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D G Braun

Publications and source records attributed to D G Braun.

At least 37 records · Page 2Linked to original sources
PubMed1987

The proliferative response of human peripheral blood lymphocytes to group A streptococcal vaccine.

The proliferative response of human peripheral blood mononuclear cells to group A streptococcal vaccine (strain J17A4) was assessed by measurement of [3H]thymidine incorporation. This was maximal after 7 days in cultures containing 1 X 10(5) lymphocytes. Cells from 33 out of 38 adult donors (87%) yielded stimulation indices between 2.0 and 81. Responding cells were predominantly of the OKT4+, Ia+, Tac+ phenotype expressed by activated helper T lymphocytes. In contrast, no response was observed in 10 out of 12 samples of cord blood lymphocytes. Addition of conventional rabbit or monoclonal murine anti-Ia (HLA-DR) antibodies to the culture medium abrogated the vaccine-induced proliferative response. Frequencies of responding cells estimated by limiting dilution analysis were between 1 in 2370 and 1 in 4580 lymphocytes. The results suggest that lymphocyte stimulations induced with streptococcal group A vaccine represented an in vitro anamnestic cellular immune response.

Antibody-Producing Cells↗
PubMed1987

Monoclonal antibodies to the multienzyme enniatin synthetase. Production and use in structural studies.

Monoclonal antibodies have been prepared against the multifunctional enzyme enniatin synthetase, which catalyses the biosynthesis of the cyclodepsipeptide antibiotic enniatin. Five different antibodies (designated 1.56, 21.1, 25.91, 28.7 and 28.34) were characterized. 1.56, 21.1 and 25.91 were of IgG1 and 28.7 and 28.34 of IgM subclass. Binding studies showed that 21.1 and 25.91 are obviously directed against determinants based on the primary structure of the enzyme, whereas 28.7, 28.34 and 1.56 bind to the native enzyme. All antibodies inhibited enniatin formation. Based on their ability to inhibit different partial reactions of the multienzyme the antibodies could be divided into three groups: 21.1 and 25.91 inhibit valyl thioester formation, 1.56 additionally inhibits D-2-hydroxyisovaleric acid thioesterification, and 28.7 and 28.34 block both thioester sites as well as the N-methylation step. None of the antibodies affected the formation of L-valyl or D-hydroxyisovaleryl adenylate by the enzyme. The results indicate that there must be distinct thioester activation sites for valine and D-hydroxyisovalerate close to each other and in the neighbourhood of the methyltransferase site. The adenylation sites for D-hydroxy-isovalerate and L-valine are obviously located at some distance.

Affinity Labels↗
PubMed1986

Epitope mapping of human recombinant interferon alpha molecules by monoclonal antibodies.

The epitopes of six recombinant human interferon alpha (IFN alpha) subtypes have been analysed using 22 monoclonal antibodies (MAbs) obtained from different sources. The IFN alpha subtype specificity of each MAb was determined by a combined immunoprecipitation-bioassay. Eight different epitopes were identified; the number of epitopes on a given IFN alpha subtype varied between four and eight. Each subtype possessed a unique combination of epitopes. Using the best pair of monoclonal antibodies, predicted from epitope mapping studies, subtype-specific two-site (tandem) assays were developed. It was observed that some non-cross-reactive MAbs influenced each other's binding, indicating the flexible nature of IFN molecules. Competitive radioimmunoassay and the combined immunoprecipitation-bioassay were used to identify a common epitope, present on IFN alpha-A, B, C, F and J but not on IFN alpha-D. In neutralization studies, all MAbs that identified this common epitope inhibited the antiviral activity of all IFN alpha molecules tested. It was concluded that the epitope is located within the receptor-binding region of IFN molecules and is important for biological activity. A tentative localization of the common epitope and the other identified epitopes is proposed.

Antibodies, Monoclonal↗
PubMed1986

Characterization of monoclonal antibodies to a crystal protein of Bacillus thuringiensis subsp. kurstaki.

