Isolation and characterization of monoclonal antibodies to proline dehydrogenase from Escherichia coli K-12

1987 ◽  
Vol 65 (6) ◽  
pp. 507-513 ◽  
Author(s):  
Janet M. Wood ◽  
Kimberley A. C. C. Taylor ◽  
Denise J. McClellan ◽  
G. Gregg Lawrie ◽  
Richard L. Krogsrud ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Monoclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tween 20 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Proline Dehydrogenase ◽  
Western Blots ◽  
Isolation And Characterization ◽  
K 12

The PutA protein of Escherichia coli K-12 serves as both proline dehydrogenase and the repressor controlling the expression of genes putP and putA. Thirty-eight hybridoma cell lines were isolated using mice immunized with proline dehydrogenase purified from a bacterial membrane extract. The monoclonal antibodies secreted by those cells showed varying affinities for proline dehydrogenase by enzyme-linked immunosorbent assay (ELISA). Nine antibodies labelled the PutA protein in Western blots after sodium dodecyl sulfate – Polyacrylamide gel electrophoresis and two of the five tested also labelled the undenatured PutA protein. Three antibodies bound proteins present in a peripheral membrane protein fraction from both putA+ bacteria and a putA::Tn5 mutant strain. Urea denaturation eliminated the proline: 2,6-dichloroindophenol (DCIP) oxidoreductase activity, but did not alter the immunoreactivity of the PutA protein. Tween 20, which caused 1.8-fold increases in Km (proline) and Vmax for proline:DCIP oxidoreductase, increased the avidity of the antibody from hybridoma line 2.1C10.3 fivefold. The antibodies from hybridoma lines 2.1C10.2, 1.2C10.3, and 1.1B07.1 were shown by electron microscopy of immunogold-labelled preparations or by ELISA to bind the membrane-associated PutA protein, whereas those from hybridoma lines 2.1A08.2 and 1.4C09.1 failed to recognize that antigen form. These antibodies will serve as probes of the relationships among protein domain, conformation, and function for the PutA 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.

scholarly journals High-molecular weight kininogen is present in cultured human endothelial cells: localization, isolation, and characterization

Blood ◽  
1988 ◽  
Vol 71 (5) ◽  
pp. 1268-1276 ◽  
Author(s):  
F van Iwaarden ◽  
PG de Groot ◽  
JJ Sixma ◽  
M Berrettini ◽  
BN Bouma
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
High Molecular Weight ◽  
Light Chain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Enzyme Linked Immunosorbent Assay ◽  
Human Endothelial Cells ◽  
Isolation And Characterization

Abstract The presence of high-molecular weight (mol wt) kininogen was demonstrated in cultured human endothelial cells derived from the umbilical cord by immunofluorescence techniques. Cultured human endothelial cells contain 58 +/- 11 ng (n = 16) high-mol wt kininogen/10(6) cells as determined by an enzyme-linked immunosorbent assay (ELISA) specific for high-mol wt kininogen. High-mol wt kininogen was isolated from cultured human endothelial cells by immunoaffinity chromatography. Nonreduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that endothelial cell high-mol wt kininogen consisted of five protein bands with mol wts of 95,000, 85,000, 65,000, 46,000, and 30,000 daltons. Immunoblotting of the endothelial cell high-mol wt kininogen by using specific antisera against the heavy and light chain indicated that the 95,000-, 85,000-, and 65,000-dalton bands consisted of the heavy and light chain whereas the 46,000- and 30,000-dalton bands reacted only with the anti-light chain antiserum. Immunoprecipitation studies performed with lysed, metabolically labeled endothelial cells and monospecific antisera directed against high-mol wt kininogen suggested that high-mol wt kininogen is not synthesized by the endothelial cells. Endothelial cells cultured in high-mol wt kininogen-free medium did not contain high-mol wt kininogen. These studies indicate that endothelial cell high-mol wt kininogen was proteolytically cleaved in the culture medium and subsequently internalized by the endothelial cells. Binding and internalization studies performed with 125I-labeled, proteolytically cleaved, high-mol wt kininogen showed that endothelial cells can indeed bind and internalize proteolytically cleaved high-mol wt kininogen in a specific and saturable way.

scholarly journals Cloning, Expression, and Characterization of Fimbrial Operon F9 from Enterohemorrhagic Escherichia coli O157:H7

2006 ◽  
Vol 74 (4) ◽  
pp. 2233-2244 ◽  
Author(s):  
Alison S. Low ◽  
Francis Dziva ◽  
Alfredo G. Torres ◽  
Jessenya L. Martinez ◽  
Tracy Rosser ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Enterohemorrhagic Escherichia Coli ◽  
Wild Type ◽  
Induced Expression ◽  
E Coli ◽  
Extracellular Matrix Components ◽  
K 12

