scholarly journals New Skin Test for Detection of Bovine Tuberculosis on the Basis of Antigen-Displaying Polyester Inclusions Produced by Recombinant Escherichia coli

2014 ◽  
Vol 80 (8) ◽  
pp. 2526-2535 ◽  
Author(s):  
Shuxiong Chen ◽  
Natalie A. Parlane ◽  
Jason Lee ◽  
D. Neil Wedlock ◽  
Bryce M. Buddle ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Skin Test ◽  
Skin Testing ◽  
Cross Reactivity ◽  
Pha Synthase ◽  
Enzyme Linked Immunosorbent Assay ◽  
Content Type ◽  
Flight Mass Spectrometry ◽  
Environmental Mycobacteria ◽  
Test Specificity

ABSTRACTThe tuberculin skin test for diagnosing tuberculosis (TB) in cattle lacks specificity if animals are sensitized to environmental mycobacteria, as some antigens in purified protein derivative (PPD) prepared fromMycobacterium bovisare present in nonpathogenic mycobacteria. Three immunodominant TB antigens, ESAT6, CFP10, and Rv3615c, are present in members of the pathogenicMycobacterium tuberculosiscomplex but absent from the majority of environmental mycobacteria. These TB antigens have the potential to enhance skin test specificity. To increase their immunogenicity, these antigens were displayed on polyester beads by translationally fusing them to a polyhydroxyalkanoate (PHA) synthase which mediated formation of antigen-displaying inclusions in recombinantEscherichia coli. The most common form of these inclusions is poly(3-hydroxybutyric acid) (PHB). The respective fusion proteins displayed on these PHB inclusions (beads) were identified using tryptic peptide fingerprinting analysis in combination with matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). The surface exposure and accessibility of antigens were assessed by enzyme-linked immunosorbent assay (ELISA). Polyester beads displaying all three TB antigens showed greater reactivity with TB antigen-specific antibody than did beads displaying only one TB antigen. This was neither due to cross-reactivity of antibodies with the other two antigens nor due to differences in protein expression levels between beads displaying single or three TB antigens. The triple-antigen-displaying polyester beads were used for skin testing of cattle and detected all cattle experimentally infected withM. boviswith no false-positive reactions observed in those sensitized to environmental mycobacteria. The results suggested applicability of TB antigen-displaying polyester inclusions as diagnostic reagents for distinguishing TB-infected from noninfected animals.

scholarly journals Recombinant Escherichia coli Strain Produces a ZZ Domain Displaying Biopolyester Granules Suitable for Immunoglobulin G Purification

2006 ◽  
Vol 72 (11) ◽  
pp. 7394-7397 ◽  
Author(s):  
Jane A. Brockelbank ◽  
Verena Peters ◽  
Bernd H. A. Rehm
Keyword(s):  
Escherichia Coli ◽  
Immunoglobulin G ◽  
Binding Capacity ◽  
Protein A ◽  
Coli Strain ◽  
Pha Synthase ◽  
Enzyme Linked Immunosorbent Assay ◽  
Ionization Time ◽  
Flight Mass Spectrometry ◽  
Igg Binding

ABSTRACT The immunoglobulin G (IgG) binding ZZ domain of protein A from Staphylococcus aureus was fused to the N terminus of the polyhydroxyalkanoate (PHA) synthase from Cupriavidus necator. The fusion protein was confirmed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry and mediated formation of ZZ domain-displaying PHA granules in recombinant Escherichia coli. The IgG binding capacity of isolated granules was assessed using enzyme-linked immunosorbent assay and could be enhanced by the overproduction of the ZZ-PHA synthase. ZZ-PHA granules enabled efficient purification of IgG from human serum.

scholarly journals Limitations of the BP26 Protein-Based Indirect Enzyme-Linked Immunosorbent Assay for Diagnosis of Brucellosis

2013 ◽  
Vol 20 (9) ◽  
pp. 1410-1417 ◽  
Author(s):  
Ting Xin ◽  
Hongjun Yang ◽  
Nan Wang ◽  
Fang Wang ◽  
Peng Zhao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Beef Cattle ◽  
Yersinia Enterocolitica ◽  
Immunosorbent Assay ◽  
Indirect Elisa ◽  
Cross Reactivity ◽  
Infected Sheep ◽  
Enzyme Linked Immunosorbent Assay ◽  
Content Type ◽  
Experimental Animals