Ten monoclonal antibodies were produced against a k-1-type crystal protein of Bacillus thuringiensis subsp. kurstaki. Eight of the antibodies belong to the immunoglobulin G1 (IgG1) subclass, with pI values ranging from 5.5 to 8.6, one could be assigned to the IgG2b subclass, and one could be assigned to the IgM class. Competitive antibody-binding assays and analysis of antibody specificity indicated that the 10 antibodies recognized at least nine distinct antigenic determinants. Eight antibodies bound to both protoxin and toxin, whereas the other two interacted with protoxin only. One antibody completely inhibited the biological activity of the delta-endotoxin, five antibodies reduced it by 15 to 82%, and four antibodies did not affect it at all. Based on cross-reaction studies, homologies and differences in the crystal protein structures of different B. thuringiensis subspecies were revealed. All of the monoclonal antibodies strongly cross-reacted with crystal proteins from strains of B. thuringiensis subsp. tolworthi, B. thuringiensis subsp. galleriae, B. thuringiensis subsp. dendrolimus, B. thuringiensis subsp. sotto, and B. thuringiensis subsp. subtoxicus. Some antibodies interacted only weakly with crystal proteins from strains of B. thuringiensis subsp. morrisoni and B. thuringiensis subsp. entomocidus, and some of these did not interact with B. thuringiensis subsp. kenyae and B. thuringiensis subsp. darmstadiensis. No cross-reaction was found with the parasporal inclusion protein of B. thuringiensis subsp. israelensis. Studies with the monoclonal antibodies also disclosed that crystal proteins from strains of the same subspecies can exhibit substantial differences in antigenic structure. In particular, striking strain-specific differences in the protoxins of B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. thuringiensis were observed.

Antibodies, Monoclonal↗
PubMed1986

Combinatorial diversity in the generation of antibody molecules. The complete amino-acid sequence of the variable domain of a monoclonal anti-streptococcal group A polysaccharide antibody.

The first complete amino-acid sequence of the variable region of the gamma 3 heavy chain from a murine anti-streptococcal group A polysaccharide (A-CHO) immunoglobulin (monoclonal antibody 2S1.3) is described. Therefore, in conjunction with the previously published 2S1.3 light chain sequence, a V kappa 25 structure, the entire variable domain of this antibody has been determined. In addition, four partial amino-terminal heavy chain sequences of other antibodies with the same specificity are reported. These heavy chains share a high degree of homology with heavy chains from fructosan-binding murine myeloma proteins with the exception of those positions known to be encoded by the D (diversity) segment in germ line DNA. The light chains associated with the heavy chains reported here are products of the V kappa 25, V kappa 27, and J kappa 5 genes. Up to date three VH and four V kappa subgroups have been shown to contribute genetic material to the assembly of antibodies specific for the A-CHO. Unlike other experimental systems employing structurally completely resolved full antigens the antistreptococcal immune response uses V genes previously shown to be involved in the formation of antibodies with different specificities. This provides further experimental evidence for the physiological relevance of heavy/light chain association as a posttranscriptional diversification mechanism in the generation of the antibody repertoire in addition to those somatic diversifiers acting directly upon the genes encoding the variable regions of individual chains.

Amino Acid Sequence↗
PubMed1985

Thrombin specificity. Selective cleavage of antibody light chains at the joints of variable with joining regions and joining with constant regions.

