Search

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.

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.

Previously, four epitope specificities on the O chain of Brucella species were reported: M, A, C, and C/Y. In this work, according to monoclonal antibody binding to smooth lipopolysaccharides of Yersinia enterocolitica 0:9, Brucella abortus W99 (A-dominant strain), and B. melitensis Rev1 (M-dominant strain), seven O-chain epitope specificities were defined: M, A, C (M > A), C (M = A), C/Y (M > A), C/Y (M = A) and C/Y (A > M). Competitive binding assays between these monoclonal antibodies suggested that these different epitopes are probably overlapping structures.

Monoclonal antibodies and polyclonal antisera recognizing a 39-kDa protein (P39) of brucellin, a cytoplasmic extract from Brucella melitensis rough strain B115, were produced. The P39 was purified by anion-exchange chromatography. Eleven of fourteen Brucella-infected cows whose infections had been detected by the delayed-type hypersensitivity (DTH) test with brucellergen also developed a DTH reaction when purified P39 was used as the trigger. The T-cell proliferative responses to P39 of peripheral blood lymphocytes from Brucella-infected cows were also positive. None of the animals infected with other bacterial species that are presumed to induce immunological cross-reactions with Brucella spp. reacted to P39, either in DTH tests or in lymphocyte proliferation assays. A lambda gt11 genomic library of Brucella abortus was screened with a monoclonal antibody specific for P39, and the gene coding for this protein was subsequently isolated. The nucleotide sequence of the P39 gene was determined, and the deduced amino acid sequence is in accordance with the sequence of an internal peptide isolated from P39.

The reactivity of monoclonal antibody (MAb) 12G12 was analyzed in regard to the main biovars of Brucella species and some members of the families Enterobacteriaceae and Vibrionaceae which present serological cross-reactions with the smooth lipopolysaccharide (S-LPS) of Brucella species. This MAb was strictly directed against the common specific epitope of the Brucella S-LPS. It recognized all of the smooth Brucella strains and biovars except B. suis biovar 2. In order to improve the specificity of the serological diagnosis of brucellosis, a competitive enzyme-linked immunosorbent assay (cELISA) was developed with the horseradish peroxidase-conjugated MAbs 12G12 and S-LPS of B. melitensis Rev1. The specificity of the cELISA was analyzed with 936 serum samples from healthy cattle. The assay was evaluated with sera from heifers (n = 18) experimentally infected with B. abortus 544. After infection, the performance of the cELISA was in agreement with those of the complement fixation test and the rose Bengal plate test. Finally, the specificity of the assay was also evaluated in regard to false-positive serological reactions by using sera from heifers experimentally infected with Yersinia enterocolitica 0:9 (n = 4) and with field sera presenting false-positive reactions (n = 74). The specificity of the cELISA was greater than the specificities of the complement fixation test and the rose Bengal plate test. Indeed, the new assay detected only 31 of the 101 false-positive serum samples detected by at least one serological test.

Screening of a Brucella abortus genomic library with two sets of monoclonal antibodies allowed the isolation of the genes corresponding to two minor outer membrane proteins (OMP10 and OMP19) found in this bacterial species. Sequence analysis of the omp10 gene revealed an open reading frame capable of encoding a protein of 126 amino acids. The nucleotide sequence of the insert producing the OMP19 protein contains two overlapping open reading frames, the largest of which (177 codons) was shown to encode the protein of interest. Analysis of the N-terminal sequences of both putative proteins revealed features of a bacterial signal peptide, and homology to the bacterial lipoprotein processing sequence was also observed. Immunoblotting with monoclonal antibodies specific for OMP10 or OMP19 showed that both proteins are present in the 34 Brucella strains tested, representing all six Brucella species and all their biovars. The OMP19 detected in the five Brucella ovis strains examined migrated at an apparent molecular weight that is slightly higher than those of the other Brucella species, confirming the divergence of B. ovis from these species. OMP10 and OMP19 were produced in recombinant Escherichia coli and purified to homogeneity for serological analysis. A large fraction of sera from sheep naturally infected with Brucella melitensis were reactive with these proteins in an enzyme-linked immunosorbent assay, whereas sera from B. abortus-infected cattle were almost completely unreactive in this assay.

