Publications by authors named "Hervé Volland"

49 Publications

Differentiation, Quantification and Identification of Abrin and Agglutinin.

Toxins (Basel) 2021 04 18;13(4). Epub 2021 Apr 18.

Biological Toxins, Centre for Biological Threats and Special Pathogens, Robert Koch Institute, Seestr. 10, 13353 Berlin, Germany.

Abrin, the toxic lectin from the rosary pea plant has gained considerable interest in the recent past due to its potential malevolent use. However, reliable and easy-to-use assays for the detection and discrimination of abrin from related plant proteins such as agglutinin or the homologous toxin ricin from are sparse. To address this gap, a panel of highly specific monoclonal antibodies was generated against abrin and the related agglutinin. These antibodies were used to establish two sandwich ELISAs to preferentially detect abrin or agglutinin (limit of detection 22 pg/mL for abrin; 35 pg/mL for agglutinin). Furthermore, an abrin-specific lateral flow assay was developed for rapid on-site detection (limit of detection ~1 ng/mL abrin). Assays were validated for complex food, environmental and clinical matrices illustrating broad applicability in different threat scenarios. Additionally, the antibodies turned out to be suitable for immuno-enrichment strategies in combination with mass spectrometry-based approaches for unambiguous identification. Finally, we were able to demonstrate for the first time how the developed assays can be applied to detect, identify and quantify abrin from a clinical sample derived from an attempted suicide case involving .
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http://dx.doi.org/10.3390/toxins13040284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8073929PMC
April 2021

Ricin Antibodies' Neutralizing Capacity against Different Ricin Isoforms and Cultivars.

Toxins (Basel) 2021 01 29;13(2). Epub 2021 Jan 29.

Paris-Saclay University, CEA, INRAE, Medicines and Healthcare Technologies Department (DMTS), SPI, 91191 Gif-sur-Yvette, France.

Ricin, a highly toxic protein from , is considered a potential biowarfare agent. Despite the many data available, no specific treatment has yet been approved. Due to their ability to provide immediate protection, antibodies (Abs) are an approach of choice. However, their high specificity might compromise their capacity to protect against the different ricin isoforms (D and E) found in the different cultivars. In previous work, we have shown the neutralizing potential of different Abs (43RCA-G1 (anti ricin A-chain) and RB34 and RB37 (anti ricin B-chain)) against ricin D. In this study, we evaluated their protective capacity against both ricin isoforms. We show that: (i) RB34 and RB37 recognize exclusively ricin D, whereas 43RCA-G1 recognizes both isoforms, (ii) their neutralizing capacity in vitro varies depending on the cultivar, and (iii) there is a synergistic effect when combining RB34 and 43RCA-G1. This effect is also demonstrated in vivo in a mouse model of intranasal intoxication with ricin D/E (1:1), where approximately 60% and 40% of mice treated 0 and 6 h after intoxication, respectively, are protected. Our results highlight the importance of evaluating the effectiveness of the Abs against different ricin isoforms to identify the treatment with the broadest spectrum neutralizing effect.
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http://dx.doi.org/10.3390/toxins13020100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7911099PMC
January 2021

Development and Evaluation of an Immuno-MALDI-TOF Mass Spectrometry Approach for Quantification of the Abrin Toxin in Complex Food Matrices.

Toxins (Basel) 2021 01 13;13(1). Epub 2021 Jan 13.

CEA, INRAE, Département Médicaments et Technologies pour la Santé (DMTS), Université Paris Saclay, SPI, 91191 Gif-sur-Yvette, France.

The toxin abrin found in the seeds of has attracted much attention regarding criminal and terroristic misuse over the past decade. Progress in analytical methods for a rapid and unambiguous identification of low abrin concentrations in complex matrices is essential. Here, we report on the development and evaluation of a MALDI-TOF mass spectrometry approach for the fast, sensitive and robust abrin isolectin identification, differentiation and quantification in complex food matrices. The method combines immunoaffinity-enrichment with specific abrin antibodies, accelerated trypsin digestion and the subsequent MALDI-TOF analysis of abrin peptides using labeled peptides for quantification purposes. Following the optimization of the workflow, common and isoform-specific peptides were detected resulting in a ~38% sequence coverage of abrin when testing ng-amounts of the toxin. The lower limit of detection was established at 40 ng/mL in milk and apple juice. Isotope-labeled versions of abundant peptides with high ionization efficiency were added. The quantitative evaluation demonstrated an assay variability at or below 22% with a linear range up to 800 ng/mL. MALDI-TOF mass spectrometry allows for a simple and fast (<5 min) analysis of abrin peptides, without a time-consuming peptide chromatographic separation, thus constituting a relevant alternative to liquid chromatography-tandem mass spectrometry.
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http://dx.doi.org/10.3390/toxins13010052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7828309PMC
January 2021

Development and validation of a lateral flow immunoassay for rapid detection of VanA-producing enterococci.

