scholarly journals Characterization of two monoclonal antibodies against cytochrome b558 of human neutrophils

Blood ◽  
1989 ◽  
Vol 73 (6) ◽  
pp. 1686-1694 ◽  
Author(s):  
AJ Verhoeven ◽  
BG Bolscher ◽  
LJ Meerhof ◽  
R van Zwieten ◽  
J Keijer ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Transmembrane Protein ◽  
Autosomal Gene ◽  
Human Neutrophils ◽  
Western Blots ◽  
Binding Characteristics ◽  
Cytochrome B558 ◽  
Spectrophotometric Methods ◽  
Membrane Bound

Monoclonal antibodies (MoAbs) were raised against cytochrome b558, a membrane-bound component of the NADPH:O2 oxidoreductase in human neutrophils. This cytochrome consists of a low-molecular-weight (low- mol-wt) subunit of 22 to 23 Kd, probably encoded by an autosomal gene, and a high-mol-wt subunit of 75 to 90 Kd, encoded on the X-chromosome. MoAb 449 reacts with the low-mol-wt subunit and MoAb 48 with the high- mol-wt subunit on Western blots of purified cytochrome b558 and on blots of whole neutrophil extracts. In extracts of neutrophils from patients with chronic granulomatous disease (CGD) in which cytochrome b558 is not detectable by spectrophotometric methods, the low-mol-wt subunit is present, albeit in a much smaller amount. The high-mol-wt subunit is not detected by MoAb 48 in neutrophils of patients with X- linked CGD and in neutrophils of patients with the autosomal, cytochrome-b558-negative form of the disease. These results can be explained by a marked instability of these subunits when the synthesis of either of the two is disturbed. In differentiated HL-60 cells, the high-mol-wt subunit appears to be present in a different form. Cloning of the low-mol-wt subunit with the help of MoAb 449 suggests the presence of a heme-binding site on this subunit. By comparison of the binding characteristics of MoAb 449 to intact and permeabilized neutrophils with those of MoAb 7D5, recently isolated by Nakamura et al (Blood 69:1404, 1987), the low-mol-wt subunit was established as a transmembrane protein.

scholarly journals Characterization of two monoclonal antibodies against cytochrome b558 of human neutrophils

Blood ◽  
1989 ◽  
Vol 73 (6) ◽  
pp. 1686-1694 ◽  
Author(s):  
AJ Verhoeven ◽  
BG Bolscher ◽  
LJ Meerhof ◽  
R van Zwieten ◽  
J Keijer ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Transmembrane Protein ◽  
Autosomal Gene ◽  
Human Neutrophils ◽  
Western Blots ◽  
Binding Characteristics ◽  
Cytochrome B558 ◽  
Spectrophotometric Methods ◽  
Membrane Bound

Abstract Monoclonal antibodies (MoAbs) were raised against cytochrome b558, a membrane-bound component of the NADPH:O2 oxidoreductase in human neutrophils. This cytochrome consists of a low-molecular-weight (low- mol-wt) subunit of 22 to 23 Kd, probably encoded by an autosomal gene, and a high-mol-wt subunit of 75 to 90 Kd, encoded on the X-chromosome. MoAb 449 reacts with the low-mol-wt subunit and MoAb 48 with the high- mol-wt subunit on Western blots of purified cytochrome b558 and on blots of whole neutrophil extracts. In extracts of neutrophils from patients with chronic granulomatous disease (CGD) in which cytochrome b558 is not detectable by spectrophotometric methods, the low-mol-wt subunit is present, albeit in a much smaller amount. The high-mol-wt subunit is not detected by MoAb 48 in neutrophils of patients with X- linked CGD and in neutrophils of patients with the autosomal, cytochrome-b558-negative form of the disease. These results can be explained by a marked instability of these subunits when the synthesis of either of the two is disturbed. In differentiated HL-60 cells, the high-mol-wt subunit appears to be present in a different form. Cloning of the low-mol-wt subunit with the help of MoAb 449 suggests the presence of a heme-binding site on this subunit. By comparison of the binding characteristics of MoAb 449 to intact and permeabilized neutrophils with those of MoAb 7D5, recently isolated by Nakamura et al (Blood 69:1404, 1987), the low-mol-wt subunit was established as a transmembrane protein.

