoferrin; LSZ, lysozyme; MEL, melittin; MF6p, synthetic FhHDM-1/MF6p; MYO, myoglobin; P3L, A. simplex peptide: MCQCVQKYGTEFCKKRLA; PMX, polymyxin B.
Table 1.
LPS-binding properties of proteins and peptides investigated in the study.