J Antimicrob Chemother 2021 01;76(1):146-151

Team ReSIST, INSERM U1184, School of Medicine, Université Paris-Saclay, LabEx LERMIT, Le Kremlin-Bicêtre, France.

Background: VRE are nosocomial pathogens with an increasing incidence in recent decades. Rapid detection is crucial to reduce their spread and prevent infections and outbreaks.

Objectives: To evaluate a lateral flow immunoassay (LFIA) (called NG-Test VanA) for the rapid and reliable detection of VanA-producing VRE (VanA-VRE) from colonies and broth.

Methods: NG-Test VanA was validated on 135 well-characterized enterococcal isolates grown on Mueller-Hinton (MH) agar (including 40 VanA-VRE). Different agar plates and culture broths widely used in routine laboratories for culture of enterococci were tested.

Results: All 40 VanA-VRE clinical isolates were correctly detected in less than 15 min irrespective of the species expressing the VanA ligase and the medium used for bacterial growth. No cross-reaction was observed with any other clinically relevant ligases (VanB, C1, C2, D, E, G, L, M and N). Overall, the sensitivity and specificity of the assay were 100% for VanA-VRE grown on MH agar plates. NG-Test VanA accurately detects VanA-VRE irrespective of the culture medium (agar and broth). Band intensity was increased when using bacteria grown on vancomycin-containing culture media or on MH close to the vancomycin disc as a consequence of VanA induction. The limit of detection of the assay was 6.3 × 106 cfu per test with bacteria grown on MH plates and 4.9 × 105 cfu per test with bacteria grown on ChromID® VRE plates.

Conclusions: NG-Test VanA is efficient, rapid and easy to implement in the routine workflow of a clinical microbiology laboratory for the confirmation of VanA-VRE.
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http://dx.doi.org/10.1093/jac/dkaa413DOI Listing
January 2021

A Lateral Flow Immunoassay for the Rapid Identification of CTX-M-Producing Enterobacterales from Culture Plates and Positive Blood Cultures.

Diagnostics (Basel) 2020 Sep 28;10(10). Epub 2020 Sep 28.

Team Resist, UMR1184, School of Medicine of Université Paris-Saclay-INSERM-CEA, LabEx Lermit, 94276 Le Kremlin-Bicêtre, France.

We have developed a lateral flow immunoassay (LFIA), named NG-Test CTX-M MULTI (NG-Test), to detect group 1, 2, 8, 9, 25 CTX-M producers from agar plates and from positive blood cultures in less than 15 min. The NG-Test was validated retrospectively on 113 well-characterized enterobacterial isolates, prospectively on 102 consecutively isolated ESBL-producers from the Bicêtre hospital and on 100 consecutive blood cultures positive with a gram-negative bacilli (GNB). The NG-Test was able to detect all CTX-M producers grown on the different agar plates used in clinical microbiology laboratories. No false positive nor negative results were observed. Among the 102 consecutive ESBL isolates, three hyper mucous isolates showed an incorrect migration leading to invalid results (no control band). Using an adapted protocol, the results could be validated. The NG-Test detected 99/102 ESBLs as being CTX-Ms. Three SHV producers were not detected. Among the 100 positive blood cultures with GNB tested 10/11 ESBL-producers were detected (8 CTX-M-15, 2 CTX-M-27). One SHV-2-producing- was missed. The NG-Test CTX-M MULTI showed 100% sensitivity and specificity with isolates cultured on agar plates and was able to detect 98% of the ESBL-producers identified in our clinical setting either from colonies or from positive blood cultures.
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http://dx.doi.org/10.3390/diagnostics10100764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7600033PMC
September 2020

Improvement of the Immunochromatographic NG-Test Carba 5 Assay for the Detection of IMP Variants Previously Undetected.

