Cloning and sequencing of the bacterioferritin gene of Brucella melitensis 16M strain.

The 40 N-terminal amino acids of the 20 kDa antigen A2 from Brucella melitensis were sequenced and showed important similarities with 4 bacterioferrins. A monoclonal antibody raised against this antigen cross-reacted with Escherichia coli bacterioferritin. Hybridization of two sets of degenerate primers with B. melitensis HindIII-digested genomic DNA identified a 3.8 kb fragment. This fragment was shown to contain a bacterioferritin gene (bfr) encoding a 161-amino acid protein. The sequence of the Brucella bacterioferritin is 69% similar to that of E. coli, and many of the ferroxidase centre and haem-ligation residues are conserved.

Latex agglutination assay for human anti-Brucella IgM antibodies.

We report here the development of a homogeneous, easy, precise and rapid anti-Brucella IgM antibody (Ab) latex agglutination assay based on particle counting. The interference of IgG Ab was eliminated by the addition of anti-gamma Fc and free Bru-LPS. Anti-mu Fc mAb enhanced the agglutinating activity of IgM antibodies and improved the sensitivity of the test. The possible interference of rheumatoid factor was eliminated by adding human aggregated IgG. The assay is complete in 45 min, with an interassay variation of 9%. The assay correlates well (r = 0.94) with the accurate but time consuming capture ELISA.

Particle counting immunoassay. IV. The use of F(ab')2 fragments and N epsilon-chloroacetyl lysine N-carboxyanhydride for their coupling to polystyrene latex particles.

Improved performance with the latex agglutination immunoassay, PACIA, is possible when the F(ab')2 fragments of antibody are chemically coupled at their hinge region to a protein-coated latex using a new coupling reagent, N epsilon-chloroacetyl lysine N-carboxyanhydride. This reagent, whether by orientating the complete antibody molecule or its F(ab')2 fragment or by preventing antibody denaturation, increased the sensitivity of the PACIA method. The use of F(ab')2 fragments eliminated most serum interference with the test.

Homogeneous latex immunoassay for thyroid hormone testing. Determination of thyroxine and triiodothyronine.

We describe here two latex immunoassays for total thyroxine (T4) and total triiodothyronine (T3). These homogeneous 60 min assays are quantified by optically counting the monomeric particles remaining after agglutination. When precision is assessed, both methods display coefficients of variation of 3-7% for within-run assays and 4-10% for between-run assays. The accuracy of the methods, as tested by dilution and spike recovery experiments, was found to be satisfactory. Two correlation studies were carried out to compare the present method with leading commercial methods. The coefficients obtained were: r = 0.92 and r = 0.93 with 150 sera for T3, and r = 0.95 (150 sera) and r = 0.93 (108 sera) for T4.

Monoclonal antibodies to Brucella rough lipopolysaccharide: characterization and evaluation of their protective effect against B. abortus.

We characterized 4 monoclonal antibodies (mAb) specific for rough lipopolysaccharide (R-LPS) of Brucella. mAb were selected by enzyme-linked immunosorbent assay (ELISA) on whole B. abortus 45/20 rough cells and R-LPS from B. melitensis B115 rough cells. Specificity was confirmed by immunoblot analysis using R-LPS and smooth LPS (S-LPS) preparations. Anti-R-LPS revealed the low molecular mass R-LPS molecules below 20.1 kDa in the R-LPS and S-LPS preparations as well as the typical A and M patterns in high molecular mass S-LPS molecules (between 21.5 and 66 kDa) in the S-LPS preparations. An O-polysaccharide-specific mAb revealed only high molecular mass S-LPS molecules in the S-LPS preparation. In ELISA the anti-R-LPS mAb bound better on rough than on smooth B. abortus 544 whole cells, and this was confirmed by immunoelectron microscopy. Protective activity of anti-R-LPS mAb of different isotypes was tested on mice and compared with an S-LPS-specific mAb. Only the IgG3 mAb reduced significantly the splenic infection but did not reach the level of protection conferred by the S-LPS-specific mAb.

Protection conferred on mice by combinations of monoclonal antibodies directed against outer-membrane proteins or smooth lipopolysaccharide of Brucella.

