scholarly journals Immunomagnetic Separation and Coagglutination of Vibrio parahaemolyticus with Anti-Flagellar Protein Monoclonal Antibody

2008 ◽  
Vol 15 (10) ◽  
pp. 1541-1546 ◽  
Author(s):  
S. Datta ◽  
M. E. Janes ◽  
J. G. Simonson
Keyword(s):  
Staphylococcus Aureus ◽  
Monoclonal Antibody ◽  
Cell Suspension ◽  
Vibrio Parahaemolyticus ◽  
Vibrio Vulnificus ◽  
Immunomagnetic Separation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Selective Isolation ◽  
Flagellar Protein ◽  
Phosphate Buffered Saline

ABSTRACT Mice were immunized by injection of Vibrio parahaemolyticus ATCC 17802 polar flagellin in order to produce monoclonal antibodies (mAbs). mAbs were analyzed by anti-H enzyme-linked immunosorbent assay using V. parahaemolyticus polar flagellar cores. The mAb exhibiting the highest anti-H titer was coated onto Cowan I Staphylococcus aureus cells at a concentration of 75 μg/ml cell suspension and used for slide coagglutination. Of 41 isolates identified genetically as V. parahaemolyticus, 100% coagglutinated with the anti-H mAb within 30 s, and the mAb did not react with 30 isolates identified as Vibrio vulnificus. A strong coagglutination reaction with V. parahaemolyticus ATCC 17802 was still observed when the S. aureus cells were armed with as little as 15 μg of mAb/ml S. aureus cell suspension. At this concentration, the mAb cross-reacted with three other Vibrio species, suggesting that they share an identical H antigen or antigens. The anti-H mAb was then used to optimize an immunomagnetic separation protocol which exhibited from 35% to about 45% binding of 102 to 103 V. parahaemolyticus cells in phosphate-buffered saline. The mAb would be useful for the rapid and selective isolation, concentration, and detection of V. parahaemolyticus cells from environmental sources.

Immunomagnetic Separation of Vibrio vulnificus with Antiflagellar Monoclonal Antibody

2010 ◽  
Vol 73 (7) ◽  
pp. 1288-1293 ◽  
Author(s):  
R. JADEJA ◽  
M. E. JANES ◽  
J. G. SIMONSON
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Vibrio Vulnificus ◽  
Detection Method ◽  
Immunomagnetic Separation ◽  
Immunomagnetic Beads ◽  
Seafood Industry ◽  
Assay Time ◽  
Indispensable Tool ◽  
Different Strains ◽  
Phosphate Buffered Saline

Raw oysters are primary vectors for Vibrio vulnificus infections, and a rapid detection method for V. vulnificus in raw oysters before distribution would be an indispensable tool for the seafood industry. One approach to improving the recovery and detection of V. vulnificus without sacrificing assay time is through the use of immunomagnetic separation (IMS). The aim of this study was to develop and optimize an IMS protocol using anti-H (antiflagellar) antibody for determining the level of V. vulnificus in phosphate-buffered saline (PBS) suspensions and spiked oyster homogenate. Six monoclonal antibodies were produced by immunizing mice at 2-week intervals by injection of 50 μg of purified V. vulnificus ATCC 27562 flagellin. Antibodies that exhibited high anti-H titers were coated onto Cowan I Staphylococcus aureus cells and sheep anti-mouse immunoglobulin G immunomagnetic beads. The two reagents were used to determine the species specificity of the selected antibodies, which positively identified and coagglutinated 70 isolates identified genetically as V. vulnificus and did not react with 40 Vibrio parahaemolyticus isolates or nine other Vibrio species. The IMS protocol was optimized for PBS and oyster homogenate spiked with three different strains of V. vulnificus. IMS with V. vulnificus–spiked PBS yielded binding of 19 to 57%, and IMS with spiked oyster homogenate carried out at two V. vulnificus levels exhibited binding of 25 to 57%. The IMS protocol for V. vulnificus could be used to concentrate and detect V. vulnificus in seawater and shellfish homogenate.

scholarly journals Characterization of a Monoclonal Antibody That Binds to an Epitope on Soluble Bacterial Peptidoglycan Fragments