ABSTRACTBrucellosis is a serious zoonosis that occurs worldwide, and its diagnosis is typically based on the detection of antibodies againstBrucellalipopolysaccharide (LPS). However, the specificity of the LPS-based test is compromised by cross-reactivity withEscherichia coliO157:H7 andYersinia enterocoliticaO:9. Also, diagnosis based on the LPS test cannot differentiate between vaccinated and infected individuals. The detection of the 26-kDa cytosoluble protein (BP26) antibody is considered an alternative that circumvents these drawbacks because it is exclusively expressed by infectiousBrucella. A BP26-based enzyme-linked immunosorbent assay (ELISA) has been tried for the diagnosis ofBrucella-infected animals and humans, but a few results showed that BP26 couldn't react with allBrucella-positive sera. In order to explore whether different animals could produce antibodies against BP26 after being infected with variousBrucellaspecies, we infected sheep, goats, and beef cattle with common virulent referenceBrucellaspecies. All sera were collected from the experimental animals and tested using both LPS-based ELISAs and BP26-based ELISAs. The results showed that allBrucella-infected individuals could produce high levels of antibodies against LPS, but onlyB. melitensis16M- andB. melitensisM28-infected sheep andB. melitensis16M- andB. abortus2308-infected goats could produce antibodies against BP26. Therefore, we concluded that the BP26-based indirect ELISA (i-ELISA) showed bothBrucellaspecies and host specificity, which obviously limits its reliability as a substitute for the traditional LPS-based ELISA for the detection of brucellosis.

scholarly journals Display of Antigens on Polyester Inclusions Lowers the Antigen Concentration Required for a Bovine Tuberculosis Skin Test

2015 ◽  
Vol 23 (1) ◽  
pp. 19-26 ◽  
Author(s):  
Natalie A. Parlane ◽  
Shuxiong Chen ◽  
Gareth J. Jones ◽  
H. Martin Vordermeier ◽  
D. Neil Wedlock ◽  
...  
Keyword(s):  
Skin Test ◽  
Bovine Tuberculosis ◽  
Low Cost ◽  
High Sensitivity ◽  
Content Type ◽  
Environmental Mycobacteria ◽  
Low Concentrations ◽  
Polyhydroxyalkanoate Synthase ◽  
Test Specificity ◽  
Tuberculosis Skin Test

ABSTRACTThe tuberculin skin test is the primary screening test for the diagnosis of bovine tuberculosis (TB), and use of this test has been very valuable in the control of this disease in many countries. However, the test lacks specificity when cattle have been exposed to environmental mycobacteria or vaccinated withMycobacterium bovisbacille Calmette-Guérin (BCG). Recent studies showed that the use of three or four recombinant mycobacterial proteins, including 6-kDa early secretory antigenic target (ESAT6), 10-kDa culture filtrate protein (CFP10), Rv3615c, and Rv3020c, or a peptide cocktail derived from those proteins, in the skin test greatly enhanced test specificity, with minimal loss of test sensitivity. The proteins are present in members of the pathogenicMycobacterium tuberculosiscomplex but are absent in or not expressed by the majority of environmental mycobacteria and the BCG vaccine strain. To produce a low-cost skin test reagent, the proteins were displayed at high density on polyester beads through translational fusion to a polyhydroxyalkanoate synthase that mediates the formation of antigen-displaying inclusions in recombinantEscherichia coli. Display of the proteins on the polyester beads greatly increased their immunogenicity, allowing for the use of very low concentrations of proteins (0.1 to 3 μg of mycobacterial protein/inoculum) in the skin test. Polyester beads simultaneously displaying all four proteins were produced in a single fermentation process. The polyester beads displaying three or four mycobacterial proteins were shown to have high sensitivity for detection ofM. bovis-infected cattle and induced minimal responses in animals exposed to environmental mycobacteria or vaccinated with BCG.

scholarly journals Serological Response of Shiga Toxin-Producing Escherichia coli Type III Secreted Proteins in Sera from Vaccinated Rabbits, Naturally Infected Cattle, and Humans

2011 ◽  
Vol 18 (7) ◽  
pp. 1052-1057 ◽  
Author(s):  
David J. Asper ◽  
Mohamed A. Karmali ◽  
Hugh Townsend ◽  
Dragan Rogan ◽  
Andrew A. Potter
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Cross Reactivity ◽  
Secreted Proteins ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serological Response ◽  
Infected Cattle ◽  
Serum Samples ◽  
Content Type ◽  
Type Iii

