Detection and Isolation of Yersinia pestis Without Fraction 1 Antigen by Monoclonal Antibody in Foods and Water

Most available immunoassays for Yersinia pestis are based on the detection of fraction 1 antigen (F1) when yersiniae are grown at 37°C. A monoclonal antibody (MAb) was developed based on the detection of surface antigens that are not F1. F1-deficient Y. pestis cells were induced and used to immunize BALB/c mice from which MAb (immunoglobulin G1), which specifically recognizes Y. pestis, with or without F1, was obtained. This MAb (6B5) did not cross-react with enteric bacteria, including Yersinia enterocolitica. Enzyme-linked immunosorbent assay results revealed that MAb 6B5 is specific for Y. pestis, with the exception of a minor cross-reaction with Yersinia pseudotuberculosis. Western immunoblot analysis revealed that MAb 6B5 recognizes a Y. pestis outer membrane protein of ca. 30 kDa. Magnetic beads that were coated with MAb 6B5 were compared with beads coated with polyclonal antibody (PAb; rabbit) against Y. pestis for the isolation of Y. pestis in food and water samples by using a PATHATRIX cell concentration apparatus. Enrichment cultures of Y. pestis in different foods by using two different times (6 and 24 h) in brain heart infusion broth at 37°C were evaluated. Results revealed MAb 6B5–coated magnetic beads were equivalent to magnetic beads coated with PAb against Y. pestis A1122 whole cells in concentrating Y. pestis for isolation, especially when samples were enriched for 6 h. However, the selectivity for Y. pestis of the magnetic beads coated with MAb 6B5 was greater than that coated with PAb.

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Colonic Bacteria Express an Ulcerative Colitis pANCA-Related Protein Epitope

Infection and Immunity ◽

10.1128/iai.68.3.1542-1548.2000 ◽

2000 ◽

Vol 68 (3) ◽

pp. 1542-1548 ◽

Cited By ~ 140

Author(s):

O. Cohavy ◽

D. Bruckner ◽

L. K. Gordon ◽

R. Misra ◽

B. Wei ◽

...

Keyword(s):

Ulcerative Colitis ◽

Monoclonal Antibody ◽

Immune Response ◽

Disease Process ◽

Immunoblot Analysis ◽

Culture Conditions ◽

Enzyme Linked Immunosorbent Assay ◽

E Coli ◽

Colonic Bacteria ◽

Human Sera

ABSTRACT Bacteria are a suspected pathogenic factor in inflammatory bowel disease, but the identity of the relevant microbial species remains unresolved. The pANCA autoantibody is associated with most cases of ulcerative colitis (UC) and hence reflects an immune response associated with the disease process. This study addresses the hypothesis that pANCA identifies an antigen(s) expressed by bacteria resident in the human colonic mucosa. Libraries of colonic bacteria were generated using aerobic and anaerobic microbiologic culture conditions, and bacterial pools and clonal isolates were evaluated for cross-reactive antigens by immunoblot analysis using the pANCA monoclonal antibody Fab 5-3. Two major species of proteins immunoreactive to pANCA monoclonal antibodies were detected in bacteria from the anaerobic libraries. Colony isolates of the expressing bacteria were identified as Bacteroides caccae andEscherichia coli. Isolation and partial sequencing of theB. caccae antigen identified a 100-kDa protein without database homologous sequences. The E. coli protein was biochemically and genetically identified as the outer membrane porin OmpC. Enzyme-linked immunosorbent assay with human sera demonstrated elevated immunoglobulin G anti-OmpC in UC patients compared to healthy controls. These findings demonstrate that a pANCA monoclonal antibody detects a recurrent protein epitope expressed by colonic bacteria and implicates colonic bacterial proteins as a target of the disease-associated immune response.

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Immunochemical localization of the epitope for a monoclonal antibody that neutralizes human platelet-derived growth factor mitogenic activity

Molecular and Cellular Biology ◽

10.1128/mcb.9.8.3538-3542.1989 ◽

1989 ◽

Vol 9 (8) ◽

pp. 3538-3542

Author(s):

W J LaRochelle ◽

K C Robbins ◽

S A Aaronson

Keyword(s):