Antimicrob Agents Chemother 2019 12 20;64(1). Epub 2019 Dec 20.

EA7361 "Structure, Dynamic, Function and Expression of Broad Spectrum β-Lactamases," Université Paris Sud, Université Paris Saclay, LabEx Lermit, Faculty of Medicine, Le Kremlin-Bicêtre, France

Here, we evaluated the immunochromatographic assay NG-Test Carba 5v2 (NG-Biotech), with improved IMP variant detection on 31 IMP producers, representing the different branches of the IMP phylogeny, including 32 OXA-48, 19 KPC, 12 VIM, 14 NDM, and 13 multiple carbapenemase producers (CPs), 13 CPs that were not targeted, and 13 carbapenemase-negative isolates. All tested IMP variants were accurately detected without impairing detection of the other carbapenemases. Thus, NG-Test Carba 5v2 is now well adapted to countries with high IMP prevalence and to the epidemiology of CP-, where IMPs are most frequently detected.
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http://dx.doi.org/10.1128/AAC.01940-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7187612PMC
December 2019

Development and Multicentric Validation of a Lateral Flow Immunoassay for Rapid Detection of MCR-1-Producing .

J Clin Microbiol 2019 05 26;57(5). Epub 2019 Apr 26.

Service de Pharmacologie et Immuno-analyse (SPI), JOLIOT, CEA, INRA, Université Paris-Saclay, Gif sur Yvette, France.

Colistin has become a last-resort antibiotic for the treatment of infections caused by highly drug-resistant Gram-negative bacteria. Moreover, it has been widely used in the livestock sector. As a consequence, colistin resistance is emerging worldwide. Among the colistin resistance mechanisms, the spread of the plasmid-encoded colistin resistance gene (mostly in ) is of particular concern due to its increased transferability compared to that of chromosome-encoded resistance. The early detection of MCR-1-producing bacteria is essential to prevent further spread and provide appropriate antimicrobial therapy. Lateral flow immunoassays (LFIAs) were manufactured with selected monoclonal antibodies. A collection of 177 human and 121 animal enterobacterial isolates was tested in a multicentric study. One bacterial colony grown on agar plates was suspended in extraction buffer and dispensed on the cassette. Migration was allowed for 15 min, and the results were monitored by the appearance of a specific band. The positive results showed a pink line resulting in an unambiguous interpretation. All MCR-1-producing isolates were found to be positive by the LFIA, and no false-negative results were observed. Three out of four MCR-2-producing isolates were also found to be positive. Our test does not detect MCR-3-, MCR-4-, or MCR-5-producing isolates. LFIA allows the detection of MCR-1 with 100% sensitivity and 98% specificity. This test is fast, sensitive, specific, easy to use, and cost-effective and can therefore be implemented in any microbiology laboratory worldwide. LFIA is a major tool for the rapid detection and monitoring of MCR-1 producers in humans and animals.
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http://dx.doi.org/10.1128/JCM.01454-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6498016PMC
May 2019

Evaluation of the NG-Test CARBA 5 multiplex immunochromatographic assay for the detection of KPC, OXA-48-like, NDM, VIM and IMP carbapenemases.

J Antimicrob Chemother 2018 12;73(12):3523-3526

Antimicrobial Resistance and Healthcare Associated Infections (AMRHAI) Reference Unit, National Infection Service, Public Health England, London, UK.

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http://dx.doi.org/10.1093/jac/dky342DOI Listing
December 2018

A multiplex lateral flow immunoassay for the rapid identification of NDM-, KPC-, IMP- and VIM-type and OXA-48-like carbapenemase-producing Enterobacteriaceae.

J Antimicrob Chemother 2018 04;73(4):909-915

Service de Pharmacologie et Immunoanalyse (SPI), CEA, INRA, Laboratoire d'Etudes et de Recherches en Immunonalyse, Université Paris-Saclay, F-91191, Gif-sur-Yvette, France.

Objectives: The global spread of carbapenemase-producing Enterobacteriaceae represents a substantial challenge in clinical practice and rapid and reliable detection of these organisms is essential. The aim of this study was to develop and validate a lateral flow immunoassay (Carba5) for the detection of the five main carbapenemases (KPC-, NDM-, VIM- and IMP-type and OXA-48-like).

Methods: Carba5 was retrospectively and prospectively evaluated using 296 enterobacterial isolates from agar culture. An isolated colony was suspended in extraction buffer and then loaded on the manufactured Carba5.

