Use of monoclonal antibodies specific for the a determinant of K88 pili for detection of enterotoxigenic Escherichia coli in pigs

Westerman, R B; Fortner, G W; Mills, Ken; Phillips, R. E.; Greenwood, J. M.

doi:10.1128/jcm.31.2.311-314.1993

Cited by 6 publications

(3 citation statements)

References 19 publications

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“…Production of hybridomas. The methods used for the production and screening of hybridomas have been described previously (39)(40)(41). Briefly, microtiter plates were prepared for the fusion by adding 3 ϫ 10 3 mouse peritoneal macrophages in Dulbecco's modified Eagle medium (Gibco BRL, Grand Island, N.Y.) supplemented with hypoxanthine-aminopterin-thymidine (ATCC), 10% hybridoma cloning factor (Sigma Chemical Company), and 15% heat-inactivated (56ЊC, 30 min) fetal bovine serum (Hyclone Laboratories, Logan, Utah) to each well on the day before the fusion.…”

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Production and characterization of monoclonal antibodies specific for the lipopolysaccharide of Escherichia coli O157

Westerman

Keen

et al. 1997

J Clin Microbiol

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Identification of the O157 antigen is an essential part of the detection of Escherichia coli O157:H7, which is recognized as a major etiologic agent of hemorrhagic colitis. However, polyclonal antibodies produced against E. coli O157:H7 lipopolysaccharide (LPS) may react with several other bacteria including Brucella abortus, Brucella melitensis, Yersinia enterocolitica O9, Escherichia hermannii, and Stenotrophomonas maltophilia. We produced eight monoclonal antibodies (MAbs) specific for the LPS of E. coli O157. Western blots (immunoblots) of both the phenol phase (smooth) and the aqueous phase (rough) of hot phenol-water-purified LPS indicated that three of the MAbs were specific for the O antigen and five were reactive with the LPS core. The eight MAbs could be further differentiated by their reactivities to Salmonella O30 LPS (group N), which is reported to be identical to the E. coli O157 antigen. All eight MAbs reacted strongly to all of the 64 strains of E. coli O157 tested, which included 47 isolates of O157:H7 and 17 other O157 strains. None of the eight MAbs cross-reacted with any of the 38 other E. coli serotypes tested, which consisted of 29 different O-antigen serotypes, or with 38 strains (22 genera) of nonE. coli gram-negative enteric bacteria. Escherichia coli O157:H7 has been identified as a major etiologic agent of hemorrhagic colitis (31), which is occasionally accompanied by serious complications such as hemolyticuremic syndrome (18, 24) or thrombic thrombocytopenic purpura (7). Recent studies have shown that beef and dairy cattle are natural reservoirs of E. coli O157:H7 (2, 43) and that E. coli O157:H7 can be isolated from the feces of asymptomatic cattle (2, 22), from raw milk (2, 9), and from poultry, pork, and lamb (12, 30). However, undercooked ground beef is the major source of E. coli O157:H7 in food-borne outbreaks (8). The E. coli O157:H7 serotype has traditionally been identified by agglutination tests (14, 21) or immunofluorescence assays (25) with polyclonal antibodies. However, use of polyclonal antisera may result in false-positive identification of E. coli O157 (3, 20). For example, polyclonal anti-O157 E. coli antisera may cross-react with E. coli O7 and O116 (13, 27). In addition, other bacteria possess cross-reacting epitopes which mimic epitopes on the lipopolysaccharide (LPS) of E. coli

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Production and characterization of monoclonal antibodies specific for the lipopolysaccharide of Escherichia coli O157

Westerman

Keen

et al. 1997

J Clin Microbiol

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“…ELISA for screening of hybridomas. The ELISA method for the screening of hybridomas has previously been described in detail (27,28). Briefly, 0.1 ml of spent hybridoma medium was added to wells coated with spirochetes.…”

Section: Methodsmentioning

confidence: 99%

Production and characterization of monoclonal antibodies specific for lipooligosaccharide of Serpulina hyodysenteriae