The cloning and sequencing of the Brucella abortus major 25-kDa outer membrane protein (OMP) is reported. The 25-kDa (group 3) OMP has been proposed, on the basis of amino acid composition, to be the counterpart of OmpA (D. R. Verstraete, M. T. Creasy, N. T. Caveney, C. L. Baldwin, M. W. Blab, and A. J. Winter, Infect. Immun. 35:979-989, 1982). However, the amino acid sequence predicted from the cloned B. abortus gene did not reveal significant homology with either OmpA sequences from different members of the family Enterobacteriaceae or other known protein sequences.

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.

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)

Recombinant lambda gt11 phages were selected by screening a genomic library of Brucella abortus DNA with monoclonal antibodies specific for a 16.5-kDa Brucella outer membrane protein (Omp16). The corresponding gene, named pal, was subcloned on a 0.7-kb AluI fragment. Immunoblotting confirmed the expression of a recombinant Omp16 in the transformants. DNA sequence analysis revealed an open reading frame of 168 codons. The deduced amino acid sequence agrees with an internal peptide sequence of native Omp16 and contains a potential lipoprotein signal peptide cleavage site, giving rise to a predicted mature protein of 144 amino acids. The predicted sequence of Omp16 also shows a remarkable degree of similarity to the sequences of three peptidoglycan-associated bacterial lipoproteins. In immunoblotting with a monoclonal antibody specific for Omp16, we demonstrated that Omp16 was expressed in the 34 Brucella strains tested, representing all six species and known biovars.

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.

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.

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.

Twenty-two monoclonal antibodies (mAbs) specific for smooth lipopolysaccharide (S-LPS) were generated by fusion of spleen cells from mice infected with the rough Brucella melitensis strain B115 with the NSO myeloma. According to reactivity in enzyme-linked immunosorbent assay (ELISA) with O-polysaccharide (O-PS) and absence of reactivity with rough lipopolysaccharide (R-LPS), it was postulated that these mAbs recognized epitopes present on the O-PS. Most of the mAbs reacted equally well in ELISA and immunoblotting with S-LPS types of Brucella A and M dominant strains and were designated as specific for common (C) epitopes. Three mAbs were highly specific for M dominant S-LPS. All these mAbs, in contrast to a mAb specific for the A epitope, showed little or no cross-reactivity with Yersinia enterocolitica O:9 S-LPS. S-LPS of B. melitensis B115 was extracted and analysed by immunoblotting and ELISA with mAbs specific for A, M and C epitopes. Reactivity of the mAbs with this S-LPS was compared to reactivity with S-LPS of A and M dominant smooth Brucella strains. The results suggest that S-LPS of B. melitensis B115 bears mainly C epitopes and a few M epitopes. The very weak reactivity of this S-LPS with the mAb specific for the A epitope and the fact that the mAbs specific for C and M epitopes showed little or no cross-reactivity with Y. enterocolitica O:9 S-LPS suggest that O-PS from this rough strain could be used to distinguish Y. enterocolitica O:9 infection from Brucella infection.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

