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Biomedical subjects

B R Brodeur

Publications and source records attributed to B R Brodeur.

At least 55 records · Page 3Linked to original sources
PubMed1988

Heterohybridomas secreting human monoclonal antibodies against Haemophilus influenzae type b.

Seven human monoclonal antibodies (HmAb) directed against outer membrane antigens of Haemophilus influenzae type b (Hib) were produced by fusing Sp2/HPT heteromyeloma cells with human tonsillar lymphocytes sensitized in vitro for 6 days. The heterohybridomas were maintained in culture for at least one year and secreted, when cultured in Dulbecco's modified Eagle's medium without fetal calf serum, between 1 and 15 micrograms/10(6) cells/ml/24 h. All of the HmAb were IgGs except HiH-12 which is an IgM. Antibodies directed against the lipopolysaccharide and proteins of apparent molecular masses of 43, 37 and 27 kDa were identified by immunoblotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of outer membrane. Binding radioimmunoassay with live bacteria showed that five out of seven HmAb adsorbed to cell surface-exposed antigenic determinants. HmAb HiH-6, HiH-7 and HiH-10 reacted with a surface-accessible determinant on the 43-kDa outer membrane protein. In a dot enzyme immunoassay, these HmAb recognized 103 out of 111 Hib strains isolated worldwide. The strains were selected to represent the most common genotypic variations among Hib. None of these HmAb reacted with other bacterial species tested. These HmAb may serve to study the bacterial surface antigens implicated in the human humoral response and protection to Hib infections.

Antibodies, Bacterial↗
PubMed1987

A monoclonal antibody directed against a serotype-specific, outer-membrane protein of Haemophilus influenzae type b.

Monoclonal antibodies (Mabs) were produced against outer-membrane proteins (OMPs) of Haemophilus influenzae type b. The clones were screened by ELISA with outer-membrane preparations of H. influenzae type b and untypable strains as coating antigens. Antibodies directed against the proteins of mol. wt (10(3)) 43, 37 and 13 were identified by immunoblotting of SDS-PAGE patterns of OMPs. Proteolytic enzyme treatments of the OMPs resulted in reduction of Mab reactivity as measured by ELISA. Furthermore, the absence of reactivity of Mab Hb-2 with a preparation of lipopolysaccharide confirmed the protein nature of its corresponding epitope. Binding assays with live bacteria showed that Hb-2 reacted with a cell surface-exposed antigenic determinant. Mab Hb-2 was bactericidal in vitro in the presence of complement. The characterisation of Hb-2 (IgG2a) by Western immunoblotting analysis revealed that it was directed against the 37 X 10(3)-mol. wt OMP. In a dot-enzyme immunoassay, Hb-2 reacted specifically with 326 strains of H. influenzae type b. It did not cross-react with the other serotypes or untypable strains of H. influenzae or with other bacterial species. This is the first report of a monoclonal antibody identifying a serotype-specific surface-exposed OMP of H. influenzae type b.

Antibodies, Monoclonal↗
PubMed1987

Identification of Haemophilus influenzae type b by a monoclonal antibody coagglutination assay.

A coagglutination assay using monoclonal antibody is described for the identification of Haemophilus influenzae type b. An immunoglobulin G2a monoclonal antibody, Hb-2, directed against a serotype-specific outer membrane protein of H. influenzae type b was adsorbed to Staphylococcus aureus Cowan 1 cells. In a dot enzyme immunoassay, Hb-2 reacted with 453 of 455 H. influenzae type b isolates and did not react with H. influenzae of other serotypes, untypeable H. influenzae strains, or other bacterial species. The Hb-2 coagglutination assay was evaluated by testing 136 H. influenzae type b strains selected on the basis of multilocus enzyme genotypes, 5 strains of another serotype, and 94 untypeable H. influenzae strains. The specificity of the coagglutination assay was demonstrated by the inhibition of the reaction by free Hb-2 monoclonal antibodies. The coagglutination assay was as specific as the dot enzyme immunoassay and can be rapidly performed and easily interpreted.

Agglutination Tests↗
PubMed1986

High yield monoclonal antibody production in ascites.