2001 ◽  
Vol 8 (3) ◽  
pp. 647-651 ◽  
Author(s):  
Glenn J. Merkel ◽  
Barbara A. Scofield
Keyword(s):  
Staphylococcus Aureus ◽  
Monoclonal Antibody ◽  
Immunosorbent Assay ◽  
Enzymatic Digestion ◽  
Immunoglobulin M ◽  
Enzyme Linked Immunosorbent Assay ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Bacterial Peptidoglycan

ABSTRACT We employed an inhibition-type enzyme-linked immunosorbent assay (ELISA) to characterize a murine immunoglobulin M monoclonal antibody (MAb) that bound soluble macromolecular peptidoglycan (PG). With this ELISA, the MAb was capable of detecting soluble PG concentrations of less than 10 ng/ml. Enzymatic digestion of PG reduced binding by more than 100-fold, implying that the epitope recognized by this antibody depended on repeating subunits within the glycan backbone. Additionally, the MAb bound to epitopes on both O-acetylated and non-O-acetylated PG fragments from gram-negative bacteria, as well as PG fragments from Staphylococcus aureus and PG fragments released into the medium by a number of gram-positive and gram-negative bacteria.

Sensitivity of Vibrio Species in Phosphate-Buffered Saline and in Oysters to High-Pressure Processing

2003 ◽  
Vol 66 (12) ◽  
pp. 2276-2282 ◽  
Author(s):  
DAVID W. COOK
Keyword(s):  
High Pressure ◽  
Vibrio Parahaemolyticus ◽  
Vibrio Vulnificus ◽  
High Pressure Processing ◽  
Decimal Reduction Time ◽  
Log Reduction ◽  
Naturally Occurring ◽  
Multiple Strains ◽  
Phosphate Buffered Saline ◽  
Vibrio Spp

Multiple strains of Vibrio vulnificus, Vibrio parahaemolyticus, and Vibrio cholerae non-O1 were tested in phosphate-buffered saline for their sensitivity to high-pressure processing (HPP). Variability in sensitivity among strains was observed for all species; this variability decreased at higher pressures. V. vulnificus was the species that was most sensitive to treatment at 200 MPa (decimal reduction time [D] = 26 s), and V. cholerae was the species that was most resistant to treatment at 200 MPa (D = 149 s). The O3:K6 serotype of V. parahaemolyticus was more resistant to pressure than other serotypes of V. parahaemolyticus were. The results of studies involving V. vulnificus naturally occurring in oysters revealed that a pressure treatment of 250 MPa for 120 s achieved a >5-log reduction in the levels of this bacterium. V. parahaemolyticus serotype O3:K6 in oysters required a pressure of 300 MPa for 180 s for a comparable 5-log reduction. When properly applied, HPP can be effective in improving the safety of shellfish with respect to Vibrio spp.

scholarly journals Characterization of Alpha-ToxinhlaGene Variants, Alpha-Toxin Expression Levels, and Levels of Antibody to Alpha-Toxin in Hemodialysis and Postsurgical Patients with Staphylococcus aureus Bacteremia

2014 ◽  
Vol 53 (1) ◽  
pp. 227-236 ◽  
Author(s):  
Batu K. Sharma-Kuinkel ◽  
Yuling Wu ◽  
David E. Tabor ◽  
Hoyin Mok ◽  
Bret R. Sellman ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Monoclonal Antibody ◽  
Clinical Outcome ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Enzyme Linked Immunosorbent Assay ◽  
Alpha Toxin ◽  
Content Type ◽  
Antibody Levels

