Development of an immunoassay test system based on monoclonal antybodies and immunomagnetic particles for the detection of F. tularensis cells

Tularemia is an especially dangerous infection caused by the gram-negative bacterium Francisella tularensis. It belongs to natural focal infections, and therefore is under continuous control by quarantine services. When carrying out their activities they use a whole range of diagnostic tools. The objective of this research is to develop an enzyme immunoassay based on highly specific monoclonal antibodies and immunomagnetic particles for monitoring the tularemia pathogen. To produce hybridomas mice were immunized with cells of the vaccine strain F. tularensis subsp. holarctica 15 NIIEG. After cell fusion hybridomas were selected by a solid-phase enzyme immunoassay (ELISA) using lipopolysaccharide (LPS) of the tularemia microbe. As a result, two hybridomas, 1C2 and 3F5, were produced. MABs of the hybridomas were obtained by using BALB / c mice. The MABs were purified by sepharose A affinity chromatography and used for conjugation with magnetic particles, and for biotinylation followed by matching a pair for ELISA. The pair of IMPs and MABs 3F5 as well as biotinylated FB11-x MABs was the best in detecting tularemia cells. The use of this MAB pair in ELISA allowed the identification of 105 microbial cells/ml in a 4 ml sample and 5×103 microbial cells/ml in a 45ml sample. Interaction with F. tularensis subsp. novicida Utah112 cells was absent.

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Evaluation of the Accelerate Pheno System for Fast Identification and Antimicrobial Susceptibility Testing from Positive Blood Cultures in Bloodstream Infections Caused by Gram-Negative Pathogens

Journal of Clinical Microbiology ◽

10.1128/jcm.00181-17 ◽

2017 ◽

Vol 55 (7) ◽

pp. 2116-2126 ◽

Cited By ~ 84

Author(s):

Matthias Marschal ◽

Johanna Bachmaier ◽

Ingo Autenrieth ◽

Philipp Oberhettinger ◽

Matthias Willmann ◽

...

Keyword(s):

Blood Culture ◽

Antimicrobial Susceptibility ◽

Susceptibility Testing ◽

Bloodstream Infections ◽

Test System ◽

Blood Cultures ◽

Antimicrobial Susceptibility Testing ◽

Diagnostic Tools ◽

Gram Negative ◽

Content Type

ABSTRACT Bloodstream infections (BSI) are an important cause of morbidity and mortality. Increasing rates of antimicrobial-resistant pathogens limit treatment options, prompting an empirical use of broad-range antibiotics. Fast and reliable diagnostic tools are needed to provide adequate therapy in a timely manner and to enable a de-escalation of treatment. The Accelerate Pheno system (Accelerate Diagnostics, USA) is a fully automated test system that performs both identification and antimicrobial susceptibility testing (AST) directly from positive blood cultures within approximately 7 h. In total, 115 episodes of BSI with Gram-negative bacteria were included in our study and compared to conventional culture-based methods. The Accelerate Pheno system correctly identified 88.7% (102 of 115) of all BSI episodes and 97.1% (102 of 105) of isolates that are covered by the system's identification panel. The Accelerate Pheno system generated an AST result for 91.3% (95 of 104) samples in which the Accelerate Pheno system identified a Gram-negative pathogen. The overall category agreement between the Accelerate Pheno system and culture-based AST was 96.4%, the rates for minor discrepancies 1.4%, major discrepancies 2.3%, and very major discrepancies 1.0%. Of note, ceftriaxone, piperacillin-tazobactam, and carbapenem resistance was correctly detected in blood culture specimens with extended-spectrum beta-lactamase-producing Escherichia coli ( n = 7) and multidrug-resistant Pseudomonas aeruginosa ( n = 3) strains. The utilization of the Accelerate Pheno system reduced the time to result for identification by 27.49 h ( P < 0.0001) and for AST by 40.39 h ( P < 0.0001) compared to culture-based methods in our laboratory setting. In conclusion, the Accelerate Pheno system provided fast, reliable results while significantly improving turnaround time in blood culture diagnostics of Gram-negative BSI.

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Bioelectrochemical enzyme immunoassay of human choriogonadotropin with magnetic electrodes.