Isolation, Cultivation, and Characterization of Borrelia burgdorferi from Rodents and Ticks in the Charleston Area of South Carolina

2000 ◽  
Vol 38 (1) ◽  
pp. 120-124
Author(s):  
J. H. Oliver ◽  
K. L. Clark ◽  
F. W. Chandler ◽  
L. Tao ◽  
A. M. James ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Borrelia Burgdorferi ◽  
Reference Strain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Western Blots ◽  
Cotton Rat ◽  
Cotton Mouse ◽  
B 31

ABSTRACT Twenty-eight Borrelia burgdorferi isolates from the Charleston, S.C., area are described. This represents the first report and characterization of the Lyme disease spirochete from that state. The isolates were obtained from December 1994 through December 1995 from the tick Ixodes scapularis , collected from vegetation, and from the rodents Peromyscus gossypinus (cotton mouse), Neotoma floridana (eastern wood rat), and Sigmodon hispidus (cotton rat). All isolates were screened immunologically by indirect immunofluorescence with monoclonal antibodies to B. burgdorferi -specific outer surface protein A (OspA) (antibodies H5332 and H3TS) and B. burgdorferi -specific OspB (antibodies H6831 and H614), a Borrelia (genus)-specific antiflagellin antibody (H9724), Borrelia hermsii -specific antibodies (H9826 and H4825), and two polyclonal antibodies (one to Borrelia species and another to B. burgdorferi ). Six of the isolates were analyzed by exposing Western blots to monoclonal antibodies H5332, H3TS, H6831, and H9724. All isolates were also analyzed by PCR with five pairs of primers known to amplify selected DNA target sequences specifically reported to be present in the reference strain, B. burgdorferi B-31. The protein profiles of six of the isolates (two from ticks, one from a cotton mouse, two from wood rats, and one from a cotton rat) also were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We conclude that the 28 Charleston isolates are B. burgdorferi sensu stricto based on their similarities to the B. burgdorferi B-31 reference strain.

Characterization of murine monoclonal antibodies againstHelicobacter pylorilipopolysaccharide specific for Lexand Leyblood group determinants

2005 ◽  
Vol 83 (5) ◽  
pp. 589-596 ◽  
Author(s):  
Eleonora Altman ◽  
Blair A Harrison ◽  
Tomoko Hirama ◽  
Vandana Chandan ◽  
Rebecca To ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Monoclonal Antibodies ◽  
Blood Group ◽  
Cell Envelope ◽  
Mucosal Cell ◽  
Blood Group Antigens ◽  
Binding Characteristics ◽  
Igg3 Subclass ◽  
H Pylori

The cell envelope of Helicobacter pylori contains lipopolysaccharide (LPS), the O-chain of which expresses type 2 Lexand Leyblood group antigens, which mimic human gastric mucosal cell-surface glycoconjugates and may contribute to the survival of H. pylori in gastric mucosa. Here we describe the generation of monoclonal antibodies specific for Lexand Leyblood group determinants and the characterization of their binding properties using purified, structurally defined H. pylori LPS, synthetic glycoconjugates, and H. pylori cells. Analysis of oligosaccharide binding by SPR provided a rapid and reliable means for characterization of antibody affinities. One of the antibodies, anti-Lex, was of IgG3 subclass and had superior binding characteristics as compared with the commercially available anti-LexIgM. These antibodies could have potential in the immunodiagnosis of certain types of cancer, in serotyping of H. pylori isolates, and in structure–function studies.Key words: Helicobacter pylori, lipopolysaccharide, monoclonal antibodies, Lewis determinants, immunodiagnosis.

scholarly journals Production and characterization of polyclonal and monoclonal antibodies against human thromboxane synthase

Blood ◽  
1990 ◽  
Vol 76 (1) ◽  
pp. 80-85 ◽  
Author(s):  
R Nusing ◽  
MP Wernet ◽  
V Ullrich
Keyword(s):  
Enzyme Activity ◽  
Monoclonal Antibodies ◽  
Western Blot ◽  
Human Lung ◽  
Human Platelet ◽  
Human Lung Tissue ◽  
Western Blots ◽  
Conformational Epitopes ◽  
Platelet Thromboxane