The effect of monoclonal antibodies (MAbs) injected alone or in combination on brucella splenic infection in CD-1 mice was tested 7 and 21 days after a challenge with virulent Brucella abortus 544. Passive immunisation of mice with anti-25-27-kDa MAb alone, or mixed with protective anti-16.5 and anti-36-38-kDa MAbs, or with MAbs of the same specificity which were previously demonstrated to have no activity on CD-1 mice, produced a significant reduction of spleen counts of B. abortus (p less than 0.01). Other combinations of MAbs did not reduce splenic infection in comparison with the untreated control group. BALB/c mice were used to test the possible interference of the immune response of CD-1 mice against MAbs that were produced in BALB/c mice. No reduction of splenic infection was shown with anti-25-27- or -36-38-kDa MAbs, whereas anti-lipopolysaccharide (LPS) MAb which was produced in CBA mice was effective. Combination of anti-protein MAbs with the anti-LPS MAb produced only the effect of the anti-LPS MAb at 7 and 21 days after challenge.

Immunogenic properties of Brucella melitensis cell-wall fractions in BALB/c mice.

The immunogenicity of several Brucella melitensis cell-wall (CW) fractions was tested in BALB/c mice. These CW fractions were smooth lipopolysaccharide (S-LPS) fraction from smooth (S) B. melitensis strain 16M, sodium dodecyl sulphate-insoluble (SDS-I) CW fraction from B. melitensis strain 16M (S) undigested or digested with pepsin, and SDS-I CW fraction from rough (R) B. melitensis strain H38. The B. melitensis SDS-I CW fraction contained two major outer-membrane proteins (OMPs) of 25-27 kDa and 31-34 kDa, peptidoglycan (PG) and a small quantity (1.5%) of LPS. One month after immunisation, mice were challenged with virulent B. melitensis strain H38 (S) and Brucella spleen counts were recorded on days 28 and 49 after challenge. Before challenge, as measured by ELISA, the highest antibody responses to S-LPS were observed in mice immunised with SDS-I CW fraction from B. melitensis strain 16M (S), whether digested with pepsin or undigested. All immunised mice, except those immunised with the SDS-I CW fraction from the R strain, showed higher IgG1 than IgG2a antibody responses to S-LPS (IgG1:IgG2a ratio 3.64-7.71). Antibody responses to the 25-27-kDa OMP were very low, with the highest responses in the mice immunised with the SDS-I CW fraction from the R strain. These results indicated that, in BALB/c mice, these CW fractions probably induced Th2-dependent more than Th1-dependent antibody responses.(ABSTRACT TRUNCATED AT 250 WORDS)

Protection conferred on mice by monoclonal antibodies directed against outer-membrane-protein antigens of Brucella.

Twenty-six monoclonal antibodies (MAbs) directed against seven brucella outer-membrane proteins (OMPs) of 10, 16.5, 19, 25-27, 31-34, 36-38 and 89 Kda were screened for passive protection of mice; three MAbs (directed against 16.5, 25-27 and 36-38 Kda) reduced significantly the initial colonisation of the spleen measured 7 days after challenge. Although significant, the reduction in numbers of Brucella organisms in the spleen was low compared with that conferred by MAbs against lipopolysaccharide of smooth specificity (S-LPS). The three most protective MAbs belonged to two isotypes (IgG1 and IgG2a) and were specific for three different OMPs. No relationship between protection and binding of MAbs to the challenge S strain or its rough mutant was observed in the mouse model. The humoral protection depended mainly on antibodies directed against S-LPS, although some MAbs against OMPs had weak protective activity.

Longitudinal study of brucellosis in mice by immunoassay of lipopolysaccharide-related antigens in blood and urine.