ABSTRACTEscherichia coliO157:H7 is an important zoonotic pathogen, causing hemolytic uremic syndrome (HUS). The colonization of cattle and human hosts is mediated through the action of effectors secreted via a type III secretion system (T3SS). The structural genes for the T3SS and many of the secreted effectors are located on a pathogenicity island called the locus of enterocyte effacement (LEE). We cloned and expressed the genes coding for 66 effectors and purified each to measure the cross-reactivity of type III secreted proteins from Shiga toxin-producingEscherichia coli(STEC) serotypes. These included 37 LEE-encoded proteins and 29 non-LEE effectors. The serological response against each protein was measured by Western blot analysis and enzyme-linked immunosorbent assay (ELISA) using sera from rabbits immunized with type III secreted proteins (T3SPs) from four STEC serotypes, experimentally infected cattle, and human sera from six HUS patients. Twenty proteins were recognized by at least one of the STEC T3SP-vaccinated rabbits by Western blotting. Several structural proteins (EspA, EspB, and EspD) and a number of effectors (Tir, NleA, and TccP) were recognized by O26-, O103-, O111-, and O157-specific sera. Sera from experimentally infected cattle and HUS patients were tested using an ELISA against each of the proteins. Tir, EspB, EspD, EspA, and NleA were recognized by the majority of the samples tested. A number of other proteins also were recognized by individual serum samples. Overall, proteins such as Tir, EspB, EspD, NleA, and EspA were highly immunogenic in vaccinated and naturally infected subjects and could be candidates for a cross-protective STEC vaccine.

scholarly journals Subtractive Phage Display Selection from Canine Visceral Leishmaniasis Identifies Novel Epitopes That Mimic Leishmania infantum Antigens with Potential Serodiagnosis Applications

2013 ◽  
Vol 21 (1) ◽  
pp. 96-106 ◽  
Author(s):  
Lourena E. Costa ◽  
Mayara I. S. Lima ◽  
Miguel A. Chávez-Fumagalli ◽  
Daniel Menezes-Souza ◽  
Vivian T. Martins ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Phage Display ◽  
Leishmania Infantum ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Phage Display Technology ◽  
Predictive Values ◽  
Content Type ◽  
Phage Elisa

ABSTRACTVisceral leishmaniasis (VL) is a zoonotic disease that is endemic to Brazil, where dogs are the main domestic parasite reservoirs, and the percentages of infected dogs living in regions where canine VL (CVL) is endemic have ranged from 10% to 62%. Despite technological advances, some problems have been reported with CVL serodiagnosis. The present study describes a sequential subtractive selection through phage display technology from polyclonal antibodies of negative and positive sera that resulted in the identification of potential bacteriophage-fused peptides that were highly sensitive and specific to antibodies of CVL. A negative selection was performed in which phage clones were adhered to purified IgGs from healthy andTrypanosoma cruzi-infected dogs to eliminate cross-reactive phages. The remaining supernatant nonadhered phages were submitted to positive selection against IgG from the blood serum of dogs that were infected withLeishmania infantum. Phage clones that adhered to purified IgGs from the CVL-infected serum samples were selected. Eighteen clones were identified and their reactivities tested by a phage enzyme-linked immunosorbent assay (phage-ELISA) against the serum samples from infected dogs (n= 31) compared to those from vaccinated dogs (n= 21), experimentally infected dogs with cross-reactive parasites (n= 23), and healthy controls (n= 17). Eight clones presented sensitivity, specificity, and positive and negative predictive values of 100%, and they showed no cross-reactivity withT. cruzi- orEhrlichia canis-infected dogs or with dogs vaccinated with two different commercial CVL vaccines in Brazil. Our study identified eight mimotopes ofL. infantumantigens with 100% accuracy for CVL serodiagnosis. The use of these mimotopes by phage-ELISA proved to be an excellent assay that was reproducible, simple, fast, and inexpensive, and it can be applied in CVL-monitoring programs.

scholarly journals Rearrangement of Gene Order in thephaCABOperon Leads to Effective Production of Ultrahigh-Molecular-Weight Poly[(R)-3-Hydroxybutyrate] in Genetically Engineered Escherichia coli

2012 ◽  
Vol 78 (9) ◽  
pp. 3177-3184 ◽  
Author(s):  
Ayaka Hiroe ◽  
Kenji Tsuge ◽  
Christopher T. Nomura ◽  
Mitsuhiro Itaya ◽  
Takeharu Tsuge
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Gene Order ◽  
Ralstonia Eutropha ◽  
Genetically Engineered ◽  
Pha Synthase ◽  
Limiting Factor ◽  
Expression Plasmid ◽  
Content Type ◽  
Ultrahigh Molecular Weight