Alpha-toxin is a majorStaphylococcus aureusvirulence factor. This study evaluated potential relationships betweenin vitroalpha-toxin expression ofS. aureusbloodstream isolates, anti-alpha-toxin antibody in serum of patients withS. aureusbacteremia (SAB), and clinical outcomes in 100 hemodialysis and 100 postsurgical SAB patients. Isolates underwentspatyping andhlasequencing. Serum anti-alpha-toxin IgG and neutralizing antibody levels were measured by using an enzyme-linked immunosorbent assay and a red blood cell (RBC)-based hemolysis neutralization assay. Neutralization of alpha-toxin by an anti-alpha-toxin monoclonal antibody (MAb MEDI4893) was tested in an RBC-based lysis assay. Most isolates encodedhla(197/200; 98.5%) and expressed alpha-toxin (173/200; 86.5%).In vitroalpha-toxin levels were inversely associated with survival (cure, 2.19 μg/ml, versus failure, 1.09 μg/ml;P< 0.01). Both neutralizing (hemodialysis, 1.26 IU/ml, versus postsurgical, 0.95;P< 0.05) and IgG (hemodialysis, 1.94 IU/ml, versus postsurgical, 1.27;P< 0.05) antibody levels were higher in the hemodialysis population. Antibody levels were also significantly higher in patients infected with alpha-toxin-expressingS. aureusisolates (P< 0.05). Levels of both neutralizing antibodies and IgG were similar among patients who were cured and those not cured (failures). Sequence analysis ofhlarevealed 12 distincthlagenotypes, and all genotypic variants were susceptible to a neutralizing monoclonal antibody in clinical development (MEDI4893). These data demonstrate that alpha-toxin is highly conserved in clinicalS. aureusisolates. Higherin vitroalpha-toxin levels were associated with a positive clinical outcome. Although patients infected with alpha-toxin-producingS. aureusexhibited higher anti-alpha-toxin antibody levels, these levels were not associated with a better clinical outcome in this study.

scholarly journals LM6-M: a high avidity rat monoclonal antibody to pectic α-1,5-L-arabinan

2017 ◽  
Author(s):  
Valérie Cornuault ◽  
Fanny Buffetto ◽  
Susan E Marcus ◽  
Marie-Jeanne Crépeau ◽  
Fabienne Guillon ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
High Sensitivity ◽  
In Vitro Assays ◽  
Enzyme Linked Immunosorbent Assay ◽  
High Avidity ◽  
Plant Cell Walls ◽  
Rhamnogalacturonan I ◽  
Phosphate Buffered Saline

Abstract1,5-arabinan is an abundant structural feature of side chains of pectic rhamnogalacturonan-I which is a matrix constituent of plant cell walls. The study of arabinan in cells and tissues is driven by putative roles for this polysaccharide in the generation of cell wall and organ mechanical properties. The biological function(s) of arabinan is still uncertain and high quality molecular tools are required to detect its occurrence and monitor its dynamics. Here we report a new rat monoclonal antibody, LM6-M, similar in specificity to the published rat monoclonal antibody LM6 (Willats et al. (1998) Carbohydrate Research 308: 149-152). LM6-M is of the IgM immunoglobulin class and has a higher avidity for α-1-5-L-arabinan than LM6. LM6-M displays high sensitivity in its detection of arabinan in in-vitro assays such as ELISA and epitope detection chromatography and in in-situ analyses.AbbreviationsAraArabinoseBSABovine Serum AlbuminGalGalactoseGalAGalacturonic acidEDCEpitope detection chromatographyELISAEnzyme-Linked Immunosorbent AssaymAbMonoclonal antibodyPBSPhosphate-buffered salineRhaRhamnoseRG-IRhamnogalacturonan-I

An ELISA-on-a-Chip Biosensor System Coupled with Immunomagnetic Separation for the Detection of Vibrio parahaemolyticus within a Single Working Day

2010 ◽  
Vol 73 (8) ◽  
pp. 1466-1473 ◽  
Author(s):  
SUNG-MIN SEO ◽  
IL-HOON CHO ◽  
JIN-WOO JEON ◽  
HYUN-KYU CHO ◽  
EUN-GYOUNG OH ◽  
...  
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Rapid Detection ◽  
Magnetic Particles ◽  
Detection System ◽  
Foodborne Pathogen ◽  
Rapid Test ◽  
Immunomagnetic Separation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Biosensor System ◽  
Test Kit

In this study, we constructed a rapid detection system for a foodborne pathogen, Vibrio parahaemolyticus, by using enzyme-linked immunosorbent assay (ELISA)-on-a-chip (EOC) biosensor technology to minimize the risk of infection by the microorganism. The EOC results showed a detection capability of approximately 6.2 × 105 cells per ml, which was significantly higher than that of the conventional rapid test kit. However, this high level of sensitivity required cultivation of the pathogen prior to analysis, which typically exceeded a day. To shorten the test period, we combined the EOC technology with immunomagnetic separation (IMS), which could enhance the sensitivity of the biosensor. IMS was carried out with magnetic particles coated with a monoclonal antibody specific to the microbe. To test the performance of the IMS-EOC method, fish intestine samples were prepared by artificially inoculating less than 1 or 5 CFU/10 g, allowing for enrichment over predetermined times, and analyzing the sample by using the EOC sensor after concentrating the culture 86-fold via IMS. Using this approach, the bacterium was detected after (at most) 9 h, which approximately corresponds to standard working hours. Thus, the IMS-EOC method allowed for the rapid detection of V. parahaemolyticus, which is responsible for foodborne diseases, and this method could be used for early isolation of contaminated foods before distribution.