ABSTRACT Recent transposon mutagenesis studies with two enterohemorrhagic Escherichia coli (EHEC) strains, a sero- type O26:H- strain and a serotype O157:H7 strain, led to identification of a putative fimbrial operon that promotes colonization of young calves (1 to 2 weeks old). The distribution of the gene encoding the major fimbrial subunit present in O-island 61 of EHEC O157:H7 in a characterized set of 78 diarrheagenic E. coli strains was determined, and this gene was found in 87.2% of the strains and is therefore not an EHEC-specific region. The cluster was amplified by long-range PCR and cloned into the inducible expression vector pBAD18. Induced expression in E. coli K-12 led to production of fimbriae, as demonstrated by transmission electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The fimbriae were purified, and sera to the purified major subunit were raised and used to demonstrate expression from wild-type E. coli O157:H7 strains. Induced expression of the fimbriae, designated F9 fimbriae, was used to characterize binding to bovine epithelial cells, bovine gastrointestinal tissue explants, and extracellular matrix components. The fimbriae promoted increases in the levels of E. coli K-12 binding only to bovine epithelial cells. In contrast, induced expression of F9 fimbriae in E. coli O157:H7 significantly reduced adherence of the bacteria to bovine gastrointestinal explant tissue. This may have been due to physical hindrance of type III secretion-dependent attachment. The main F9 subunit gene was deleted in E. coli O157:H7, and the resulting mutant was compared with the wild-type strain for colonization in weaned cattle. While the shedding levels of the mutant were reduced, the animals were still colonized at the terminal rectum, indicating that the adhesin is not responsible for the rectal tropism observed but may contribute to colonization at other sites, as demonstrated previously with very young animals.

scholarly journals High-molecular weight kininogen is present in cultured human endothelial cells: localization, isolation, and characterization

Blood ◽  
1988 ◽  
Vol 71 (5) ◽  
pp. 1268-1276 ◽  
Author(s):  
F van Iwaarden ◽  
PG de Groot ◽  
JJ Sixma ◽  
M Berrettini ◽  
BN Bouma
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
High Molecular Weight ◽  
Light Chain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Enzyme Linked Immunosorbent Assay ◽  
Human Endothelial Cells ◽  
Isolation And Characterization

The presence of high-molecular weight (mol wt) kininogen was demonstrated in cultured human endothelial cells derived from the umbilical cord by immunofluorescence techniques. Cultured human endothelial cells contain 58 +/- 11 ng (n = 16) high-mol wt kininogen/10(6) cells as determined by an enzyme-linked immunosorbent assay (ELISA) specific for high-mol wt kininogen. High-mol wt kininogen was isolated from cultured human endothelial cells by immunoaffinity chromatography. Nonreduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that endothelial cell high-mol wt kininogen consisted of five protein bands with mol wts of 95,000, 85,000, 65,000, 46,000, and 30,000 daltons. Immunoblotting of the endothelial cell high-mol wt kininogen by using specific antisera against the heavy and light chain indicated that the 95,000-, 85,000-, and 65,000-dalton bands consisted of the heavy and light chain whereas the 46,000- and 30,000-dalton bands reacted only with the anti-light chain antiserum. Immunoprecipitation studies performed with lysed, metabolically labeled endothelial cells and monospecific antisera directed against high-mol wt kininogen suggested that high-mol wt kininogen is not synthesized by the endothelial cells. Endothelial cells cultured in high-mol wt kininogen-free medium did not contain high-mol wt kininogen. These studies indicate that endothelial cell high-mol wt kininogen was proteolytically cleaved in the culture medium and subsequently internalized by the endothelial cells. Binding and internalization studies performed with 125I-labeled, proteolytically cleaved, high-mol wt kininogen showed that endothelial cells can indeed bind and internalize proteolytically cleaved high-mol wt kininogen in a specific and saturable way.

Some Characteristics of the Outer Membrane Material Released by Growing Enterotoxigenic Escherichia coli

1980 ◽  
Vol 29 (2) ◽  
pp. 704-713 ◽  
Author(s):  
Henk Gankema ◽  
Jan Wensink ◽  
Pieter A. M. Guinée ◽  
Wim H. Jansen ◽  
Bernard Witholt
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Freeze Fracture ◽  
Sucrose Density Gradient ◽  
Enterotoxigenic Escherichia Coli ◽  
E Coli ◽  
Freeze Fracture Electron Microscopy ◽  
K 12