Abstract Polyclonal and monoclonal antibodies (MoAbs) were raised against human platelet thromboxane (Tx) synthase. Neither the antiserum nor the MoAbs inhibited the enzyme activity significantly. Three MoAbs, Tu 300, Kon 6, and Kon 7, were purified and further characterized. They are monospecific as shown by activity precipitation or Western blot analysis, and recognized different epitopes on Tx-synthase. Tu 300 could precipitate the enzyme and recognized conformational epitopes, whereas Kon 6 and Kon 7 only reacted in Western blots. Antibody Tu 300 can be used in immunohistology but shows no crossreactivity with Tx- synthase from other species. In human lung tissue staining with peroxidase, coupled Tu 300 was only found in alveolar macrophages.

scholarly journals Characterization of monoclonal antibodies that recognize band 4.5 polypeptides associated with nucleoside transport in pig erythrocytes

1987 ◽  
Vol 244 (3) ◽  
pp. 749-755 ◽  
Author(s):  
A H Good ◽  
J D Craik ◽  
S M Jarvis ◽  
F Y P Kwong ◽  
J D Young ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Specific Inhibitor ◽  
Nucleoside Transport ◽  
Erythrocyte Membranes ◽  
Prolonged Treatment ◽  
Western Blots ◽  
Reversible Binding ◽  
Transport Inhibitor ◽  
Neonatal Pig

Three monoclonal antibodies have been raised against partially purified band 4.5 polypeptides [Steck (1974) J. Cell Biol. 62, 1-19] from pig erythrocyte membranes. The antibodies were capable of binding to both intact pig erythrocytes and protein-depleted membrane preparations and recognized detergent-solubilized polypeptides from adult and neonatal pig erythrocytes that were photolabelled with [G-3H]nitrobenzylthioinosine (NBMPR), a potent specific inhibitor of nucleoside transport. The antibodies did not recognize polypeptides from neonatal pig erythrocytes that were photolabelled with the glucose-transport inhibitor [3H]cytochalasin B. Reactivity with polypeptides of apparent Mr 64,000 [10% (w/v) acrylamide gels] was demonstrated by Western-blot analysis. The antibodies recognized pig band 4.5 polypeptides after prolonged treatment with endoglycosidase F, a finding consistent with reactivity against polypeptide, rather than carbohydrate, determinants. Trypsin digestion of NBMPR-labelled protein-depleted pig erythrocyte membranes generated two labelled polypeptide fragments (Mr 43,000 and 26,000). Two of the antibodies recognized both fragments on Western blots, whereas the third bound to the larger, but not to the smaller, fragment. The antibodies had no significant effect on reversible binding of NBMPR to protein-depleted pig erythrocyte membranes and did not bind to NBMPR-labelled polypeptides in human, rabbit or mouse erythrocytes.

scholarly journals Identification and characterization of the 2D6 and Mr 23000 antigens on the plasma membrane of rat spermatozoa

1987 ◽  
Vol 241 (2) ◽  
pp. 353-360 ◽  
Author(s):  
R Jones ◽  
C R Brown
Keyword(s):  
Plasma Membrane ◽  
Antigenic Determinant ◽  
Egg Recognition ◽  
Western Blots ◽  
One Dimensional ◽  
Antigen Present ◽  
Membrane Bound ◽  
Species Specific ◽  
Identification And Characterization