Results: All 185 isolates expressing a carbapenemase related to one of the Carba5 targets were correctly and unambiguously detected in <15 min. All other isolates gave negative results except those producing OXA-163 and OXA-405, which are considered low-activity carbapenemases. No cross-reaction was observed with non-targeted carbapenemases, ESBLs, AmpCs or oxacillinases (OXA-1, -2, -9 and -10). Overall, this assay reached 100% sensitivity and 95.3% (retrospectively) to 100% (prospectively) specificity.

Conclusions: Carba5 is efficient, rapid and easy to implement in the routine workflow of a clinical microbiology laboratory for confirmation of the five main carbapenemases encountered in Enterobacteriaceae.
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http://dx.doi.org/10.1093/jac/dkx521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5890661PMC
April 2018

Bimodal fluorescence/Xe NMR probe for molecular imaging and biological inhibition of EGFR in Non-Small Cell Lung Cancer.

Bioorg Med Chem 2017 12 6;25(24):6653-6660. Epub 2017 Nov 6.

SCBM, CEA, Université Paris-Saclay, F-91191 Gif-sur-Yvette, France. Electronic address:

Although Non-Small Cell Lung Cancer (NSCLC) is one of the main causes of cancer death, very little improvement has been made in the last decades regarding diagnosis and outcomes. In this study, a bimodal fluorescence/Xe NMR probe containing a xenon host, a fluorescent moiety and a therapeutic antibody has been designed to target the Epidermal Growth Factor Receptors (EGFR) overexpressed in cancer cells. This biosensor shows high selectivity for the EGFR, and a biological activity similar to that of the antibody. It is detected with high specificity and high sensitivity (sub-nanomolar range) through hyperpolarized Xe NMR. This promising system should find important applications for theranostic use.
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http://dx.doi.org/10.1016/j.bmc.2017.11.002DOI Listing
December 2017

Rapid Detection of Abrin Toxin and Its Isoforms in Complex Matrices by Immuno-Extraction and Quantitative High Resolution Targeted Mass Spectrometry.

Anal Chem 2017 11 20;89(21):11719-11727. Epub 2017 Oct 20.

Service de Pharmacologie et Immunoanalyse (SPI), Laboratoire d'Etude du Métabolisme des Médicaments, CEA, INRA, Université Paris Saclay , F-91191 Gif-sur-Yvette cedex, France.

Abrin expressed by the tropical plant Abrus precatorius is highly dangerous with an estimated human lethal dose of 0.1-1 μg/kg body weight. Due to the potential misuse as a biothreat agent, abrin is in the focus of surveillance. Fast and reliable methods are therefore of great importance for early identification. Here, we have developed an innovative and rapid multiepitope immuno-mass spectrometry workflow which is capable of unambiguously differentiating abrin and its isoforms in complex matrices. Toxin-containing samples were incubated with magnetic beads coated with multiple abrin-specific antibodies, thereby concentrating and extracting all the isoforms. Using an ultrasonic bath for digestion enhancement, on-bead trypsin digestion was optimized to obtain efficient and reproducible peptide recovery in only 30 min. Improvements made to the workflow reduced total analysis time to less than 3 h. A large panel of common and isoform-specific peptides was monitored by multiplex LC-MS/MS through the parallel reaction monitoring mode on a quadrupole-Orbitrap high resolution mass spectrometer. Additionally, absolute quantification was accomplished by isotope dilution with labeled AQUA peptides. The newly established method was demonstrated as being sensitive and reproducible with quantification limits in the low ng/mL range in various food and clinical matrices for the isoforms of abrin and also the closely related, less toxic Abrus precatorius agglutinin. This method allows for the first time the rapid detection, differentiation, and simultaneous quantification of abrin and its isoforms by mass spectrometry.
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http://dx.doi.org/10.1021/acs.analchem.7b03189DOI Listing
November 2017

Highly sensitive sandwich immunoassay and immunochromatographic test for the detection of Clostridial epsilon toxin in complex matrices.

PLoS One 2017 11;12(7):e0181013. Epub 2017 Jul 11.

Service de Pharmacologie et Immunoanalyse (SPI), CEA, INRA, Université Paris-Saclay, Gif-sur-Yvette, France.