The high-molecular-weight material released into the medium by Escherichia coli AP1, an enterotoxigenic strain of porcine origin, has been isolated and resolved into two clearly distinct fractions, based on sucrose density gradient and differential centrifugation, chemical analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and freeze-fracture electron microscopy. These two fractions, referred to as “medium vesicles” and “medium lipopolysaccharides”, were compared with the cellular outer and cytoplasmic membranes, the periplasmic fraction, and the cytoplasmic fraction. The medium vesicles closely resembled outer membrane and accounted for 3 to 5% of the total cellular outer membrane. They contained most of the heat-labile enterotoxin (LT) activity released into the medium by E. coli AP1. The medium lipopolysaccharide consisted mostly of lipopolysaccharide and a small amount of outer membrane and contained relatively little LT activity. Based on experiments with E. coli K-12 strains, in which about 5% of the newly synthesized outer membrane is lost from areas of outer membrane synthesis, it is proposed that enterotoxigenic E. coli strains release LT as part of such newly synthesized outer membrane fragments and that released outer membrane fragments may function as physiologically significant LT carriers.

Expression and characterization of single-chain variable fragment antibody against staphylococcal enterotoxin A in Escherichia coli

2014 ◽  
Vol 60 (11) ◽  
pp. 737-743 ◽  
Author(s):  
Weifeng Chen ◽  
Li Hu ◽  
Aiping Liu ◽  
Jinquan Li ◽  
Fusheng Chen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Food Poisoning ◽  
Staphylococcal Enterotoxin ◽  
Enzyme Linked Immunosorbent Assay ◽  
Staphylococcal Enterotoxin A ◽  
Single Chain Variable Fragment ◽  
Single Chain

The staphylococcal enterotoxins (SEs) are potent gastrointestinal exotoxins synthesized by Staphylococcus aureus, which is responsible for various diseases including septicemia, food poisoning, and toxic shock syndrome, as well as bovine mastitis. Among them, staphylococcal enterotoxin A (SEA) is one of the most commonly present serotypes in staphylococcal food poisoning cases. In this study, the stable hybridoma 3C12 producing anti-SEA monoclonal antibody was established with an equilibrium dissociation constant (KD) of 1.48 × 10−8 mol·L−1, its ScFv-coding genes were obtained and then the anti-SEA single chain variable fragment (ScFv) protein was expressed in Escherichia coli. Characterization of the expressed target ScFv protein was analyzed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis, Western blot, and enzyme-linked immunosorbent assay. The results demonstrated that the recombinant anti-SEA ScFv protein retained a specific binding activity for SEA, and the KD value of the soluble ScFv was about 3.75 × 10−7 mol·L−1. The overall yield of bioactive anti-SEA ScFv in E. coli flask culture was more than 10 mg·L−1.

scholarly journals Role of interleukin-15 and interleukin-18 in the secretion of sIL-6R and sgp130 by human neutrophils

2003 ◽  
Vol 12 (3) ◽  
pp. 179-183 ◽  
Author(s):  
E. Jablonska ◽  
M. Marcinczyk
Keyword(s):  
Monoclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Neutrophils ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cytoplasmic Proteins ◽  
Wide Range ◽  
Interleukin 15 ◽  
Culture Supernatants

Background:Available data indicate that neutrophils (PMN) produce a wide range of cytokines with the potential to modulate immune response. Recent investigation have shown that interleukin (IL)-15 and IL-18 potentiated several functions of normal neutrophils. It has been reported that IL-18-induced cytokine production may be significantly enhanced by coincident addition of IL-15.Aims:In the present study we compared the effect of recombinant human (rh)IL-15 and rhIL-18 as well as effect of a rhIL-15 and rhIL-18 combination on the induction secretion of sIL-6Rα and sgp130 by human neutrophils. Methods: PMN were isolated from heparinized whole blood of healthy persons. The PMN were cultured for 18 h at 37°C in a humidified incubator with 5% CO2. rhIL-15 and/or rhIL-18 and lipopolysaccharide were tested to PMN stimulation. The culture supernatants of PMN were removed and examined for the presence of sIL-6R and sgp130 by human enzyme-linked immunosorbent assay kits. Cytoplasmic protein fractions of PMN were analysed for the presence of sIL-6R and sgp130 by western blotting using monoclonal antibodies capable of detecting these proteins. Cells were lysed and cytoplasmic proteins were electrophoresed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The resolved proteins were transferred onto nitrocellulose and incubated with the primary monoclonal antibodies anti-sIL-6R and anti-sgp130. The membranes were incubated at room temperature with alkaline phosphatase anti-mouse immunoglobulin G. Immunoreactive protein bans were visualized by an AP Conjugate Substrate Kit.Results and conclusion:The results of our investigation revealed that IL-15 alone, similarly to IL-18, has no significant ability for the regulation of both soluble IL-6 receptors, sIL-6R and sgp130, released by human neutrophils. It is interesting to note that the secretion of sgp130 was changed after PMN stimulation with rhIL-15 in the presence of rhIL-18. The combination of rhIL-15 and rhIL-18 was shown to induce PMN to secretion relatively higher amounts of sgp130 compared with the stimulation of PMN with rhIL-15 alone and rhIL-18 alone. The results obtained suggest that IL-15 and IL-18, belonging to the inflammatory cytokines, through the regulation of sgp130 secretion must be also considered as anti-inflammatory mediators that may influence the balance reactions mediated by the IL-6 cytokine family.

scholarly journals Variable major proteins of Borrellia hermsii.