Previous investigations [Jones, Brown, von Glos & Gaunt (1985) Exp. Cell Res. 156, 31-44] have demonstrated the appearance of a new antigenic determinant (recognized by monoclonal antibody 2D6) on the plasma membrane of rat spermatozoa during post-testicular maturation in the epididymis. Identification of the 2D6 antigen on Western blots from one-dimensional SDS/polyacrylamide gels revealed that it co-migrated with a membrane protein (designated Mr 23,000 antigen) present on testicular and immature germ cells, suggesting that one antigen might be a modified version of the other. In the present work, however, we demonstrate that, although they have similar Mr and are present in soluble and membrane-bound forms, the 2D6 and Mr 23,000 antigens are biochemically and immunologically distinct molecules. The properties of the antigens are described and compared. The Mr 23,000 antigen is present on both testicular and cauda epididymidal spermatozoa, has a pI of 6.1, contains no detectable carbohydrate, is not tissue-specific and is degraded by V8 protease. By contrast, the 2D6 antigen is glycosylated, has a broad pI from 4.5 to 6.1, is tissue- and species-specific and is resistant to digestion with V8 protease. Its role in sperm-egg recognition is discussed.

scholarly journals Isolation and characterization of gelatinase granules from human neutrophils

Blood ◽  
1994 ◽  
Vol 83 (6) ◽  
pp. 1640-1649 ◽  
Author(s):  
L Kjeldsen ◽  
H Sengelov ◽  
K Lollike ◽  
MH Nielsen ◽  
N Borregaard
Keyword(s):  
Plasma Membrane ◽  
Qualitative Difference ◽  
Human Neutrophils ◽  
Immunogold Electron Microscopy ◽  
Double Labeling ◽  
Intact Cells ◽  
Cytochrome B558 ◽  
Isolation And Characterization ◽  
Specific Granules

We recently confirmed the existence of gelatinase granules as a subpopulation of peroxidase-negative granules by double-labeling immunogold electron microscopy on intact cells and by subcellular fractionation. Further characterization of gelatinase granules has been hampered by poor separation of specific and gelatinase granules on both two-layer Percoll gradients and sucrose gradients. We have developed a three-layer Percoll density gradient that allows separation of the different granules and vesicles from human neutrophils; in particular, it allows separation of specific and gelatinase granules. This allows us to characterize these two granule populations with regard to their content of membrane proteins, which become incorporated into the plasma membrane during exocytosis. We found that gelatinase granules, defined as peroxidase-negative granules containing gelatinase but lacking lactoferrin, contain 50% of total cell gelatinase, with the remaining residing in specific granules. Furthermore, we found that 20% to 25% of both the adhesion protein Mac-1 and the NADPH-oxidase component cytochrome b558 is localized in gelatinase granules. Although no qualitative difference was observed between specific granules and gelatinase granules with respect to cytochrome b558 and Mac-1, stimulation of the neutrophil with FMLP resulted in a selective mobilization of the least dense peroxidase-negative granules, ie, gelatinase granules, which, in concert with secretory vesicles, furnish the plasma membrane with Mac-1 and cytochrome b558. This shows that gelatinase granules are functionally important relative to specific granules in mediating early inflammatory responses.

scholarly journals Characterization of Chemotactic Responses and Flagella of Hyphomicrobium Strain W1-1B

1998 ◽  
Vol 180 (11) ◽  
pp. 3003-3006 ◽  
Author(s):  
Laura Tuhela ◽  
Jayne B. Robinson ◽  
Olli H. Tuovinen
Keyword(s):  
Monoclonal Antibodies ◽  
Enzyme Immunoassay ◽  
Electron Micrographs ◽  
Smooth Surface ◽  
Rhizobium Meliloti ◽  
Surface Pattern ◽  
Polyacrylamide Gels ◽  
Western Blots

ABSTRACT Motile swarmer cells of Hyphomicrobium strain W1-1B displayed positive chemotactic responses toward methylamine, dimethylamine, and trimethylamine but did not display significant chemotactic responses towards methanol and arginine. Electron micrographs of negatively stained intact flagellar filaments indicated a novel striated surface pattern. The flagella were composed of two proteins of 39 and 41 kDa. Neither protein was a glycoprotein as determined by Schiff’s staining and by enzyme immunoassay. Protein fingerprints visualized from silver-stained polyacrylamide gels and Western blots of protease-digested samples indicated that the two proteins were similar but not identical. Monoclonal antibodies prepared to the complex flagella of Rhizobium meliloti cross-reacted with the striated flagella of Hyphomicrobium strain W1-1B; however, these antibodies did not cross-react with smooth-surface flagella. These results suggest that complex and striated flagella possess homologous epitope regions.

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