Immunochemical identification of Brucella abortus lipopolysaccharide epitopes

Abstract

Sera from Brucella abortus-infected and -vaccinated bovines recognized four lipopolysaccharide (LPS) determinants: two in the O-polysaccharide (A and C), one in the core oligosaccharide from rough Brucella LPS (R), and one in lipid A (LA). From 46 different hybridomas secreting monoclonal antibodies (MAbs) against various LPS moieties, 9 different specificities were identified. Two epitopes, A and C/Y, were present in the O-polysaccharide. Two epitopes were found in the core oligosaccharide (R1 and R2) of rough Brucella LPS. MAbs against R1 and R2 epitopes reacted against LPS from different rough Brucella species; however, MAbs directed to the R2 epitope also reacted against enterobacterial LPS from deep rough mutants. Three epitopes (LA1, LA2, and LA3) were located in the lipid A backbone. Different sets of MAbs recognized two epitopes in the lipid A-associated outer membrane protein (LAOmp3-1 and LAOmp3-2). LPS preparations from smooth brucellae had small amounts of rough-type LPS. Although LPS from rough brucellae did not show smooth-type LPS in western blots (immunoblots), two hybridomas generated from mice immunized with rough B. abortus produced antibodies against smooth B. abortus LPS. Results are discussed in relation to the structure and function of B. abortus LPS and to previous findings on the epitopic density of the molecule.

Characterization of Brucella abortus lipopolysaccharide macrodomains as mega rafts

“…The monoclonal antibody (mAb) (Baps C/Y) directed against B. abortus LPS C/Y epitope coupled to peroxidase and antiserum from infected cows were previously described (Rojas et al, 1994). The antiserum from infected cows was used to label Y. enterocolitica 0:9 LPS (same O-chain as B. abortus; for review see Moriyon, 2004).…”

A two‐component regulatory system playing a critical role in plant pathogens and endosymbionts is present in Brucella abortus and controls cell invasion and virulence

“…For quantification analyses, extracellular bacteria were detected by incubating the cells for 30 min with a serum (diluted 1:10 000 in 10% mouse serum in PBS) from a B. abortus-infected cow (Rojas et al, 1994) and by revealing the antibodies with Texas red-conjugated goat anti-cow IgG antibodies (Immunotech). Intracellular bacteria were detected by permeabilization of cells with 0.05% saponin (Sigma), incubation for 30 min with serum (at 1:5000 in 10% mouse serum in PBS) from a B. abortus-infected rabbit (Rojas et al, 1994) revealed by FITCconjugated donkey anti-rabbit IgG (Immunotech). For confocal studies regarding the intracellular localization of internalized bacteria, two protocols were used.…”

“…The presence of cathepsin D (a well-known marker for lysosomes) was detected with rabbit anti-cathepsin D antibodies (Meresse et al, 1995) revealed by FITC-conjugated donkey anti-rabbit IgG. For triple immunofluorescence analysis, extracellular bacteria were detected by incubating the cells for 30 min with a mouse monoclonal anti-Brucella antibody (diluted 1:100 in 10% horse serum in PBS) (Rojas et al, 1994) revealed by cyanin 5-conjugated donkey anti-mouse IgG (Immunotech). Cells were then permeabilized with 0.05% saponin and intracellular bacteria were detected by incubating cells for 30 min with the cow anti-Brucella serum revealed by Texas red-conjugated goat anti-cow IgG.…”

Comparison of the Antibody Response in Adult Cattle Against Different Epitopes of Brucella abortus Lipopolysaccharide

“…Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) for the separation of LPS and lipid A was carried out in 12 and 15 % discontinuous gels in a Mini Protean II unit (Bio‐Rad, Richmond, CA, USA). The transfer of the antigens to nitrocellulose membranes was performed as previously described (Rojas et al., 1994). Briefly, the gels were electrotransferred in a semidry transblot cell (Bio‐Rad) for 15 min at 15 V. After blocking overnight at 4°C with PBS–0.05 % Tween 20–2 % casein, the membranes were incubated for 2 h at 37°C with sera diluted 1:500, followed by washing as above.…”

“…Because LPS is the major antigenic component of the outer membrane in Brucella , a large proportion of the humoral response of cattle is directed to LPS, particularly to its more exposed region, the O‐chain. However, little is known about the recognition of ‘deeper’ domains in the LPS molecule, such as the core oligosaccharide and lipid A (Rojas et al., 1994). In order to investigate the specificity of bovine sera to the different domains of B. abortus LPS, several immunochemical tests were performed with positive sera, and the results were evaluated considering previous findings on the epitopic distribution of the molecule.…”