A monoclonal antibody (3D6) was produced which reacted only with Brucella sonicated cell extracts that had been lysozyme-treated after sonication. The monoclonal antibody (mAb) reacted with the three major outer-membrane proteins (OMPs) of B. melitensis B115 in Western blots. A large number of reactive bands ranging from 12 to 43 kDa were present in lysozyme-treated Escherichia coli and Yersinia enterocolitica sonicated cell extracts. In a latex agglutination inhibition immunoassay, mAb 3D6 showed better reactivity with purified peptidoglycan (PG) of B. melitensis B115 than with that of Escherichia coli. This mAb was also used in immunogold electron microscopy with whole Brucella cells and sections. No binding was observed on whole cells and immunogold labelling in sections was observed close to the outer membrane, in the periplasmic space and in the cytoplasm. These findings indicate that mAb 3D6 is specific for PG subunits. Immunoblot analysis of B. melitensis B115 rough sonicated cell extracts after SDS-PAGE, with or without lysozyme treatment, was performed using mAbs specific for Brucella OMPs of molecular masses of 10, 16.5, 19, 25-27, 31-34, 36-38 and 89 kDa, for PG and for rough lipopolysaccharide (R-LPS) and smooth lipopolysaccharide (S-LPS). mAbs specific for the 25-27, 31-34 and 36-38 kDa OMPs reacted with three to six bands. All of them except the band of lowest molecular mass reacted with the PG-specific mAb and not with R-LPS- and S-LPS-specific mAbs.(ABSTRACT TRUNCATED AT 250 WORDS)

Spleen cells from mice infected with the rough Brucella melitensis strain B115 were fused with NSO myeloma cells. Hybridoma supernatants were screened in ELISA with cell walls (CW), sonicated cell extracts (CE) and rough lipopolysaccharide (R-LPS) of B. melitensis strain B115 and whole B. melitensis B115 cells. Surprisingly, 22 monoclonal antibodies (mAbs) reacting in ELISA with both CW and CE but not with R-LPS and bacterial cells were shown by immunoblot analysis and ELISA to react with smooth lipopolysaccharide (S-LPS). These mAbs also reacted in ELISA with O polysaccharides (OPS) from the smooth Brucella abortus strain 99 and the smooth B. melitensis strain 16M and thus recognize epitopes present on the O-chain. Proteinase K LPS preparations from B. melitensis B115 analysed by immunoblotting with one mAb (12G12) recognizing S-LPS of both A and M specificity displayed the typical S-LPS high-molecular-mass ladder pattern but no S-LPS was detected in the phenol/water/chloroform/light petroleum LPS preparation of the same strain. mAb 12G12, specific for S-LPS, and a mAb (A68/03F03/D05) specific for R-LPS were used to localize the O-chain and R-LPS expressed in B. melitensis strain B115 by immunoelectron microscopy. Immunogold labelling was observed at the surface of B. melitensis B115 cells with the anti-R-LPS mAb but not with the anti-S-LPS mAb. In ultrathin sections, immunogold labelling with the S-LPS specific mAb was observed in the cytoplasm and in the periphery of the cytoplasm, probably at the cytoplasmic membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

We previously integrated the luxAB gene into the Yersinia enterocolitica chromosome. In this article, we assessed, by luminometry, the survival of the engineered strain KNG1024 in the digestive tracts of mice and cows. In situ detection and a count of the released strain were performed on feces from orally inoculated BALB/c mice for 24 days. This method is a rapid and reliable system for long-term monitoring of genetically engineered bacteria. In cow feces, the count of Y. enterocolitica ranged from 210 to 6,000 CFU/g of feces. This very low count was not detectable by direct luminometry.

Mice passively immunized prior to a challenge infection with immunoglobulin G (IgG) and IgM monoclonal antibodies (MAbs) specific for a common epitope of both A- and M-dominant strains had viable Brucella abortus 544 or Brucella melitensis H38 counts in the spleen reduced to the same extent as did mice passively immunized with MAbs specific for either the A or the M epitope. The IgA MAb was not effective.

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.