Epsilon toxin is one of the four major toxins of Clostridium perfringens. It is the third most potent clostridial toxin after botulinum and tetanus toxins and is thus considered as a potential biological weapon classified as category B by the Centers for Disease Control and Prevention (CDC). In the case of a bioterrorist attack, there will be a need for a rapid, sensitive and specific detection method to monitor food and water contamination by this toxin, and for a simple human diagnostic test. We have produced and characterized five monoclonal antibodies against common epitopes of epsilon toxin and prototoxin. Three of them neutralize the cytotoxic effects of epsilon toxin in vitro. With these antibodies, we have developed highly sensitive tests, overnight and 4-h sandwich enzyme immunoassays and an immunochromatographic test performed in 20 min, reaching detection limits of at least 5 pg/mL (0.15 pM), 30 pg/mL (0.9 pM) and 100 pg/mL (3.5 pM) in buffer, respectively. These tests were also evaluated for detection of epsilon toxin in different matrices: milk and tap water for biological threat detection, serum, stool and intestinal content for human or veterinary diagnostic purposes. Detection limits in these complex matrices were at least 5-fold better than those described in the literature (around 1 to 5 ng/mL), reaching 10 to 300 pg/mL using the enzyme immunoassay and 100 to 2000 pg/mL using the immunochromatographic test.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0181013PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5507444PMC
September 2017

Histone variant H2A.J accumulates in senescent cells and promotes inflammatory gene expression.

Nat Commun 2017 05 10;8:14995. Epub 2017 May 10.

Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette cedex, France.

The senescence of mammalian cells is characterized by a proliferative arrest in response to stress and the expression of an inflammatory phenotype. Here we show that histone H2A.J, a poorly studied H2A variant found only in mammals, accumulates in human fibroblasts in senescence with persistent DNA damage. H2A.J also accumulates in mice with aging in a tissue-specific manner and in human skin. Knock-down of H2A.J inhibits the expression of inflammatory genes that contribute to the senescent-associated secretory phenotype (SASP), and over expression of H2A.J increases the expression of some of these genes in proliferating cells. H2A.J accumulation may thus promote the signalling of senescent cells to the immune system, and it may contribute to chronic inflammation and the development of aging-associated diseases.
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http://dx.doi.org/10.1038/ncomms14995DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5436145PMC
May 2017

Detection of Yersinia pestis in Complex Matrices by Intact Cell Immunocapture and Targeted Mass Spectrometry.

Methods Mol Biol 2017 ;1600:69-83

CEA, iBiTec-S, Service de Pharmacologie et d'Immunoanalyse, Bât. 136, 91191, Gif-sur-Yvette, France.

We describe an immunoaffinity-liquid chromatography-tandem mass spectrometry (immuno-LC-MS/MS) protocol for the direct (i.e., without prior culture), sensitive and specific detection of Yersinia pestis in complex matrices. Immunoaffinity enables isolation and concentration of intact bacterial cells from food and environmental samples. After protein extraction and digestion, suitable proteotypic peptides corresponding to three Y. pestis-specific protein markers (murine toxine, plasminogen activator and pesticin) are monitored by targeted LC-MS/MS using the selected reaction monitoring (SRM) mode. This immuno-LC-MS/MS assay has a limit of detection of 2 × 10 CFU/mL in milk or tap water, and 4.5 × 10 CFU in 10 mg of soil.
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http://dx.doi.org/10.1007/978-1-4939-6958-6_7DOI Listing
February 2018

Development and Validation of a Lateral Flow Immunoassay for Rapid Detection of NDM-Producing Enterobacteriaceae.

J Clin Microbiol 2017 07 12;55(7):2018-2029. Epub 2017 Apr 12.

EA7361, Université Paris-Sud, Université Paris-Saclay, LabEx Lermit, Bacteriology-Hygiene Unit, APHP, Hôpital Bicêtre, Le Kremlin-Bicêtre, France