ABSTRACTUltrahigh-molecular-weight poly[(R)-3-hydroxybutyrate] [UHMW-P(3HB)] synthesized by genetically engineeredEscherichia coliis an environmentally friendly bioplastic material which can be processed into strong films or fibers. An operon of three genes (organized asphaCAB) encodes the essential proteins for the production of P(3HB) in the native producer,Ralstonia eutropha. The three genes of thephaCABoperon arephaC, which encodes the polyhydroxyalkanoate (PHA) synthase,phaA, which encodes a 3-ketothiolase, andphaB, which encodes an acetoacetyl coenzyme A (acetoacetyl-CoA) reductase. In this study, the effect of gene order of thephaCABoperon (phaABC,phaACB,phaBAC,phaBCA,phaCAB, andphaCBA) on an expression plasmid in genetically engineeredE. coliwas examined in order to determine the best organization to produce UHMW-P(3HB). The results showed that P(3HB) molecular weights and accumulation levels were both dependent on the order of thephagenes relative to the promoter. The most balanced production result was achieved in the strain harboring thephaBCAexpression plasmid. In addition, analysis of expression levels and activity for P(3HB) biosynthesis enzymes and of P(3HB) molecular weight revealed that the concentration of active PHA synthase had a negative correlation with P(3HB) molecular weight and a positive correlation with cellular P(3HB) content. This result suggests that the level of P(3HB) synthase activity is a limiting factor for producing UHMW-P(3HB) and has a significant impact on P(3HB) production.

scholarly journals Towards Rational Design of a Toxoid Vaccine against the Heat-Stable Toxin of Escherichia coli

2016 ◽  
Vol 84 (4) ◽  
pp. 1239-1249 ◽  
Author(s):  
Arne M. Taxt ◽  
Yuleima Diaz ◽  
Rein Aasland ◽  
John D. Clements ◽  
James P. Nataro ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rational Design ◽  
Diarrheal Disease ◽  
Cross Reactivity ◽  
Single Amino Acid ◽  
Minimal Risk ◽  
Content Type ◽  
Heat Stable ◽  
Low Toxicity ◽  
Mutant Forms

EnterotoxigenicEscherichia coli(ETEC) is an important cause of diarrheal disease and death in children <5 years old. ETEC strains that express the heat-stable toxin (ST), with or without the heat-labile toxin, are among the four most important diarrhea-causing pathogens. This makes ST an attractive target for an ETEC vaccine. An ST vaccine should be nontoxic and elicit an immune response that neutralizes native ST without cross-reacting with the human endogenous guanylate cyclase C receptor ligands. To identify variants of ST with no or low toxicity, we screened a library of all 361 possible single-amino-acid mutant forms of ST by using the T84 cell assay. Moreover, we identified mutant variants with intact epitopes by screening for the ability to bind neutralizing anti-ST antibodies. ST mutant forms with no or low toxicity and intact epitopes are termed toxoid candidates, and the top 30 candidates all had mutations of residues A14, N12, and L9. The identification of nontoxic variants of L9 strongly suggests that it is a novel receptor-interacting residue, in addition to the previously identified N12, P13, and A14 residues. The screens also allowed us to map the epitopes of three neutralizing monoclonal antibodies, one of which cross-reacts with the human ligand uroguanylin. The common dominant epitope residue for all non-cross-reacting antibodies was Y19. Our results suggest that it should be possible to rationally design ST toxoids that elicit neutralizing immune responses against ST with minimal risk of immunological cross-reactivity.

scholarly journals Tuberculin Skin Testing Boosts Interferon Gamma Responses to DIVA Reagents in Mycobacterium bovis-Infected Cattle

2017 ◽  
Vol 24 (5) ◽  
Author(s):  
Gareth J. Jones ◽  
Mick Coad ◽  
Bhagwati Khatri ◽  
Javier Bezos ◽  
Natalie A. Parlane ◽  
...  
Keyword(s):  
Skin Test ◽  
Mycobacterium Bovis ◽  
Interferon Gamma ◽  
Skin Testing ◽  
Interferon Response ◽  
Tuberculin Skin Testing ◽  
Test Scenario ◽  
Positive Skin ◽  
Content Type ◽  
Bcg Vaccination

