Characterization of Periplasmic Protein BP26 Epitopes of Brucella melitensis Reacting with Murine Monoclonal and Sheep Antibodies

More than 35,000 new cases of human brucellosis were reported in 2010 by the Chinese Center for Disease Control and Prevention. An attenuated B. melitensis vaccine M5-90 is currently used for vaccination of sheep and goats in China. In the study, a periplasmic protein BP26 from M5-90 was characterized for its epitope reactivity with mouse monoclonal and sheep antibodies. A total of 29 monoclonal antibodies (mAbs) against recombinant BP26 (rBP26) were produced, which were tested for reactivity with a panel of BP26 peptides, three truncated rBP26 and native BP26 containing membrane protein extracts (NMP) of B. melitensis M5-90 in ELISA and Western-Blot. The linear, semi-conformational and conformational epitopes from native BP26 were identified. Two linear epitopes recognized by mAbs were revealed by 28 of 16mer overlapping peptides, which were accurately mapped as the core motif of amino acid residues 93DRDLQTGGI101 (position 93 to 101) or residues 104QPIYVYPD111, respectively. The reactivity of linear epitope peptides, rBP26 and NMP was tested with 137 sheep sera by ELISAs, of which the two linear epitopes had 65–70% reactivity and NMP 90% consistent with the results of a combination of two standard serological tests. The results were helpful for evaluating the reactivity of BP26 antigen in M5-90

Evaluation for reactivity of peptides and proteins of <i>B. melitensis</i> to sheep sera in ELISAs.

<p>Reactivity of epitope peptides and proteins of <i>B. melitensis</i> to 137 sheep sera were tested, and the predictive values in ELISAs were evaluated in line with the combined SAT and RBPT. PPV, positive predictive value; NPV, negative predictive value. Sensitivity = a/(a+c); Specificity = d/(b+d); agreement = (a+d)/(a+b+c+d); PPV = a/(a+b); NPV = d/(c+d).</p

Peptides derived from BP26 of <i>B. melitensis</i> M5-90.

<p>Four groups of synthesized peptides are listed. Group A, 27 of 16mer peptides and 2 of 12 or 11mer peptides with 7mer overlapping deriving from BP26 of <i>B. melitensis</i> M5-90. Group B, 12 of 9mer peptides with 8mer overlapping shortening from P11 or P12. Group C, The mutated peptides basing on P11 and P12′, in which two site mutations are presented in underline bold letters. Group D, an HCV NS3 peptide deriving from an HCV 1b strain in the laboratory. A single letter was used for encoding amino acid (aa) sequence and aa position was numbered from BP26 protein.</p

Reactivity of sheep sera to linear epitope peptides, rBP26 and NMP of <i>B. melitensis</i> in ELISAs.

<p>The receiver operating characteristic (ROC) curves were plotted using the SPSS software version 13.0. According to optimal cutoff values by the highest sum of sensitivity and specificity, the areas under ROC curves for antibody responses in sheep sera by ELISAs were compared with the results by a combination of two standard serological tests. <i>P</i> value was calculated for comparing the differences between areas under ROC curve and reference (0.5) by the Kolmogorov-Smirnov Z analysis.</p

Classification of mAbs reactive to the epitopes of native BP26.

<p>MAbs were individually tested to react with the synthetic peptides by Peptide-ELISA, denatured and non-denatured native BP26 containing membrane protein extracts of M5-90 (NMP) by Western-Blot or Dot-ELISA, respectively. According to the nature of antigen, the epitopes recognized by mAbs were classified into three statuses of linear (L), semi-conformational (SC) and conformational (C) epitopes. Un-classified indicates artificial epitopes of recombinant BP26.</p

Competitive binding of mAb to the 16mer peptide inhibited by 9mer or mutated peptides in Peptide-ELISA.

<p>(A) P11 and mAb 3H5; (B) P12 and mAb 2A4; (C) P12 and mAb 5A5. A volume of 50 µl of supernatants of mAb 2A4, 3H5 or 5A5 cell cultures was pre-mixed with 50 µl of each 9mer peptide dilution (P1101 to P1106 or P1201 to P1206 and control peptides) at concentration of 0, 2, 4, 8, 16 and 32 µg/ml. A volume of 100 µl of the mAb and peptide mixture was added to the 16mer peptides P11 or P12 coated wells. P11 or P12 was used as an inhibitive (positive) control (PC), respectively. One HCV NS3 peptide was used a non-inhibitive (negative) control (NC). (D) Binding of mAbs to P11 and P12′ compared with their mutated peptides MutP11 and MutP12′.</p