A panel of monoclonal antibodies (MAbs) to seven Brucella outer membrane proteins were characterized. These antibodies were obtained by immunizing mice with sodium dodecyl sulfate-insoluble (SDS-I) fractions, cell walls, or whole bacterial cells of Brucella abortus or B. melitensis. Enzyme-linked immunosorbent assays were used to screen the hybridoma supernatants and to determine their binding at the surface of rough and smooth B. abortus and B. melitensis cells. The outer membrane proteins (OMPs) recognized by these antibodies were the proteins with molecular masses of 25 to 27 kDa and 36 to 38 kDa (porin) (major proteins) and the proteins with molecular masses of 10, 16.5, 19, 31 to 34, and 89 kDa (minor proteins). Surface exposure of these OMPs was visualized by electron microscopy by using the MAbs and immunogold labeling. Binding of the MAbs on whole rough bacterial cells indicates that the 10-, 16.5-, 19-, 25- to 27-, 31- to 34-, 36- to 38-, and 89-kDa OMPs are exposed at the cell surface. However, enzyme-linked immunosorbent assay results indicate a much better binding of the anti-OMP MAbs on rough strains than on the corresponding smooth strains except for the anti-19-kDa MAb. Immunoelectron microscopy showed that on smooth B. abortus cells only the 89- and 31- to 34-kDa OMPs were not accessible to the MAbs tested. Binding of the anti-31- to 34-kDa MAb at the cell surface was observed for the rough B. abortus cells and for the rough and smooth B. melitensis cells. These results indicate the importance of steric hindrance due to the presence of the long lipopolysaccharide O side chains in the accessibility of OMPs on smooth Brucella strains and should be considered when undertaking vaccine development.

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.

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.

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.

Latex particles coated with F(ab)'2 fragments of anti-protein C IgG antibodies are agglutinated by protein C, and the quantity of particles agglutinated is proportional to the concentration of protein C. The reaction can be quantitated by optical particle counting. Based on this system, we designed an immunoassay for protein C. Precision measured at low, medium and high levels of protein C varied from 3.3% to 13.7%. Specificity was evaluated by dilution recovery. A correlation coefficient of r = 0.959 was found when the new method was compared with a chromogenic technique on 131 plasmas.

We set up an immunoassay by particle counting for theophylline. Theophylline concentration is assayed by its capacity to inhibit the agglutination of theophylline coated latex particles by a specific monoclonal antibody, the agglutination being enhanced by a rabbit anti-mouse IgG antiserum. The dose range is 2-64 mg/L. The cross-reactions observed with caffeine (0.3%), theobromine (0.2%), 3-methylxanthine (0.7%) and 8-chlorotheophylline (2%) are very good when compared with other published methods. Within and between-run precisions measured at low, medium and high level of the calibration curve show coefficients of variation ranging from 3.9% to 9.5%. Our assay was correlated with the Fluorescence Polarization Immunoassay (FPIA) and a correlation coefficient of 0.96 was determined for 89 samples.

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).

In this latex immunoassay for human placental lactogen, microtiter plates are used as the reaction vessel and the absorbance at 405 nm is measured to quantify the reactions. This 30-min assay necessitates only one serum dilution and two pipetting steps. The calibration curve extends from 0.5 to 15 mg/L. CVs range from 4.2% to 7.0% for within-run determination and from 7.0% to 11.2% for between-run determinations. A correlation coefficient of 0.949 was obtained for 84 sera when the method was compared with a commercial radioimmunoassay.