Selective cleavage of polypeptides by alpha-thrombin can be reasonably predicted [Chang, J.Y. (1985) Eur. J. Biochem. 151,217-224]. This knowledge was applied to the selective cleavage of antibody light chains with the aim of producing intact fragments of both variable region and constant region. (a) Mouse kappa light chains 10K26 and 10K44 from anti-(azobenzene arsonate) antibodies contain 20 Arg/Lys-Xaa bonds. Only two of them, one ProArg-Thr bond located at the joint of the variable region with the joining peptide and one ValLys-Ser bond located near the carboxyl-terminal end of the constant region, were selectively cleaved by alpha-thrombin. The ProArg-Thr bond has a 50% cleavage time of about 10 min under the designated conditions, whereas the ValLys-Ser has a 50% cleavage time approx. 9-10 h. A single selective cleavage at the joining position of the variable region and joining peptide can be achieved by short-time thrombin digestion. Fragments containing intact variable region (1-96) and intact joining peptide-constant region (97-214) obtained from both denatured and native light chains of 10K26 can be separated by gel filtration. (b) lambda light chains from both human and mouse all begin with the GlnProLys-(Ala/Ser) structure (positions 108-111) at their constant regions. This ProLys-Ala/Ser bond is also susceptible to specific thrombin cleavage. Four human lambda chain (KERN, NEI, NEW, VOR) and one mouse lambda chain (RPC20) were shown to be selectively cleaved by thrombin at these ProLys-Ala/Ser bonds. For human lambda chains, the 50% cleavage time at this ProLys-Ala bond was approx. 3-4 h under the designated conditions. Six additional thrombin specific cleavages were also detected within the variable regions of NEI, VOR and RPC-20. (c) Heparin inhibits thrombin cleavage of Arg/Lys-Xaa bonds located near the center of the antibody light chain, but slightly activates thrombin cleavage of those located near the amino or carboxyl-terminal ends of the protein. The significance of these findings is threefold. (a) It demonstrates that selective cleavage of large polypeptides by alpha-thrombin can also be reasonably predicted. (b) It provides a useful method for light chain fragmentation which can greatly facilitate amino acid sequencing of antibodies. (c) It serves to generate fragments containing intact variable regions and constant regions from antibody light chains of human and mouse. Such fragments may be useful for chemical semisynthesis of a human-mouse light chain chimeras.

Amino Acid Sequence↗
PubMed1985

Chromogenic labeling of monosaccharides using 4'-N,N-dimethylamino-4-aminoazobenzene.

Twenty-three monosaccharides, e.g., D- or L-pentoses, D- or L-hexoses, heptose, 2- or 6-deoxyhexoses, 2-deoxy-2-aminohexoses, hexuronic acids, and N-acetylmuramic acid, were coupled to the azo dye 4'-N,N-dimethylamino-4-aminoazobenzene by reductive amination using sodium cyanoborohydride as reducing agent and in the presence of pentaerythritol. The structure of the colored glycamines was established by mass spectrometry. The average yield of the reaction was more than 80%. The sugar derivatives were separated either by silica-gel thin-layer chromatography or by high-performance liquid chromatography. Spectrophotometric quantitation was performed in the visible range at the picomole level. The method was applied to the determination of the sugar composition of the glycosphingolipid globotetraosyl ceramide and the human milk oligosaccharide lacto-N-fucopentaose I.

Amination↗
PubMed1985

Biological activities of human recombinant interferon alpha/beta targeted by anti-Epstein-Barr virus monoclonal antibodies.

The requirement of high doses of interferon (IFN) during therapy severely restrict its application. Thus a model using an Epstein-Barr virus (EBV) membrane antigen (MA) specific monoclonal antibody (MAb) was developed to assess the feasibility of coupling minimal amounts of IFN to a MAb and specifically delivering the IFN to the target cells. Coupled IFN was first shown to retain fully both its anti-viral and anti-proliferative properties when tested on human tumor cell lines QIMR-WIL (EBV-MA+) and the U-266 (EBV-MA-). A series of in vitro pulsing experiments demonstrated the specific targeting of both the anti-viral and anti-proliferative properties of IFN to the EBV-MA+ QIMR-WIL cells and not EBV-MA- cell lines.

Antibodies, Monoclonal↗
PubMed1985

Rheumatic fever-associated B cell alloantigens as identified by monoclonal antibodies.

Mice immunized with B lymphocytes obtained from patients who had had well-documented rheumatic fever in the past yielded 2 monoclonal antibodies, termed 83S19.23 and 256S10, which identified certain alloantigens present on the B cells of these patients. The frequency of the B cell marker detected by clone 83S19.23 in rheumatic fever patients was found to be 59%, 77%, and 74% in India, New Mexico, and New York, respectively. Monoclonal antibody 256S10 identified 75% of those rheumatic fever patients who were nonreactive to clone 83S19.23. Thus, the 2 antibodies identify approximately 92% of all rheumatic fever patients and suggest the presence of a diallelic genetic marker for susceptibility to rheumatic fever.