Immunoassays based on latex agglutination or enzyme labelling (ELISA) were devised for the detection of lipopolysaccharide (LPS) of Brucella abortus, or its degradation products, in biological fluids of infected mice. The agglutination of latex was measured by counting of the remaining non-agglutinated particles in an automated immunoassay analyser. LPS was assayed by agglutination with antibody-coated latex and by competitive inhibition of agglutination of LPS-coated latex by anti-LPS antiserum. The inhibition system was more sensitive for the detection of degradation products of LPS. Correlation between ELISA and agglutination inhibition immunoassay was excellent (r = 0.96). Degradation of LPS occurred during storage, particularly when the samples contained specific antibodies. It could be prevented by removing cells immediately after collecting blood samples and by heating or alkaline denaturation of plasma. CBA/H mice were infected with various doses [65-(65 x 10(6) cfu] of B. abortus biovar 3 cells and the course of infection followed by immunoassay of LPS-related antigens in serum and urine, and by titration of specific antibodies and non-specific circulating immune complexes. The concentration of LPS degradation products, assayed by the agglutination inhibition assay, was related to the severity of the infection, which was assessed by viable counts of B. abortus in the spleen. A close correlation was observed between the values of antigenaemia, the number of cfu (r = 0.97), and the inoculum size (r = 0.99 at day 28).

Simultaneous expression of smooth and rough phase properties related to lipopolysaccharide in a strain of Brucella melitensis.

Brucella strains exhibit either a rough (R) or a smooth (S) colonial phase identifiable by bacteriological methods. This depends on the biosynthesis and translocation to the surface in S but not in R strains, of the O-polysaccharide chain of the lipopolysaccharide (LPS) molecule. B. melitensis biovar 1 strain EP exhibited simultaneously both S and R characteristics in relation to colonial morphology, agglutination by monospecific anti-M and anti-R sera, activity of bacteriophages lytic for rough Brucella spp. (phage R/C) and for smooth B. melitensis (phage Iz). B. melitensis strain EP expressed fewer O-chains with a similar distribution of molecular weights than B. melitensis reference strain 16M by SDS-PAGE and immunoblotting, but higher amounts of R-LPS. Quantitative determination of S-LPS by a turbidimetric latex inhibition immunoassay with monoclonal antibodies confirmed the limited expression of S-LPS in strain EP. As with other gram-negative bacteria, the phenomenon could be attributed to a deficiency in one step of the biosynthetic assembly of the O-chains.

Antibody response to the 89-kDa outer membrane protein of Brucella in bovine brucellosis.

The antibody response of cattle to the minor 89-kDa outer-membrane protein (OMP) of brucella was measured by indirect ELISA with the purified protein and compared with the antibody response to smooth lipopolysaccharide (S-LPS). Pre-incubating sera with sonicated cell extracts of Escherichia coli prevented the binding of antibodies from uninfected animals to the 89-kDa OMP, suggesting the presence of one or more cross-reactive epitopes on this protein. In cattle infected experimentally with Brucella abortus, the antibody response to the 89-kDa OMP was later and less intense than that to S-LPS. In naturally infected cattle, 68% of animals showing an antibody response to S-LPS also showed an antibody response to the 89-kDa OMP. Results indicate that specific epitopes of the 89-kDa OMP in combination with those of other OMPs could be useful for diagnosis of brucellosis in cattle.

Humoral immunity in mice mediated by monoclonal antibodies against the A and M antigens of Brucella.

All smooth strains of Brucella bear two lipopolysaccharide (LPS) antigens in a ratio that defines the classification of strains in serovars, A (A greater than M), M (M greater than A) and A.M (A = M). Anti-LPS-A monoclonal antibodies (MAb-A) were previously shown to convey protection to mice against B. abortus (A) strain 544, as shown by lower spleen counts than in controls at days 7 and 21 after challenge. Anti-LPS-M monoclonal antibodies (MAb-M) were obtained and tested for M-specificity with LPS from reference strains by ELISA, by agglutination of LPS-coated latex particles, and by inhibition of this agglutination. Antigens A and M of three strains were quantified by a homologous LPS-latex and MAb agglutination inhibition assay. Protection conferred by MAb-A and MAb-M against three strains, B. abortus 544 (A), B. abortus 292 (M) and B. melitensis H38 (M), was tested at equivalent challenge and MAb doses: intravenous challenge was adjusted to give similar infection at day 7; MAb doses were adjusted to the same specific ELISA titre. Under these conditions, MAb-A and MAb-M conferred both early and late protection, as shown at days 7 and 21, against the strains that bore the homologous major antigen, i.e., strain 544 on one hand and strains H38 and 292 on the other. In contrast, MAb directed against the minor antigen of the challenge strain conferred significant protection at day 7 only with strains 544 and H38 and no or inconsistent protection against strain 292, which expressed the lowest amount of minor antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of the major T-cell antigens present in the Brucella melitensis B115 protein preparation, Brucellergene OCB.