1995

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Serpulina (Treponema) hyodysenteriae is the causative agent of swine dysentery, a contagious mucohemorrhagic disease of the colon. Diagnosis of swine dysentery is extremely difficult because of the presence of cross-reactive antibodies to the proteins of S. hyodysenteriae and Serpulina innocens, a nonpathogenic inhabitant of the porcine large intestine. Therefore, monoclonal antibodies (MAbs) against the serotype-specific lipooligosaccharide (LOS) antigens of S. hyodysenteriae were produced to rapidly differentiate S. hyodysenteriae from S. innocens. Whole-cell preparations of S. hyodysenteriae serotypes 1 through 7 were used as antigens. MAbs were characterized by an indirect enzyme-linked immunosorbent assay with whole-cell or LOS antigen and by Western blot (immunoblot) analysis with whole-cell lysates as antigen. A total of 12 LOS-specific MAbs which could identify and differentiate the seven original serotypes of S. hyodysenteriae were produced. The MAb serospecificities are as follows: MAb 9G8, serotype 1; MAb 31D9, serotype 2; MAb 7D3, serotypes 2 and 7; MAb 24B7, serotype 3; MAb 13C2, serotype 4; MAb 18E9, serotype 4; MAb 2B7, serotype 6; MAb 1D2, serotypes 2, 5, and 7; MAb 9C5, serotypes 2, 5, and 7; MAb 11C9, serotype 7; MAb 11E10, serotype 7; and MAb 6G11, serotype 7.

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“…5 The usual methods of detecting E coli with the K88-type ®mbriae have been seroagglutinations, 6±8 immuno¯uorescence, 6,9 or enzyme-liked immunosorbent assay (ELISA) using polyclonal antibodies 10,11 or monoclonal antibodies. 12 The egg yolk of chicken is recognized as a rich source of speci®c antibodies. 13 These include the potential of producing more speci®c antibodies against mammalian antigens in birds compared to mammals because of the phylogenic distance between birds and mammals, low cost of production and convenience, and what is becoming more important, compatibility with regulations for modern animal production and animal welfare.…”

Section: Introductionmentioning

confidence: 99%

Use of chicken egg‐yolk antibodies against K88+ fimbrial antigen for quantitative analysis of enterotoxigenic Escherichia coli (ETEC) K88+ by a sandwich ELISA

Kim¹,

Jin²,

Cho³

et al. 1999

J. Sci. Food Agric.

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An enzyme-linked immunosorbent sandwich assay (ELISA) has been developed to detect and quantify both the K88 ®mbrial antigen and the concentration of bacterial cells in fecal and other samples using anti-®mbrial chicken egg-yolk and anti-®mbrial rabbit serum antibodies. The assay has a sensitivity of 40 ng ml À1 for K88 ®mbrial antigen and 10 7 CFU ml À1 for intact cells and cell homogenate. The inter-and intra-assay coef®cients of variation (CV) for intact cells, homogenates, and ®mbrial antigens were similar, suggesting that reproducible results can be obtained with any of the three preparations. In all cases the CV increased with decreasing concentration of the antigen, especially when very low concentrations of antigen were used. Fimbrial antigens had lower CV (values ranged from 2 to 37%, depending on its concentration) than standards prepared from either whole cells or homogenized cells (values ranged from 1 to 67%). The correlation between the actual number of cells as counted and the number as estimated from the ELISA using the amount of ®mbrial antigen was r = 0.98, P`0.001. The anti-K88 polyclonal antibodies from chicken serum and egg yolk had little or no cross-reactivity with K99, 987P ETEC. The correlation (r) between the fecal score for diarrhea and the number of E coli in the fecal swab was high, r =0.80 (n = 64, P`0.0001). The assay is sensitive and speci®c and provides a good estimate of the amount of ®mbrial protein or the number of K88 ETEC in the sample.

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Use of monoclonal antibodies specific for the a determinant of K88 pili for detection of enterotoxigenic Escherichia coli in pigs

Cited by 6 publications

References 19 publications

Production and characterization of monoclonal antibodies specific for the lipopolysaccharide of Escherichia coli O157

Production and characterization of monoclonal antibodies specific for the lipopolysaccharide of Escherichia coli O157

Production and characterization of monoclonal antibodies specific for lipooligosaccharide of Serpulina hyodysenteriae

Use of chicken egg‐yolk antibodies against K88+ fimbrial antigen for quantitative analysis of enterotoxigenic Escherichia coli (ETEC) K88+ by a sandwich ELISA

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