Antibodies, Monoclonal↗
PubMed1985

Induction of tumouricidal leucocytes by the intranasal application of MTP-PE, a lipophilic muramyl peptide.

Single intranasal applications of MTP-PE, a lipophilic muramyl peptide, induce tumouricidal and tumouristatic leucocytes in the lungs of rats. In ex vivo assays the tumouristatic activity was detectable for 8 days after drug administration. By separation of the effector cells on Ficoll-Hypaque gradients, it was shown that both neutrophils and macrophages are responsible for this activity. Using the B16/BL6 melanoma system in mice, there was a high survival rate after repeated intranasal applications of MTP-PE.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1984

Glycosphingolipid-blotting: an immunological detection procedure after separation by thin layer chromatography.

A method for detecting glycosphingolipids (GSL) in situ after thin layer chromatography is described. The separated GSL are transferred by diffusion to nitrocellulose. The replica is incubated with poly- or monoclonal antibodies and bound antibodies are detected with second antibodies coupled to peroxidase. Advantages of the procedure are its speed, the non-radioactive detection method, and its suitability for screening applications. In addition, small scale affinity purification of antibodies from the replicas is possible. The presence of Forssman antigen in mouse tissues and the reaction of monoclonal antibodies with human GSL is demonstrated.

Animals↗
PubMed1984

Monoclonal antibodies against simian virus 40 nuclear large T tumour antigen: epitope mapping, papova virus cross-reaction and cell surface staining.

Thirty six cloned hybridomas have been isolated which produce monoclonal antibodies directed against simian virus 40 (SV40) large T tumour antigen. They have been shown to recognize at least six different epitopes along the T antigen polypeptide according to their reaction with the various truncated forms of T antigen expressed by adenovirus-SV 40 hybrid viruses. Sixteen antibodies cross-react with cells infected by the closely related human BK virus. Only two antibodies, PAb1604 and PAb1614, directed against different epitopes of the SV40 T antigen, cross-react with polyoma large T tumour antigen which has a more limited amino acid sequence homology. This cross-reaction is rarely seen with polyclonal antibodies. Monoclonal antibody PAb1620 gave nuclear immunofluorescence only with murine cells transformed by SV40 and was found to react with a complex of T-antigen and 53 000-dalton host-coded protein. All the monoclonal antibodies react with nuclear T antigen and all but four antibodies stained the surface of SV40-transformed cells. These were four of the five antibodies directed against the central third of the T antigen. Thus the monoclonal antibodies show that cell surface T antigen differs from nuclear T antigen, either in accessibility or structure.

Animals↗
PubMed1984

Microisolation of DABITC-derivatized protein by gel electrophoresis: application to the purification of antibody heavy and light chains.

Proteins and polypeptides are derivatized with dimethylaminoazobenzene isothiocyanate (DABITC) before their separation on sodium dodecyl sulfate-polyacrylamide gels. DABITC-derivatized proteins are detected visually in the picomole range without further staining and destaining procedures. The recovered colored protein can also be used for direct sequence determinations. This method was applied to purify heavy and light chains of murine hybridoma-derived antibodies. Starting with ammonium sulfate-precipitated protein mixture, this method allows, in a one-step procedure, the isolation of pure light and heavy chains suitable for automatic sequencing.

Amino Acid Sequence↗
PubMed1984

Antiviral and antiproliferative effects of interferons delivered via monoclonal antibodies.

We present in vitro experiments demonstrating the feasibility of specifically targeting IFNs by coupling to monoclonal antibodies (MAbs). Human interferon alpha (HuIFN-alpha), both natural and recombinant, were coupled to Epstein-Barr virus (EBV) membrane antigen (MA) specific MAbs and shown to retain full antiviral and antiproliferative activities when tested on several human cell lines. A series of in vitro pulsing experiments demonstrated that a minimum amount of MAb-delivered IFN exerts pronounced antiviral and antiproliferative effects on human QIMR-WIL target cells which carry EBV (viral) membrane antigens (EBV-MA+) and not on U-266 or Hep2 cell lines (EBV-MA-). Additionally the antiproliferative action of MAb-coupled rIFN-alpha appeared to be enhanced compared with that of uncoupled.