BALB/c males were mated with Swiss Webster/HPB females to produce a first generation cross. Hybridoma cells derived from fusing SP2/0 myeloma cells and histocompatible spleen cells were injected intraperitoneally into these mice to induce formation of ascites tumors and production of ascitic fluid containing large quantities of monoclonal antibody. Mice, 80 days old weighing between 28 g to 35 g, were treated with 0.5 ml of pristane 18 days before inoculation of 3.2 X 10(6) hybridoma cells. The first generation crosses, (BALB/c male X SW/HPB female)F1, produced up to 4 times more ascitic fluid of equally high antibody level over a longer period compared to the BALB/c parent. This first generation cross is a cost effective means for monoclonal antibody production.

Animals↗
PubMed1986

Rapid diagnosis of severe Haemophilus influenzae serotype b infections by monoclonal antibody enzyme immunoassay for outer membrane proteins.

A highly sensitive and specific enzyme immunoassay (EIA) for the detection of Haemophilus influenzae serotype b antigens in body fluids and broth cultures was developed, with a polyclonal antibody directed against polyribose phosphate as the solid-phase reagent and a biotinylated monoclonal antibody directed against H. influenzae type b outer membrane protein as the liquid-phase reagent. H. influenzae type b antigens could be detected in broth cultures containing as little as 50 organisms per ml. The sensitivity and specificity of this system were compared with those of two commercial kits and counterimmunoelectrophoresis. The overall detection of H. influenzae type b antigens in clinical specimens collected from children infected with H. influenzae type b was as follows: with Phadebact, 86 and 86% in cerebrospinal fluid and urine specimens, respectively; with Bactigen, 86, 80, and 92%, with counterimmunoelectrophoresis, 78, 73, and 75%, and with biotin-avidin EIA, 100, 100, and 100% for cerebrospinal fluid, serum, and urine specimens, respectively. In the biotin-avidin EIA, no positive reactions were noted in specimens collected from patients infected with other bacteria or from patients without evidence of bacterial infection, whereas false-positive reactions were found by counterimmunoelectrophoresis and the commercial kits. These results suggest that this monoclonal antibody reacting with the outer membrane protein is more specific and sensitive than the conventional methods using polyclonal antisera for the detection of H. influenzae type b antigens during severe infections in children.

Antibodies, Monoclonal↗
PubMed1986

Mouse models of infection for Neisseria meningitidis B,2b and Haemophilus influenzae type b diseases.

A disseminated and fatal infection was established in C57BL mice, injected intraperitoneally with either Neisseria meningitidis B,2b or Haemophilus influenzae type b bacteria plus enhancement factors. The effects of mucin, hemoglobin, and iron dextran as enhancement of bacterial infectivity in mice were evaluated individually and in combination. A mixture of mucin and hemoglobin was most effective in enhancing the virulence of the pathogens. Inbred mouse lines were more susceptible than outbred ones. Relative virulence of a number of bacterial strains was also compared in one selected mouse line. Neisseria meningitidis B,2b and Haemophilus influenzae type b strains were more virulent than non-B,2b and nontypable strains. Finally, the course of bacteremia for the two infections in mice was followed by quantitative blood cultures. The animals succumbed to the generalized condition within 72 h. In the case of Neisseria meningitidis B,2b, 10 organisms with 4% mucin and 1.6% hemoglobin were sufficient to kill 50% of the animals. For Haemophilus influenzae type b, 300 bacteria with 5% mucin and 2% hemoglobin were necessary to obtain similar effects.

Animals↗
PubMed1985

Parameters affecting in vitro immunization of human lymphocytes.

A method for the in vitro generation of antibody-secreting cells from human tonsil lymphocytes is described. Several parameters affecting such in vitro immunization have been studied in order to define optimal conditions for the production of specific antibody. In the presence of 15% of T-cell growth factors, lymphocytes prepared by Ficoll-Paque centrifugation increased 2.5 times over a period of 6 days. The major factors triggering specific antibody production against Haemophilus influenzae type b outer membrane preparations were: the use of tonsil lymphocyte cultures instead of peripheral blood lymphocytes, foetal calf serum instead of human serum, T-cell growth factors as a non-specific lymphocyte activator, low concentrations of antigen ranging from 1 to 10 ng/ml, a cell concentration of 4 X 10(6) cells/culture, and finally the removal of OKT8 positive suppressor T-cells. Under these conditions, a maximum antibody production was achieved after 7 days of culture and specific antibody response was obtained after in vitro immunization of tonsil lymphocytes from 10 out of 11 individuals tested.