Monoclonal Antibody ◽

Growth Factor ◽

Receptor Binding ◽

Human Platelet ◽

Immunoblot Analysis ◽

Platelet Derived Growth Factor ◽

Enzyme Linked Immunosorbent Assay ◽

Pdgf Receptor ◽

Cognate Receptor ◽

Peptide Antibody

A monoclonal antibody (mAb), sis 1, generated against human c-sis-encoded platelet-derived growth factor (PDGF) BB, was shown by enzyme-linked immunosorbent assay and Western blot (immunoblot) analysis to recognize human PDGF BB and human platelet PDGF AB but not the human PDGF AA. This monoclonal antibody potently inhibited PDGF receptor-binding and mitogenic activities of both human PDGF BB and PDGF AB but had no effect on PDGF AA. Finally, we demonstrated that an immunoaffinity-purified anti-c-sis peptide antibody (anti-V4) which also blocked binding of PDGF BB to its cognate receptor and competed with mAb sis 1 for binding to PDGF BB. All of these results suggest that mAb sis 1 recognizes an epitope of the c-sis gene product, PDGF BB, that spatially overlaps the V4 surface domain of PDGF BB, immunochemically localizing a region of PDGF BB critical for PDGF receptor binding and activation.

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Unstable Expression and Thermal Instability of a Species-Specific Cell Surface Epitope Associated with a 66-Kilodalton Antigen Recognized by Monoclonal Antibody EM-7G1 within Serotypes of Listeria monocytogenes Grown in Nonselective and Selective Broths

Applied and Environmental Microbiology ◽

10.1128/aem.64.8.3070-3074.1998 ◽

1998 ◽

Vol 64 (8) ◽

pp. 3070-3074 ◽

Cited By ~ 18

Author(s):

Ramakrishna Nannapaneni ◽

Robert Story ◽

Arun K. Bhunia ◽

Michael G. Johnson

Keyword(s):

Monoclonal Antibody ◽

Listeria Monocytogenes ◽

Cell Surface ◽

Brain Heart Infusion ◽

Live Cells ◽

Enzyme Linked Immunosorbent Assay ◽

Specific Cell ◽

Enrichment Broth ◽

Species Specific ◽

Heat Instability

ABSTRACT Conditions that resulted in unstable expression and heat instability of a cell surface epitope associated with a 66-kDa antigen in Listeria monocytogenes serotypes were identified with the probe monoclonal antibody (MAb) EM-7G1 in an enzyme-linked immunosorbent assay. This epitope appeared to be absent in three serotypes (serotypes 3b, 4a, and 4c), which did not react with MAb EM-7G1 irrespective of the enrichment broth tested. The remaining 10 serotypes were detected by MAb EM-7G1 only when cells were grown in nonselective brain heart infusion broth (BHI) or selectiveListeria enrichment broth (LEB). When cells were grown inListeria repair broth (LRB), only 6 of the 13 serotypes were detected by MAb EM-7G1, and recognition of serogroup 4 was completely lost. None of the 13 serotypes was detected by MAb EM-7G1 when cells were grown in two other commonly usedListeria-selective media, UVM1 broth and Fraser broth (FRB), indicating that possible loss of epitope expression occurred under these conditions. MAb EM-7G1 maintained species specificity without cross-reacting with live or heat-killed cells of six otherListeria spp. (Listeria ivanovii,Listeria innocua, Listeria seeligeri,Listeria welshimeri, Listeria grayi, andListeria murrayi) irrespective of the enrichment conditions tested. Due to heat instability of the cell surface epitope when it was exposed to 80 or 100°C for 20 min, MAb EM-7G1 is suitable for detection of live cells of L. monocytogenes in BHI or LEB but not in LRB, UVM1, or FRB enrichment medium.

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Caspase-1 Activation in Macrophages Infected with Yersinia pestis KIM Requires the Type III Secretion System Effector YopJ

Infection and Immunity ◽

10.1128/iai.01695-07 ◽

2008 ◽

Vol 76 (9) ◽

pp. 3911-3923 ◽

Cited By ~ 51

Author(s):

Sarit Lilo ◽

Ying Zheng ◽

James B. Bliska

Keyword(s):