Antibodies, Monoclonal↗
PubMed1984

Murine V kappa 25 and V kappa 27 amino-acid sequences of C57B1/6 origin: monoclonal antibodies 17S29.1 and 22S25.1 specific for the group A-streptococcal polysaccharide.

Antibodies 17S29.1 and 22S25.1 are monoclonal, hybridoma-derived gamma 3 kappa murine immunoglobulins with specificity for N-acetyl-glucosamine beta 1----3-linked to the L-rhamnose backbone structure, the immunodeterminant of the streptococcal Group A polysaccharide. The VL 17S29.1 amino-acid sequence is the third complete one reported from an antibody with this specificity, the second fully determined V kappa 25 structure and the first complete V kappa sequence of C57B1/6 origin derived from a carbohydrate-specific antibody. VL22S25.1 is a member of the V kappa 27 isotype of murine immunoglobulin VL regions. V kappa 17S29.1 and the determined part of the V kappa 22S25.1 sequence are compared to the previously described V kappa regions of streptococcal Group A polysaccharide-specific antibodies and to 12 selected partial and complete V kappa regions of antibodies with other specificities, predominantly to carbohydrate antigens. Both V kappa 17S29.1 and V kappa 22S25.1 increase the variability of known murine V kappa regions. They are the most homologous to the other V kappa regions derived from antibodies with streptococcal Group A polysaccharide specificity and share with them the amino-acid residue Arg74, so far characteristic for V kappa regions from antibodies with this specificity. The analysis of groups of independently expressed, highly homologous V kappa regions, namely V kappa 17S29.1 and V kappa 2S1.3 as one and V kappa 7S34.1 and V kappa 22S25.1 as a second group, offers the possibility of estimating the minimal number of V kappa germline genes involved in the immune response to the structurally defined streptococcal Group A polysaccharide antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkylation↗
PubMed1984

Murine V kappa 21A isotype sequence: monoclonal antibody 50S10.1 specific for the group A streptococcal polysaccharide.

Antibody 50S10.1 is a hybridoma-derived gamma 3 kappa antibody of BAB-14 mouse strain origin, with specificity for N-acetylglucosamine beta 1----3 linked to L-rhamnose, the immunodeterminant of the streptococcal Group A polysaccharide. The VL50S10.1 amino acid sequence is the fourth complete one reported with this specificity and the first fully determined V kappa 21A structure. Furthermore it is the first V kappa 21A isotype sequence derived from an antibody with known antigen specificity. The V kappa region of this and the previously described monoclonal anti-streptococcal Group A polysaccharide antibodies 7S34.1, 2S1.3 and 17S29.1 are compared, showing that in monoclonal antibody 50S10.1 a V kappa germline gene is expressed which is unrelated to those previously shown to be expressed in antibodies of this specificity. V kappa 50S10.1 increases the variability of known murine V kappa regions and confirms stretches of V kappa 21A sequences previously established.

Amino Acid Sequence↗
PubMed1983

Sequence determination of eglin C using combined microtechniques of amino acid analysis, peptide isolation, and automatic Edman degradation.

The complete amino acid sequence of a proteinase inhibitor, eglin c (Mr 8100), has been determined with less than 150 micrograms of the protein using the following microtechniques: (a) amino acid analysis with a low-nanogram amount of protein hydrolysate using dimethylaminoazobenzene sulfonyl chloride, (b) peptide isolation at the picomole level using the dimethylaminoazobenzene isothiocyanate (DABITC) precolumn derivatization method, and (c) automatic Edman degradation. One amino acid residue has been corrected for the previously reported sequence. The Contribution of each technique to the microsequencing is discussed. In addition, a new high-performance liquid chromatography system that gives a complete baseline separation of all phenylthiohydantoin-amino acids is described.

Amino Acid Sequence↗