Brucellergene is a commercial allergen prepared from Brucella melitensis strain B115 and containing at least 20 cytoplasmic proteins. These proteins were separated by SDS-PAGE. The unstained gel was divided into 18 fractions and proteins were eluted from the gel fractions. The capacity of the separated proteins to elicit delayed-type hypersensitivity (DTH) in infected guinea-pigs or to induce the production of interferon-gamma (IFN-gamma) by blood cells from infected cattle was evaluated. The biological activity of the corresponding protein fractions blotted on to nitrocellulose was measured in a lymphocyte blastogenesis assay. Among the 18 fractions tested, two-spanning the mol. wt ranges 17-22 (fraction 8) and 35-42-kDa (fraction 17)-showed the maximum biological activity in the three tests. These fractions contain two antigens, the Brucella bacterioferritin (BFR) and P39 proteins. Both proteins are good candidates for the detection of cellular immunity to Brucella.

Diagnosis of bovine brucellosis by enzyme immunoassay of milk.

Enzyme-immunoassays using lipopolysaccharide as antigen were developed for the detection of bovine IgG1, IgG2 or IgA Brucella antibodies (Ab) in milk. The results of these tests were compared with those of the milk ring test (MRT) by analyzing 3212 bulk milk samples from farms located in regions where brucellosis is prevalent. Among the 105 herds detected by ELISA and/or MRT, 29 infected herds were detected by ELISA only. The 40 MRT-positive herds were also ELISA positive. Five herds became infected during the study and were detected by ELISA 15 days to 6 months prior to detection by MRT. The ELISA IgG1 titration (IgG1 ELISA) detected 92.8% of the herds found positive in the three ELISA assays. The concomitant use of IgA ELISA raised the sensitivity to 100% but slightly decreased the specificity. The IgG2 ELISA did not improve the diagnosis. The sensitivity of MRT and IgG1 ELISA was compared by testing successive dilutions in negative milk of 110 individual MRT positive milks samples. On average, IgG1 ELISA was 22 times more sensitive than MRT.

[Antibody activity of CSF oligoclonal IgG in infectious neurological diseases. Detection using immunoblotting]. / Activité anticorps des IgG oligoclonales du LCR dans les pathologies neurologiques infectieuses. Détection par "immunoblotting".

The authors describe various applications of an immunoblot technique which allows the qualitative determination of the specific antibody activity of oligoclonal IgG intrathecally synthesized in infectious diseases of the nervous system. After dilution of sera to the same IgG concentration as the paired CSF samples, 10 microliters of both fluids are applied side by side on agarose gel plates and isoelectrically focused. Precipitated IgG or specific IgG antibodies are then blotted onto a nitrocellulose sheet previously coated with either a rabbit anti-IgG antiserum or the antigen under study, respectively. The immunoblot is successively incubated with biotinylated anti-IgG antiserum and with the streptavidin-biotin-peroxidase complex before staining with 4 chloro-1-naphthol. This method was applied to samples from patients with subacute sclerosing panencephalitis, herpetic encephalitis, meningoradiculitis due to Herpes Zoster, neuro-AIDS, neurobrucellosis, meningoradiculitis or encephalomyelitis due to Borrelia burgdorferi, and tuberculous meningitis. In each case, specific oligoclonal IgG antibodies, superimposed or not on a diffuse polyclonal synthesis were detected in the CSF, but not, or more faintly, in the corresponding serum. This was taken as evidence for an intra-thecal synthesis of these antibodies. In contrast, when a "mirror effect" was observed, i.e. similar oligoclonal bands in both serum and CSF after dilution at the same IgG concentration, an intra-thecal synthesis was ruled out.

Diagnosis of bovine brucellosis by skin test: conditions for the test and evaluation of its performance.