Cell Death ◽

Yersinia Pestis ◽

Type Iii Secretion ◽

Yersinia Pseudotuberculosis ◽

Type Iii Secretion System ◽

Secretion System ◽

Host Cells ◽

Enzyme Linked Immunosorbent Assay ◽

Type Iii ◽

Caspase 1

ABSTRACT Pathogenic Yersinia species utilize a type III secretion system (T3SS) to translocate effectors called Yersinia outer proteins (Yops) into infected host cells. Previous studies demonstrated a role for effector Yops in the inhibition of caspase-1-mediated cell death and secretion of interleukin-1β (IL-1β) in naïve macrophages infected with Yersinia enterocolitica. Naïve murine macrophages were infected with a panel of different Yersinia pestis and Yersinia pseudotuberculosis strains to determine whether Yops of these species inhibit caspase-1 activation. Cell death was measured by release of lactate dehydrogenase (LDH), and enzyme-linked immunosorbent assay for secreted IL-1β was used to measure caspase-1 activation. Surprisingly, isolates derived from the Y. pestis KIM strain (e.g., KIM5) displayed an unusual ability to activate caspase-1 and kill infected macrophages compared to other Y. pestis and Y. pseudotuberculosis strains tested. Secretion of IL-1β following KIM5 infection was reduced in caspase-1-deficient macrophages compared to wild-type macrophages. However, release of LDH was not reduced in caspase-1-deficient macrophages, indicating that cell death occurred independently of caspase-1. Analysis of KIM-derived strains defective for production of functional effector or translocator Yops indicated that translocation of catalytically active YopJ into macrophages was required for caspase-1 activation and cell death. Release of LDH and secretion of IL-1β were not reduced when actin polymerization was inhibited in KIM5-infected macrophages, indicating that extracellular bacteria translocating YopJ could trigger cell death and caspase-1 activation. This study uncovered a novel role for YopJ in the activation of caspase-1 in macrophages.

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Monoclonal Antibody against Babesia equi: Characterization and Potential Application of Antigen for Serodiagnosis

Journal of Clinical Microbiology ◽

10.1128/jcm.36.7.1835-1839.1998 ◽

1998 ◽

Vol 36 (7) ◽

pp. 1835-1839 ◽

Cited By ~ 14

Author(s):

Abgaandorjiin Avarzed ◽

Ikuo Igarashi ◽

Daniel T. De Waal ◽

Satoru Kawai ◽

Yukio Oomori ◽

...

Keyword(s):

Monoclonal Antibody ◽

Specific Antigen ◽

Antibody Test ◽

Immunoblot Analysis ◽

Babesia Microti ◽

Confocal Laser ◽

Enzyme Linked Immunosorbent Assay ◽

Babesia Equi ◽

Microscopic Studies

Monoclonal antibody (MAb) BEG3 was produced against Babesia equi parasites to define a species-specific antigen for diagnostic use. The MAb reacted with single, paired, and Maltese cross forms of B. equi, and no reaction was observed with this MAb on acetone-fixed Babesia caballi, Babesia ovata, or Babesia microti parasites in the indirect immunofluorescent antibody test. Confocal laser and immunoelectron microscopic studies showed that the antigen which was recognized by this MAb was located on the surface of B. equi parasites. This MAb recognized a 19-kDa protein of B. equi antigen and did not react with B. caballi antigen or normal horse erythrocytes in immunoblot analysis. This MAb also significantly inhibited the in vitro growth of the B. equi parasite. Preliminary studies using partially purified antigen, which was separated by high-pressure liquid chromatography and recognized by the MAb, suggested that it is a suitable antigen for enzyme-linked immunosorbent assay detection of anti-B. equi antibodies in naturally infected horse sera.

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Molecular Characterization of KatY (Antigen 5), a Thermoregulated Chromosomally Encoded Catalase-Peroxidase of Yersinia pestis

Journal of Bacteriology ◽

10.1128/jb.181.10.3114-3122.1999 ◽

1999 ◽

Vol 181 (10) ◽

pp. 3114-3122 ◽

Cited By ~ 37

Author(s):

Emilio Garcia ◽

Yuri A. Nedialkov ◽

Jeffrey Elliott ◽

Vladimir L. Motin ◽

Robert R. Brubaker

Keyword(s):