Antibodies, Bacterial↗
PubMed1985

Protection against infection with Neisseria meningitidis group B serotype 2b by passive immunization with serotype-specific monoclonal antibody.

Hybridomas derived from mice immunized with Neisseria meningitidis serogroup B serotype 2b (B,2b) outer membrane preparations produced monoclonal antibodies (MAbs) specific for major outer membrane proteins of classes 1, 2, and 5. The MAbs were examined by enzyme-linked immunosorbent assay against a selected panel of seven strains of N. meningitidis (B,2b) of different sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns, a serotype 2a, and a nontypable strain. The five MAbs selected were all bactericidal and of different immunoglobulin subclasses. None of the MAbs reacted with other bacterial strains in a dot-enzyme immunoassay. The corresponding antigenic determinant for each MAb was localized on a specific outer membrane protein by immunoblotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of major outer membrane proteins. MAbs M5-11 and M5-30 bound to the class 2 protein and were serotype 2b specific. MAb M2-20 bound to the class 1 protein, and MAbs M5-16 and M5-19 bound to the class 5 protein. A mouse model of infection was established whereby a local infection progressed to lethal bacteremia over 3 days, and 50% of the animals were killed with an intraperitoneal injection of 10 meningococci plus 4% mucin and 1.6% hemoglobin. The ability of the MAbs to provide passive protection against experimental infection with N. meningitidis (B,2b) was examined. Both serotype-specific MAbs M5-11 and M5-30 were highly protective even though they were of different immunoglobulin subclasses. The class 5-specific MAb offered no protection, while the class 1-specific MAb gave limited protection. It may therefore be possible to provide protection against serotype 2b infection by using as vaccine the class 2 serotype-specific surface-exposed outer membrane protein epitopes defined by MAb M5-11 or M5-30.

Animals↗
PubMed1984

Parameters affecting ascites tumour formation in mice and monoclonal antibody production.

Hybridoma cells injected intraperitoneally into mice induce formation of ascites tumours and production of ascites fluid containing high levels of monoclonal antibody. Several parameters affecting the growth of the immunoglobulin-producing tumours have been studied in order to define optimal conditions for ascitic fluid formation and monoclonal antibody production. Using hybridomas produced by fusing SP2/0 myeloma cells with immunized mouse spleen cells we have shown: (1) that the optimal number of hybridoma cells required to induce an ascites tumour was between 6 and 32 X 10(5) cells; (2) that each mouse should be treated with a maximum of 0.5 ml of pristane; (3) that the priming period for pristane should be 14 days prior to the injection of cells; (4) that ascites formation and monoclonal antibody production is significantly better in males; and finally (5) that the age of mice used should range between 43 and 78 days. Under these conditions each mouse produces on average 7-10 ml of ascites fluid, containing a high level of antibody, over a maximum period of 6 days. The animals should start producing between the 5th and 9th day and usually survive 11-16 days after being injected with the tumour cells.

Animals↗
PubMed1983

Serotypes of Neisseria meningitidis associated with an increased incidence of meningitis cases in the Hamilton area, Ontario, during 1978 and 1979.

The distribution of serotypes among strains of Neisseria meningitidis responsible for a marked increase of meningitis cases in the Hamilton area, Ontario, in 1978 and 1979 was determined. Twenty-six serogroup B and two serogroup W135 strains isolated from cerebrospinal fluid, blood, and skin of 28 patients were serotyped by agar gel double diffusion. Twenty-one (81%) of the group B strains were serotype 2b as judged by the formation of characteristic serotype precipitin bands with the specific anti-2996 (type 2b) serum. Fourteen of the serotype 2b strains also reacted with anti-77252 serum, which suggested that one strain or several closely related strains were mainly responsible for the increase in meningitis during the 2-year period. Examination of the outer membrane complexes (OMC) of the strains by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) revealed that all 21 of the serotype 2b strains contained the class 2 protein (molecular weight 41500) which is known to be the site of the serotype 2b determinant. Further characterization of the serotype 2b, 77252 strains by enzyme-linked immunosorbent assays (ELISA) and SDS-PAGE suggested that the 77252 determinant was present in the class 1 proteins of these strains. The serotype 2b containing strains were isolated from 77.7 and 70% of males and females, respectively, from 81.8% of children less than 5 years of age, and from 75.0% of patients of all age groups. The study indicates the important role of serotype 2b meningococci in causing the increased incidence of meningitis and further substantiates the important association of the serotype 2b determinant with group B serotype 2 meningococcal disease in Canada.