ABSTRACT Mycobacterium bovis BCG vaccination sensitizes cattle to bovine tuberculin, which compromises the use of the current bovine tuberculosis (TB) surveillance tests. Although the performance of a blood test (that utilizes antigens expressed by Mycobacterium bovis but not by BCG) capable of discriminating infected from vaccinated animals (DIVA interferon gamma test [DIT]) has been evaluated in naturally infected TB field reactors, there is a need to perform similar analysis in a BCG-vaccinated M. bovis-infected population. Furthermore, we explored different scenarios under which a DIT may be implemented alongside BCG vaccination: (i) serial testing to resolve potential false-positive skin test results or (ii) a standalone test to replace the single intradermal comparative cervical tuberculin (SICCT) skin test. Our results demonstrated significantly better relative test sensitivity when the DIT was evaluated in a serial test scenario. Direct comparison of pre- and post-skin test blood samples revealed that the SICCT test induced significant boosting of the gamma interferon response in M. bovis-infected animals to both the ESAT-6–CFP-10 and Rv3615c peptide cocktails that comprise the DIT, which persisted for the ESAT-6–CFP-10 reagent for at least 14 days. Importantly, no similar boosting effects were observed in noninfected BCG vaccinates, suggesting that DIVA blood testing after a recent skin test would have minimal impact on test specificity.

scholarly journals Characterization of the Highly Active Polyhydroxyalkanoate Synthase of Chromobacterium sp. Strain USM2

2011 ◽  
Vol 77 (9) ◽  
pp. 2926-2933 ◽  
Author(s):  
Kesaven Bhubalan ◽  
Jo-Ann Chuah ◽  
Fumi Shozui ◽  
Christopher J. Brigham ◽  
Seiichi Taguchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Weight Percent ◽  
Pha Synthase ◽  
Content Type ◽  
Highly Active ◽  
Key Enzyme ◽  
Polyhydroxyalkanoate Synthase

ABSTRACTThe synthesis of bacterial polyhydroxyalkanoates (PHA) is very much dependent on the expression and activity of a key enzyme, PHA synthase (PhaC). Many efforts are being pursued to enhance the activity and broaden the substrate specificity of PhaC. Here, we report the identification of a highly active wild-type PhaC belonging to the recently isolatedChromobacteriumsp. USM2 (PhaCCs). PhaCCsshowed the ability to utilize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) monomers in PHA biosynthesis. Anin vitroassay of recombinant PhaCCsexpressed inEscherichia colishowed that its polymerization of 3-hydroxybutyryl-coenzyme A activity was nearly 8-fold higher (2,462 ± 80 U/g) than that of the synthase from the model strainC. necator(307 ± 24 U/g). Specific activity using a Strep2-tagged, purified PhaCCswas 238 ± 98 U/mg, almost 5-fold higher than findings of previous studies using purified PhaC fromC. necator. Efficient poly(3-hydroxybutyrate) [P(3HB)] accumulation inEscherichia coliexpressing PhaCCsof up to 76 ± 2 weight percent was observed within 24 h of cultivation. To date, this is the highest activity reported for a purified PHA synthase. PhaCCsis a naturally occurring, highly active PHA synthase with superior polymerizing ability.

Placental alkaline phosphatase as a tumor marker for primary intracranial germinoma

1988 ◽  
Vol 68 (5) ◽  
pp. 710-720 ◽  
Author(s):  
Jun Shinoda ◽  
Hiromu Yamada ◽  
Noboru Sakai ◽  
Takashi Ando ◽  
Toshifumi Hirata ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Tumor Marker ◽  
Cross Reactivity ◽  
Cyst Fluid ◽  
Enzyme Linked Immunosorbent Assay ◽  
Placental Alkaline Phosphatase ◽  
Intestinal Alkaline Phosphatase ◽  
Content Type ◽  
Intracranial Germinoma ◽  
Very High

✓ A sensitive enzyme-linked immunosorbent assay (ELISA) was used in a retrospective study of placental alkaline phosphatase (PLAP) levels in serum, cerebrospinal fluid (CSF), and intratumoral cyst fluid in primary intracranial germinoma. The ELISA showed no cross-reactivity with intestinal alkaline phosphatase except in very high concentrations, after samples had been heat-treated. Three patients with germinoma were studied for serum PLAP levels and in all the levels were elevated (3.78, 0.52, and 2.11 IU/liter). Two of the germinoma patients were studied for PLAP levels in the CSF, and both had elevated levels (0.83 and 9.83 IU/liter). The intratumoral cyst fluid in one case of germinoma was tested for PLAP and the level was found to be very high (603 IU/liter). These PLAP levels decreased concomitantly with the reduction in tumor size during irradiation. Serum PLAP levels were measured in 40 control adult male individuals and in the CSF of 20 nonpregnant patients with subarachnoid hemorrhage. The upper normal limits were 0.20 and 0.11 IU/liter in the serum and the CSF, respectively. All PLAP levels measured in the serum of patients with various brain tumors were 0.18 IU/liter or less. This study strongly suggests that PLAP is a clinically useful tumor marker for primary intracranial germinoma.

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