1982 ◽  
Vol 156 (5) ◽  
pp. 1312-1324 ◽  
Author(s):  
A G Barbour ◽  
S L Tessier ◽  
H G Stoenner
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibodies ◽  
Antigenic Variation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Major Protein ◽  
Relapsing Fever ◽  
Western Blots

Borrelia hermsii, a relapsing fever agent, manifests antigenic variation in vivo and in vitro. We studied three mouse-passaged serotypes of strain HS1 (7, 14, and 21) and a HS1 derivative obtained after multiple in vitro passages (C serotype). All four serotypes had two major proteins in whole cell lysates fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One major protein species (pII) had the same apparent subunit molecular weight (or approximately 3.9 X 10(4) in all the serotypes. In contrast, the other abundant protein in lysates, pI, had a different apparent molecular weight in each serotype. In one gel the molecular weights of pIc, pI7, pI14, and pI21 were 1.9, 4.2, 4.1, and 4.0 X 10(4), respectively. Serotype-specific mouse antisera bound to both hemologous and heterologous pIIs, to homologous pI, but not to heterologous pI in Western blots. Hybridomas were raised from spleens of mice infected with B. hermsii. Monoclonal antibodies were identified by immunofluorescence assays using whole organisms. Monoclonal antibodies specific for serotype 7 (H1826) or for serotype 21 (H3326) bound only to pI7 or pI21, respectively, in Western blots. The surface location of the pI was suggested not only by the immunofluorescence studies but also by the labeling of pI7 and pI21 when whole cells of serotypes 7 and 21 were incubated with 125I in the presence of Iodogen. Under the same circumstances, pII was relatively poorly labeled. These studies have identified the variable pI proteins of B. hermsii as serotype-specific antigens. A change from one pI to another may be the basis of antigenic variation of Borrelia species during relapsing fever.

scholarly journals TechLab and Alexon Giardia Enzyme-Linked Immunosorbent Assay Kits Detect Cyst Wall Protein 1

1999 ◽  
Vol 37 (3) ◽  
pp. 611-614 ◽  
Author(s):  
James H. Boone ◽  
Tracy D. Wilkins ◽  
Theodore E. Nash ◽  
Jill E. Brandon ◽  
Elizabeth A. Macias ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Cyst Wall ◽  
Fluorescent Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Acid Sequences ◽  
Enzyme Linked Immunosorbent Assay ◽  
Stool Specimens ◽  
Human Stool ◽  
Immunofluorescence Antibody

A Giardia lamblia antigen detected by the TechLabGiardia Test (TechLab, Inc., Blacksburg, Va.) and the Alexon ProSpecT Giardia microplate assay (Alexon, Inc., Sunnyvale, Calif.) was purified by immunoaffinity chromatography from supernatant fluids of encystment cultures. Two major proteins (M r 22,000 and 26,000) were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie staining that did not resemble the GSA65 antigen reportedly detected by the Alexon test. These proteins reacted intensely with the monoclonal antibodies used in both commercial enzyme-linked immunosorbent assays (ELISAs). Both proteins had identical N-terminal amino acid sequences and were identified as cyst wall protein 1 (CWP1). The 26-kDa form appeared early during encystment followed by the appearance of the 22-kDa form. Recombinant CWP1 (M r 26,000) was strongly positive in both commercial tests. CWP1 was stable in human stool specimens, resistant to degradation by proteases andN- and O-glycanases, and unaffected by oxidation with sodium periodate. Two minor proteins withM rs of 32,000 and 39,000 were detected in CWP1 preparations by using a sensitive fluorescent protein stain. Both were identified as CWP2, and neither reacted with the monoclonal antibodies from the commercial tests. We analyzed 535 stool specimens for CWP1 by using both commercial ELISAs and resolved discrepant results by using routine ova and parasite examination (O&P) and on immunofluorescence antibody assay. The presence of CWP1 correlated well between both ELISAs (98.7% correlation). Our results demonstrate that both commercial ELISAs detect CWP1, which is a useful diagnostic marker because it is highly stable, is secreted in large amounts by encysting trophozoites, and correlates well with O&P.

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