Bacterial Outer Membrane Proteins↗
PubMed1982

Enzyme-linked immunosorbent assay with polyvalent gonococcal antigen.

An indirect enzyme-linked immunosorbent assay (ELISA) with a mixture of eight different gonococcal outer-membrane proteins (OMP) as coating antigen was evaluated for detection of gonococcal antibody in 507 sera obtained from patients selected from high-risk and low-risk populations. The indirect ELISA method was more specific and sensitive when the polyvalent antigen was used than when OMP from only one serotype was used. Past episodes of the gonorrhoea had a significant influence on the seropositivity of the test. In a selected low-risk population the specificity of the assay was 94% and in a selected high-risk population the sensitivity was 78%. When sera from 24 asymptomatic individuals were tested the sensitivity was 83%. The ELISA polyvalent-antigen test should be useful as an aid for the detection of gonorrhoea in populations with a low prevalence.

Antibodies, Bacterial↗
PubMed1979

Induction of reaginic (IgE) gonococcal antibodies in the rat by a common antigen of Neisseria gonorrhoeae.

An antigen (ZAB) common to Neisseria gonorrhoeae was prepared by stepwise elution of a crude gonococcal antigen (ZA) from columns of diethylaminoethyl cellulose employing 0.02 M phosphate buffers, pH 7.6, containing increasing concentrations of sodium chloride. Rats immunized with ZAB produced reaginic (IgE) antibody which cross-reacted with ZA prepared from eight gonococcal strains by the passive cutaneous anaphylaxis (PCA) test. Heating of the sera at 56 degrees C for 4 h destroyed the PCA activity. The PCA activity of the anti-ZAB rat serum was removed after absorption with ZAB antigen or with rabbit anti-rat IgE but not after absorption with gonococcal lipopolysaccharide or with heat-killed or formalinized gonococci. Treatment of ZAB with trypsin or heating at 100 degrees C for 30 min destroyed or reduced the antigenic activity respectively. Further purification of ZAB by filtration through Sephadex G-100 gave a preparation (ZAB2) which contained the common antigen as shown by the cross-reactivity of anti-ZAB2 rat serum with seven stains of N. gonorrhoeae. Fraction ZAB2 contained material which had a molecular weight less than 13,700 and was associated with the presence of material absorbing at 260 nm. The results of this study indicate that a low molecular weight antigen, which appears to be protein in nature and associated with nuclei acid, is common to the gonococcus and is the main antigenic component inducing reaginic (IgE) antibody in the rat.

Animals↗
PubMed1978

Characterization of the mitogenic activity elicited by Neisseria gonorrhoeae ribosomal fractions.

Ribosomal preparations from Neisseria gonorrhoeae types 1 and 4 were examined for their in vitro stimulation of mouse splenocytes to determine the ribosomal moiety or contaminant responsible for the immunoproliferative activity. In immunodiffusion tests with homologous rabbit antiserum, crude 70S ribosomes formed four precipitin bands while the purified 30S and 50S subunits showed one major line. The same antiserum reacted with lysed N. gonorrhoeae and Neisseria meningitidis A cells but no precipitation occurred with Escherichia coli cells purified N. gonorrhoeae lipopolysaccharide (LPS). No membrane or LPS contaminant was detected in the purified 30S and 50S preparations. All the ribosomal preparations from virulent and non-virulent N. gonorrhoeae consistently stimulated the murine splenocytes. The mitogenic activity of the 30S and 50S ribosomal preparation was destroyed by treatment with trypsin but only slightly decreased by ribonuclease. It is suggested that the lymphoproliferative response elicited by gonococcal ribosomes is triggered by the protein moiety of the 30S or 50S subunits.