Brucellergene OCB (Rhône-Mérieux) was used as an allergen to define the intrinsic parameters of a skin test and to compare its properties with serology for the diagnosis of bovine brucellosis. The skin test was also evaluated for its capacity to solve problems associated with false positive reactions in serological tests. The optimal reading delay for the skin test was 72 hours. The brucellosis allergic reaction was two to three times less intense than the tuberculosis allergic reaction. An increase of 1.1 mm or more in the skin thickness was therefore considered to be an adequate cut-off. The specificity calculated for 1192 brucellosis-free animals (including animals from brucellosis-free herds in which false positive serological reactions had been reported) was 99-83 per cent (95 per cent confidence interval [CI] 99-40 to 99-98 per cent). The sensitivity determined from 27 experimentally infected heifers ranged from 93 per cent (95 per cent CI 76 to 100 per cent) to 78 per cent (95 per cent CI 58 to 91 per cent) when measured respectively one and six months after the infection. Allergic reactions could be detected in vaccinated animals up to four-and-a-half years after the vaccination. On the other hand, no sensitisation was recorded in naïve animals after up to eight monthly injections of the allergen. The skin test gave valuable information, in combination with the serological tests, in both acute and chronic brucellosis. The skin test discriminated brucellosis clearly from false positive serological reactions due to infections with Yersinia enterocolitica O9.

Antibody response to Brucella outer membrane proteins in bovine brucellosis: immunoblot analysis and competitive enzyme-linked immunosorbent assay using monoclonal antibodies.

Sera from Brucella-infected bovines were analyzed by immunoblotting by using sonicated cell extracts of B. melitensis or B. abortus and a competitive enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies against outer membrane proteins (OMPs) with molecular masses of 10, 16.5, 19, 25 to 27, 36 to 38, and 89 kDa. Antibody responses against OMPs were compared with antibody responses against smooth lipopolysaccharide. Immunoblot analysis indicated that the antibody response in infected animals was largely different from one animal to another. The antigens of concern were OMPs with molecular masses of 10, 16.5, 19, 25 to 27, 36 to 38, and 89 kDa and other proteins with molecular masses of between 40 and 80 kDa. According to the specificity of the competitive ELISA, OMPs useful for the detection of infected animals are the OMPs of 10, 16.5, 19, 25 to 27, and 36 to 38 kDa. A competitive ELISA with the anti-89 kDa monoclonal antibody was not specific. Results of the competitive ELISA confirmed the individual variability of the humoral immune response against OMPs. It therefore seems that a combination of several protein antigens is necessary for the development of an immunoassay with a sensitivity comparable to that of the smooth lipopolysaccharide ELISA.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting analysis of smooth-lipopolysaccharide heterogeneity among Brucella biovars related to A and M specificities.

Smooth (S)-lipopolysaccharide (LPS) preparations from reference and field strains of several biovars of Brucella abortus, B. melitensis, and B. suis were prepared by (i) the hot phenol-water method, (ii) hot sodium dodecyl sulfate extraction and proteinase K digestion, or (iii) dimethyl sulfoxide extraction. These S-LPS-enriched fractions were further analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining after periodate oxidation. Immunoblots were developed by using either monoclonal antibodies specific for Brucella A or M antigens or polyclonal polyspecific or monospecific sera from rabbits, cattle, and goats. The specificity of monoclonal antibodies reactive with Brucella unique (A or M) epitopes was demonstrated by enzyme-linked immunosorbent assay, LPS latex agglutination, or agglutination inhibition. The most-represented subunits of S-LPS ranged in Mr from 30,000 to 70,000 relative to marker proteins. According to A or M immunodominance, two sodium dodecyl sulfate-polyacrylamide gel electrophoresis banding patterns were clearly distinguished among biovars, whatever the fraction tested: a close succession of regularly spaced narrow bands for A greater than M strains and regularly spaced triplets of bands including either (i) a first thin band followed by two thick bands for B. abortus M greater than A strains or (ii) one thick band between two thin bands for B. melitensis or B. suis M greater than A strains. Moreover, A and M specificities were reaffirmed by sandwich enzyme immunoassay and latex agglutination inhibition with monoclonal antibodies and polyclonal sera.