scholarly journals Inactivation of Vibrio parahaemolyticus and Vibrio vulnificus in Phosphate-Buffered Saline and in Inoculated Whole Oysters by High-Pressure Processing

2006 ◽  
Vol 69 (3) ◽  
pp. 596-601 ◽  
Author(s):  
JAHEON KOO ◽  
MICHAEL L. JAHNCKE ◽  
PAUL W. RENO ◽  
XIAOPEI HU ◽  
PARAMESWARAKUMAR MALLIKARJUNAN
Keyword(s):  
High Pressure ◽  
Clinical Isolate ◽  
Vibrio Parahaemolyticus ◽  
Vibrio Vulnificus ◽  
High Pressure Processing ◽  
Processing Conditions ◽  
Log Reduction ◽  
Phosphate Buffered Saline

Inactivation studies for Vibrio parahaemolyticus TX-2103 (serotype O3:K6) and Vibrio vulnificus MO-624 (clinical isolate) were conducted in phosphate-buffered saline (PBS) and in inoculated oysters under high-pressure processing conditions. V. parahaemolyticus was more resistant than V. vulnificus in PBS at all pressures and times. A 6-log reduction of V. parahaemolyticus and V. vulnificus in PBS at 241 MPa required 11 and 5 min, respectively, which included a 3-min pressure come-up time. A 4.5-log reduction of V. parahaemolyticus in oysters at 345 MPa required 7.7 min, which included a 6.7-min pressure come-up time. More than a 5.4-log reduction of V. vulnificus in oysters at 345 MPa occurred during the 6-min pressure come-up time. Both V. parahaemolyticus and V. vulnificus in PBS and in oysters were reduced to nondetectable numbers at 586 MPa during the 8- and 7-min pressure come-up times, respectively.

scholarly journals A Novel Mouse Monoclonal Antibody C42 against C-Terminal Peptide of Alpha-1-Antitrypsin

2021 ◽  
Vol 22 (4) ◽  
pp. 2141
Author(s):  
Srinu Tumpara ◽  
Elena Korenbaum ◽  
Mark Kühnel ◽  
Danny Jonigk ◽  
Beata Olejnicka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Western Blotting ◽  
Complex Mixture ◽  
Immunoglobulin M ◽  
Confocal Laser ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dot Blot

The C-terminal-fragments of alpha1-antitrypsin (AAT) have been identified and their diverse biological roles have been reported in vitro and in vivo. These findings prompted us to develop a monoclonal antibody that specifically recognizes C-36 peptide (corresponding to residues 359–394) resulting from the protease-associated cleavage of AAT. The C-36-targeting mouse monoclonal Immunoglobulin M (IgM) antibody (containing κ light chains, clone C42) was generated and enzyme-linked immunosorbent assay (ELISA)-tested by Davids Biotechnologie GmbH, Germany. Here, we addressed the effectiveness of the novel C42 antibody in different immunoassay formats, such as dot- and Western blotting, confocal laser microscopy, and flow cytometry. According to the dot-blot results, our novel C42 antibody detects the C-36 peptide at a range of 0.1–0.05 µg and shows no cross-reactivity with native, polymerized, or oxidized forms of full-length AAT, the AAT-elastase complex mixture, as well as with shorter C-terminal fragments of AAT. However, the C42 antibody does not detect denatured peptide in SDS-PAGE/Western blotting assays. On the other hand, our C42 antibody, unconjugated as well as conjugated to DyLight488 fluorophore, when applied for immunofluorescence microscopy and flow cytometry assays, specifically detected the C-36 peptide in human blood cells. Altogether, we demonstrate that our novel C42 antibody successfully recognizes the C-36 peptide of AAT in a number of immunoassays and has potential to become an important tool in AAT-related studies.

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