Animals↗
PubMed1978

Enzyme-linked immunosorbent assays for the detection of Neisseria gonorrhoeae specific antibodies.

An indirect enzyme-linked immunosorbent assay (ELISA) using rigid polystyrene microtiter plates was adapted to detect specific gonococcal antibodies against outer membrane-complex antigens extracted from Neisseria gonorrhoeae. The concentration of antigen to obtain maximum coating of the well was 10 micrograms protein per millilitre. The optimal binding of the primary antibody and enzyme-conjugated antimmunoglobulin was achieved after 1 h at 37 degrees C. Under these conditions using gonococcal antisera, no cross-reactivity was observed with outer membrane antigens extracted from Neisseria meningitidis serogroups B, C, X, Y, and W135. Neisseria meningitidis serogroup A demonstrated low levels of cross-reactivity. All the non-pathogenic Neisseria spp. tested were negative (absorbance value at 400 nm/30 min less than 0.15). The reaction of immune serum against outer membrane complex absorbed to the microwells was completely inhibited with soluble-specific antigen but not with purified N. gonorrhoeae lipopolysaccharide. Quantitative inhibition permitted the measurement of low levels of antigen (0.5 microgram/ml). The detection of N. gonorrhoeae antibody with ELISA is specific and highly sensitive.

Agglutination Tests↗
PubMed1977

In vitro interaction of Neisseria gonorrhoeae type 1 and type 4 with tissue culture cells.

As a basis for studies of gonococcal pathogenicity, tissue culture cells were infected with type 1 or type 4 Neisseria gonorrhoeae to determine intracellular viability. A simple and objective means of measurement was devised, based on the uptake of tritiated protein and deoxyribonucleic acid precursors by cycloheximide-inhibited cells infected with gonococci. Cycloheximide was found to inhibit protein synthesis by over 97% tissue culture cells at a concentration of 100 microng/ml. In contrast, N, gonorrhoeae was found to be highly resistant to this antibiotic, and protein synthesis was unaffected by concentrations up to 1,000 microng/ml. Extracellular gonococci were eliminated by treatment with high concentrations of penicillin during cycloheximide inhibition and prior to the addition of radioisotope. Levels of protein and deoxyribonucleic acid synthesis by N. gonorrhoeae in the cycloheximide-treated cells were significantly higher in T1-infected cells (RE2, HeLa, or HEp-2) than in the corresponding T4-infected cells. No differences were observed in tissue cell susceptibility to gonococcal infection. Intracytoplasmic localization of N. gonorrhoeae was confirmed by electron microscopy.

Bacterial Proteins↗
PubMed1977

Mitogenic activity of Neisseria gonorrhoeae surface antigens in mouse splenic lymphocyte culture.

The mitogenic effects of Neisseria gonorrhoeae endotoxin, fractionated envelope componenents, and intact cells were examined on unsensitized mouse splenic lymphocytes in vitro. The stimulatory effect of these substances was measured by increased [3H]thymidine incorporation in spleen cell cultures. Intact cells, purified lipopolysaccharide (LPS), and cell envelope preparations were highly stimulatory and the stimulation index was dose dependent. Fractionated components of the envelope demonstrated variable stimulation when tested at identical LPS concentrations, reflecting the mitogenic activity of the protein moieties. The stimulatory dose responses for purified N. gonorrhoeae and Escherichia coli LPS were compared and mitogenicity was higher with gonococcal LPS at all concentrations tested. Alkaline detoxification or succinylation of N. gonorrhoeae LPS results in loss of ability to induce blast transformation. The mitogenicity of cell-surface components of N. gonorrhoeae is discussed in terms of LPS and protein content.

Animals↗
PubMed1977

Interferon inhibition of lymphocyte mitogenesis.

Interferon inhibits the early phase of mitogenic action of Concanavalin A on lymphocytes as measured by incorporation of labelled precursors in RNA, DNA or protein. There was no evidence for a role of cyclic AMP in the process of inhibition. Dialysis of interferon against high salt and urea did not separate cell inhibitory activity from antiviral activity. Thus, these results are compatible with the possibility that the antiviral and cell inhibitory activities are in the same molecule. However, further purification of the interferon will be required to verify this assumption.

Animals↗