Figure 1.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis and western immunoblot analysis of purified and conjugated Brucella melitensis lipopolysaccharide.
A. B. melitensis LPS was separated on a 4–12% Tris Glycine SDS-polyacrylamide gradient gel. The gel was stained for protein contamination of the lipopolysaccharide preparation with Coomassie Blue and periodic acid silver stain for LPS detection. Molecular mass (kDa) indicated at right of the panel. B. Keyhole Limpet hemocyanin (KLH) conjugated to B. melitensis LPS. B. melitenis LPS was oxidized with sodium periodate and linked to succinimidyl 4-hydrazinonicotinate (SANH)-modified KLH. Escherichia coli O55:B5 LPS (control), B. melitensis LPS alone and KLH-conjugated- KLH-conjugated B. melitensis LPS. The samples were electrophoresed on a 10% Bis-Tris polyacrylamide gel and stained with silver stain for LPS detection. Molecular mass (kDa) indicated at left of panel. C. Western immunoblot of anti-LPS monoclonal antibodies. Purified B. melitensis LPS was subjected to SDS-PAGE, transferred to nitrocellulose, and strips were probed individually with hybridoma supernatants (mAbs: 5D1, 2F1, 5H1, 2D2, 2D1 and 5E1).
Figure 2.
LPS immunoblot probed with monoclonal antibody-containing supernatants to determine cross reactivity to the main pathogenic species of Brucella and potentially cross-reactive organisms.
Upper left, silver stain for LPS. Remaining panels reacted with mAbs as indicated.
Figure 3.
ELISA-determined reactivity of a panel of monoclonal antibodies raised against KLH-conjugated B. melitensis 16M lipopolysaccharide against B. melitensis 16M and B. abortus 2308 native whole cell antigen.
Table 1.
Specificity of anti-Brucella melitensis 16M lipopolysaccharide monoclonal antibodies by indirect immunofluorescence assay.
Figure 4.
Sensitivity of B. melitensis LPS antigen detection by capture ELISA.
Monoclonal antibody 2D1 to capture antigen and biotinylated mAb 2E8 used to detect LPS in the human serum samples that were spiked with known amount of B. melitensis LPS. The average absorption value obtained from the duplicate wells were plotted in the Y-axis, and the mean value along with standard deviation in for each groups were represented as bar. Positive result defined as OD450 ≥3.0 standard deviations from mean value of negative control group (0 ng LPS). Capture ELISA using Escherichia coli 055:B5 LPS (10 ng) and Leptospira licerasiae LPS (10 ng) did not yield a signal above background and not shown in the figure.
Figure 5.
Detection of B. melitensis LPS in serum of experimentally infected mice.
A. BALB/c mice were infected with B. melitensis 16M. Sera were tested for the presence of LPS at weeks 1, 3 and 8 using the 2D1-2E8 capture ELISA. B. melitensis LPS was detected above background at weeks 1 (p≤0.003) and 3 (p≤0.004) but not at week 8. B. Colony forming units (CFU) in liver and spleen at each time point are indicated.
Figure 6.
Detection of B. melitensis LPS in human brucellosis patients from Peru.
Sera from patients presenting with a suspicion of brucellosis were either confirmed to have B. melitensis brucellosis by blood culture and serological positive (C+RB+); serologically positive but blood culture negative (C- RB+) and both serologically and blood culture negative samples (C- and RB-). Three negative control serum obtained from US blood bank (-ve USA) were also included in the assay. The average absorption value of the samples obtained by capture ELISA were plotted, and the mean value in each group are shown as bar. The signal was significantly higher in the blood culture positive than in the blood culture negative/serologically negative group (p≤0.0004) and blood culture negative/seropositive group (p≤0.001). The lower table shows the comparative result by serological tests (Rose Bengal test and Brucella IgG ELISA Kit, GenWay Biotech Inc, San Diego, CA) and the capture ELISA, which shows 70% (7/10) of the culture positive samples were detected with higher LPS antigenemia (OD450≥3.0 standard deviations from the mean value of six Peruvian C−S− samples).
Table 2.
Clinical features and diagnostic results of Peruvian patient groups.