Rat polymeric IgA (pIgA) and galactosylated bovine serum albumin (GalBSA), once injected to rats, are avidly taken up by hepatocytes via receptor-mediated endocytosis. Of injected pIgA, 64% was transferred undigested into bile within 3 h, with a peak at 30-45 min. GalBSA was essentially digested in lysosomes. By electron microscopy using ligand-peroxidase conjugates, both ligands were internalized through coated pits/coated vesicles into similar electron-lucent vesicles and tubules. Subsequently, pIgA remained mostly associated with small vesicles clustering around and fusing with bile canaliculi, while GalBSA was predominantly found in large, heterogeneous endocytic structures and in lysosomes. By subcellular fractionation, they were associated at 3 min after injection with structures that similarly sedimented in the P fraction (250 000 - 3 X 10(6) X g X min) and equilibrated at densities of about 1.13 g/ml in sucrose gradients. At 10 min and 20 min, pIgA distribution remained mostly in the P fraction at the same equilibrium density. A minor component of the pIgA distribution was found at the density of lysosomes, but contrary to lysosomal enzymes, its distribution was not affected by Triton WR 1339. In contrast to pIgA, GalBSA was progressively recovered in the L fraction (33 000 - 250 000 X g X min) with organelles equilibrating around 1.11 g/ml, and, by 20-45 min, was found in the ML fraction (10 000 - 250 000 X g X min), around 1.20 g/ml, i.e. in lysosomes. Chloroquine did not reduce the efficiency but delayed the secretion of pIgA into bile. Similarly, it did not affect the uptake of GalBSA but apparently delayed GalBSA transfer along successive populations of host organelles. The low density, GalBSA-containing structures were devoid of proteolytic activity. Anti-secretory components IgG and F(ab')2 were selectively excreted into bile, partially or totally as compounds of lower molecular mass. These antibody fragments probably result from a disulfide reduction activity along the pIgA pathway. In conclusion, our data (a) strongly suggest that pIgA and GalBSA are sorted between 3 min and 10 min after injection in non-lysosomal acidic organelles, (b) identify two successive and physically distinct endosomal populations containing GalBSA, and (c) provide the first evidence for a disulfide reduction activity along the transcytotic pathway of rat hepatocytes.

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.

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.

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)

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.

Labelled hemoglobin-haptoglobin (ham-hap), galactosylated serum albumin (gal-SA) and polymeric immunoglobulin A (p IgA) were injected intravenously to rats or mice. The labels disappeared from the plasma with a half-time of about 5 min and were almost entirely found associated with the liver where degradation products progressively appear. The uptake of hem-hap and gal-SA are partially saturable as a function of the plasmatic concentration and the uptake of gal-SA can be completely inhibited by the simultaneous injection of asialofetuin. About 45 min after injection to rats, labelled material appears in the bile in amounts corresponding to 3.9% of the injected dose (hem-hap), 2.8% (gal-SA) and 60.1% (p IgA). The molecular weight of the labelled material transferred into the bile has been characterized: it consists almost entirely of intact IgA and for about 60% of intact hem-hap and gal-SA. Cell fractionation experiments indicate that 4 min after injection, the label is associated with components which equilibrate around a density of 1.13 g/cm3 and which dissociate from marker enzymes of Golgi complex, plasma membrane and lysosomes. Longer times after injection (from 20 min for hem-hap and gal-SA to 1 h for p IgA) labelled material appears, within lysosomes. To explain all these data, we suggest that after binding to plasma membrane receptors, the ligands are rapidly interiorized into pinocytic vesicles which fuse with lysosomes. Most of the hem-hap and gal-SA molecules but only part of p IgA would be released and subsequently digested; these vesicles would dissociate from lysosomes and fuse with the biliary membrane where the molecules still bound to the membrane of the vesicles would be detached and excreted into the bile.

Particle Counting ImmunoAssay (PACIA) has been adapted to the determination of serum ferritin. Polystyrene particles (0.8 micrometers) were coated with anti-ferritin antibodies and the concentration of ferritin determined by the agglutinating activity of this protein. The agglutination was measured by the reduction of the number of non-agglutinated particles counted in an optical blood cell counter. Non-specific agglutination and inhibition of agglutination were prevented in 99% of clinical samples by the use of F(ab')2 fragments of the antiserum IgG to coat the particles and by using slightly dissociating conditions (pH 9.2, ammonium thiocyanate, EDTA). The system was automated with a sampling rate of 50/h and an incubation time of 25 min. The standard curve ranged from 1 to 100 micrograms/l; recoveries were between 93.6 to 100.2%; the correlation coefficients of PACIA with radioimmunoassays calculated respectively on 99 and 91 samples were 0.974 and 0.984; maximal within- and between assay CV were 11.2% and 7.7%.