Clinical Chemistry ◽

10.1093/clinchem/31.9.1449 ◽

1985 ◽

Vol 31 (9) ◽

pp. 1449-1452 ◽

Cited By ~ 27

Author(s):

G A Robinson ◽

H A Hill ◽

R D Philo ◽

J M Gear ◽

S J Rattle ◽

...

Keyword(s):

Enzyme Activity ◽

Enzyme Immunoassay ◽

Magnetic Particles ◽

Solid Phase ◽

Model System ◽

Antigen Binding ◽

Immunoradiometric Assay ◽

Human Choriogonadotropin ◽

Assay Time ◽

Rapid Quantification

Abstract We describe an amperometric technique for quantification of an enzyme immunoassay in which we use a magnetic working electrode, both to separate bound and free analyte and to monitor the electrochemical response. We used a "two-site" immunometric assay with monoclonal antibodies for human choriogonadotropin (hCG) as a model system in which magnetic particles were used as the solid phase. Separation of bound and free label is readily achieved by localizing the particles at the electrode. Activity of the bound enzyme in the environment of the electrode is determined electrochemically, permitting rapid quantification of the analyte without the need for a separate incubation step to measure enzyme activity. The sensitivity of the system is 150 int. units of hCG per litre (1st Int. Ref. Preparation). Correlation between the amperometric measurement of urinary hCG and data for an immunoradiometric assay was r = 0.9. The assay is rapid, requiring a total assay time for each sample of 20 min, which includes 15 min for antibody/antigen binding.

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Enzyme Linked Immunosorbent Assay (ELISA)

Natural Sciences Engineering and Technology Journal ◽

10.37275/nasetjournal.v1i2.6 ◽

2021 ◽

Vol 1 (2) ◽

pp. 29-38

Author(s):

Maya Chandra Dita

Keyword(s):

Quality Control ◽

Enzyme Immunoassay ◽

Immunosorbent Assay ◽

Solid Phase ◽

Control Measures ◽

Diagnostic Tools ◽

Enzyme Linked Immunosorbent Assay ◽

Antigen Binding ◽

Fluorogenic Substrate ◽

Specific Antibodies

Enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA) are both widely used as diagnostic tools in medicine and as quality control measures in many industries. ELISA has been the system of choice when testing soluble antigens and antibodies. EIA / ELISA uses the basic immunological concept of antigen binding to specific antibodies, which allows the detection of small amounts of antigens such as proteins, peptides, hormones or antibodies in fluid samples. In all protocols, solid-phase reagents are incubated with secondary or tertiary reactants covalently coupled with the enzyme. The unbound conjugate is washed and a chromogenic or fluorogenic substrate is added.

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Application of commercial test-systems to identify gram-negative facultatively anaerobic bacteria

Agricultural science and practice ◽

10.15407/agrisp3.01.043 ◽

2016 ◽

Vol 3 (1) ◽

pp. 43-48 ◽

Cited By ~ 1

Author(s):

V. Patyka ◽

L. Butsenko ◽

L. Pasichnyk

Keyword(s):

Erwinia Amylovora ◽

Anaerobic Bacteria ◽

Biochemical Properties ◽

Test System ◽

Phytopathogenic Bacteria ◽

Gram Negative ◽

Test Systems ◽

Facultatively Anaerobic ◽

Microbiological Methods

Aim. To validate the suitability of commercial API 20E test-system (bioMerieux) for the identifi cation and characterization of facultative gram-negative phytopathogenic bacterial isolates. Methods. Conventional mi- crobiological methods, API 20E test-system (bioMerieux) according to the manufacturer’s instructions. Re- sults. The identifi cation results for Erwinia amylovora, Pectobacterium carotovorum and Pantoea agglome- rans isolates were derived from the conventional and API 20E test systems, which, were in line with the literature data for these species. The API 20E test-system showed high suitability for P. agglomerans isolates identifi cation. Although not all the species of facultatively anaerobic phytopathogenic bacteria may be identi- fi ed using API 20E test-system, its application will surely allow obtaining reliable data about their physiologi- cal and biochemical properties, valuable for identifi cation of bacteria, in the course of 24 h. Conclusions. The results of tests, obtained for investigated species while using API 20E test-system, and those of conventional microbiological methods coincided. The application of API 20E test-system (bioMerieux) ensures fast obtain- ing of important data, which may be used to identify phytopathogenic bacteria of Erwinia, Pectobacterium, Pantoea genera.