Molecular Mass ◽

Yersinia Pestis ◽

Yersinia Pseudotuberculosis ◽

Rabbit Antiserum ◽

Deae Cellulose ◽

Recognition Sequence ◽

E Coli ◽

Catalase Peroxidase ◽

A Minor

ABSTRACT The first temperature-dependent proteins (expressed at 37°C, but not 26°C) to be identified in Yersinia pestis were antigens 3 (fraction 1), 4 (pH 6 antigen), and 5 (hereafter termed KatY). Antigens 3 and 4 are now established virulence factors, whereas little is known about KatY, except that it is encoded chromosomally, produced in abundance, possesses modest catalase activity, and is shared by Yersinia pseudotuberculosis, but notYersinia enterocolitica. We report here an improved chromatographic method (DEAE-cellulose, calcium hydroxylapatite, and Sephadex G-150) that yields enzymatically active KatY (2,423 U/mg of protein). Corresponding mouse monoclonal antibody 1B70.1 detected plasminogen activator-mediated hydrolysis of KatY, and a polyclonal rabbit antiserum raised against outer membranes of Y. pestis was enriched for anti-KatY. A sequenced ∼16-kb Y. pestis DNA insert of a positive pLG338 clone indicated thatkatY encodes an 81.4-kDa protein (pI 6.98) containing a leader sequence of 2.6 kDa; the deduced molecular mass and pI of processed KatY were 78.8 kDa and 6.43, respectively. A minor truncated variant (predicted molecular mass of 53.6 kDa) was also expressed. KatY is similar (39 to 59% identity) to vegetative bacterial catalase-peroxidases (KatG in Escherichia coli) and is closely related to plasmid-encoded KatP of enterohemorrhagic E. coli O157:H7 (75% identity). katY encoded a putative Ca2+-binding site, and its promoter contained three homologues to the consensus recognition sequence of the pCD-encoded transcriptional activator LcrF. rbsA was located upstream of katY, and cybB, cybC,dmsABC, and araD were mapped downstream. These genes are not linked to katG or katP inE. coli.

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Immunochemical localization of the epitope for a monoclonal antibody that neutralizes human platelet-derived growth factor mitogenic activity.

Molecular and Cellular Biology ◽

10.1128/mcb.9.8.3538 ◽

1989 ◽

Vol 9 (8) ◽

pp. 3538-3542 ◽

Cited By ~ 14

Author(s):

W J LaRochelle ◽

K C Robbins ◽

S A Aaronson

Keyword(s):

Monoclonal Antibody ◽

Growth Factor ◽

Receptor Binding ◽

Human Platelet ◽

Immunoblot Analysis ◽

Platelet Derived Growth Factor ◽

Enzyme Linked Immunosorbent Assay ◽

Pdgf Receptor ◽

Cognate Receptor ◽

Peptide Antibody

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Vaccination of Mice with a Yop Translocon Complex Elicits Antibodies That Are Protective against Infection with F1−Yersinia pestis

Infection and Immunity ◽

10.1128/iai.00189-08 ◽

2008 ◽

Vol 76 (11) ◽

pp. 5181-5190 ◽

Cited By ~ 30

Author(s):

Maya I. Ivanov ◽

Betty L. Noel ◽

Ryan Rampersaud ◽

Patricio Mena ◽

Jorge L. Benach ◽

...

Keyword(s):

Yersinia Pestis ◽

Yersinia Pseudotuberculosis ◽

Protective Antigen ◽

High Titer ◽

Inflammatory Factors ◽

Enzyme Linked Immunosorbent Assay ◽

Lethal Challenge ◽

Host Protection ◽

Bacterial Agent ◽

Cell Plasma

ABSTRACT Yersinia pestis, the bacterial agent of plague, secretes several proteins important for pathogenesis or host protection. The F1 protein forms a capsule on the bacterial cell surface and is a well-characterized protective antigen but is not essential for virulence. A type III secretion system that is essential for virulence exports Yop proteins, which function as antiphagocytic or anti-inflammatory factors. Yop effectors (e.g., YopE) are delivered across the host cell plasma membrane by a translocon, composed of YopB and YopD. Complexes of YopB, YopD, and YopE (BDE) secreted by Yersinia pseudotuberculosis were purified by affinity chromatography and used as immunogens to determine if antibodies to the translocon could provide protection against Y. pestis in mice. Mice vaccinated with BDE generated high-titer immunoglobulin G antibodies specific for BDE, as shown by enzyme-linked immunosorbent assay and immunoblotting, and were protected against lethal intravenous challenge with F1− but not F1+ Y. pestis. Mice passively immunized with anti-BDE serum were protected from lethal challenge with F1− Y. pestis. The YopB protein or a complex of YopB and YopD (BD) was purified and determined by vaccination to be immunogenic in mice. Mice actively vaccinated with BD or passively vaccinated with anti-BD serum were protected against lethal challenge with F1− Y. pestis. These results indicate that anti-translocon antibodies can be used as immunotherapy to treat infections by F1− Y. pestis.

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