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Isolation of d-Talomethylose (6-Deoxy-d-talose) and d-Rhamnose (6-Deoxy-d-mannose) from Capsular Polysaccharide of a Gram-negative Bacterium

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)73934-9 ◽

1962 ◽

Vol 237 (6) ◽

pp. 1767-1771

Author(s):

Alvin Markovitz

Keyword(s):

Capsular Polysaccharide ◽

Negative Bacterium ◽

Gram Negative

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Development of a noncompetitive, solid phase, bridged biotin-avidin enzyme immunoassay for measurement of human leukocyte microsomal HMG-CoA reductase protein concentration.

The Journal of Lipid Research ◽

10.1016/s0022-2275(20)38703-4 ◽

1987 ◽

Vol 28 (3) ◽

pp. 292-304

Author(s):

H J Harwood ◽

I M Alvarez ◽

Y J Greene ◽

G C Ness ◽

P W Stacpoole

Keyword(s):

Enzyme Immunoassay ◽

Protein Concentration ◽

Solid Phase ◽

Human Leukocyte ◽

Hmg Coa Reductase ◽

Coa Reductase

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Massiliamide, a cyclic tetrapeptide with potent tyrosinase inhibitory properties from the Gram-negative bacterium Massilia albidiflava DSM 17472T

The Journal of Antibiotics ◽

10.1038/s41429-020-00394-y ◽

2020 ◽

Author(s):

Andri Frediansyah ◽

Jan Straetener ◽

Heike Brötz-Oesterhelt ◽

Harald Gross

Keyword(s):

Absolute Configuration ◽

Inhibitory Activity ◽

Liquid Culture ◽

Spectroscopic Analysis ◽

2D Nmr ◽

Negative Bacterium ◽

Gram Negative ◽

The Absolute ◽

Potent Inhibitory Activity ◽

Cyclic Tetrapeptide

AbstractA cyclic tetrapeptide, designated massiliamide, was isolated from the liquid culture of the Gram-negative bacterium Massilia albidiflava DSM 17472T. The structure was elucidated through extensive spectroscopic analysis, including HR-MS and 1D and 2D NMR experiments. The absolute configuration was determined using the Marfey´s method. Massiliamide showed potent inhibitory activity towards tyrosinase with an IC50 value of 1.15 µM and no cytotoxicity.

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Porphyromonas gingivalis fimbrial protein Mfa5 contains a von Willebrand factor domain and an intramolecular isopeptide

Communications Biology ◽

10.1038/s42003-020-01621-w ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Thomas V. Heidler ◽

Karin Ernits ◽

Agnieszka Ziolkowska ◽

Rolf Claesson ◽

Karina Persson

Keyword(s):

Porphyromonas Gingivalis ◽

Von Willebrand Factor ◽

Host Cells ◽

Negative Bacterium ◽

Oral Biofilm ◽

Gram Negative ◽

Type V ◽

Von Willebrand ◽

Willebrand Factor ◽

Fimbrial Protein

AbstractThe Gram-negative bacterium Porphyromonas gingivalis is a secondary colonizer of the oral biofilm and is involved in the onset and progression of periodontitis. Its fimbriae, of type-V, are important for attachment to other microorganisms in the biofilm and for adhesion to host cells. The fimbriae are assembled from five proteins encoded by the mfa1 operon, of which Mfa5 is one of the ancillary tip proteins. Here we report the X-ray structure of the N-terminal half of Mfa5, which reveals a von Willebrand factor domain and two IgG-like domains. One of the IgG-like domains is stabilized by an intramolecular isopeptide bond, which is the first such bond observed in a Gram-negative bacterium. These features make Mfa5 structurally more related to streptococcal adhesins than to the other P. gingivalis Mfa proteins. The structure reported here indicates that horizontal gene transfer has occurred among the bacteria within the oral biofilm.

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