The global spread of carbapenemase-producing (CPE) that are often resistant to most, if not all, classes of antibiotics is a major public health concern. The NDM-1 carbapenemase is among the most worrisome carbapenemases given its rapid worldwide spread. We have developed and evaluated a lateral flow immunoassay (LFIA) (called the NDM LFIA) for the rapid and reliable detection of NDM-like carbapenemase-producing from culture colonies. We evaluated the NDM LFIA using 175 reference enterobacterial isolates with characterized β-lactamase gene content and 74 nonduplicate consecutive carbapenem-resistant clinical isolates referred for expertise to the French National Reference Center (NRC) for Antibiotic Resistance during a 1-week period (in June 2016). The reference collection included 55 non-carbapenemase producers and 120 carbapenemase producers, including 27 NDM producers. All 27 NDM-like carbapenemase producers of the reference collection were correctly detected in less than 15 min by the NDM LFIA, including 22 strains producing NDM-1, 2 producing NDM-4, 1 producing NDM-5, 1 producing NDM-7, and 1 producing NDM-9. All non-NDM-1 producers gave a negative result with the NDM LFIA. No cross-reaction was observed with carbapenemases (VIM, IMP, NDM, KPC, and OXA-48-like), extended-spectrum β-lactamases (ESBLs) (TEM, SHV, and CTX-M), AmpCs (CMY-2, DHA-2, and ACC-1), and oxacillinases (OXA-1, -2, -9, and -10). Similarly, among the 74 referred nonduplicate consecutive clinical isolates, all 7 NDM-like producers were identified. Overall, the sensitivity and specificity of the assay were 100% for NDM-like carbapenemase detection with strains cultured on agar. The NDM LFIA was efficient, rapid, and easy to implement in the routine workflow of a clinical microbiology laboratory for the confirmation of NDM-like carbapenemase-producing .
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http://dx.doi.org/10.1128/JCM.00248-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5483903PMC
July 2017

Recommended Immunological Strategies to Screen for Botulinum Neurotoxin-Containing Samples.

Toxins (Basel) 2015 Nov 26;7(12):5011-34. Epub 2015 Nov 26.

Biological Toxins, Centre for Biological Threats and Special Pathogens, Robert Koch Institute, Seestrasse 10, 13353 Berlin, Germany.

Botulinum neurotoxins (BoNTs) cause the life-threatening neurological illness botulism in humans and animals and are divided into seven serotypes (BoNT/A-G), of which serotypes A, B, E, and F cause the disease in humans. BoNTs are classified as "category A" bioterrorism threat agents and are relevant in the context of the Biological Weapons Convention. An international proficiency test (PT) was conducted to evaluate detection, quantification and discrimination capabilities of 23 expert laboratories from the health, food and security areas. Here we describe three immunological strategies that proved to be successful for the detection and quantification of BoNT/A, B, and E considering the restricted sample volume (1 mL) distributed. To analyze the samples qualitatively and quantitatively, the first strategy was based on sensitive immunoenzymatic and immunochromatographic assays for fast qualitative and quantitative analyses. In the second approach, a bead-based suspension array was used for screening followed by conventional ELISA for quantification. In the third approach, an ELISA plate format assay was used for serotype specific immunodetection of BoNT-cleaved substrates, detecting the activity of the light chain, rather than the toxin protein. The results provide guidance for further steps in quality assurance and highlight problems to address in the future.
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http://dx.doi.org/10.3390/toxins7124860DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4690110PMC
November 2015

Recommended Immunological Assays to Screen for Ricin-Containing Samples.

Toxins (Basel) 2015 Nov 26;7(12):4967-86. Epub 2015 Nov 26.

Bacteriology & Enteric Diseases Division, National Microbiology Laboratory, Public Health Agency of Canada,Winnipeg, MB R3E 3R2, Canada.

Ricin, a toxin from the plant Ricinus communis, is one of the most toxic biological agents known. Due to its availability, toxicity, ease of production and absence of curative treatments, ricin has been classified by the Centers for Disease Control and Prevention (CDC) as category B biological weapon and it is scheduled as a List 1 compound in the Chemical Weapons Convention. An international proficiency test (PT) was conducted to evaluate detection and quantification capabilities of 17 expert laboratories. In this exercise one goal was to analyse the laboratories' capacity to detect and differentiate ricin and the less toxic, but highly homologuous protein R. communis agglutinin (RCA120). Six analytical strategies are presented in this paper based on immunological assays (four immunoenzymatic assays and two immunochromatographic tests). Using these immunological methods "dangerous" samples containing ricin and/or RCA120 were successfully identified. Based on different antibodies used the detection and quantification of ricin and RCA120 was successful. The ricin PT highlighted the performance of different immunological approaches that are exemplarily recommended for highly sensitive and precise quantification of ricin.
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http://dx.doi.org/10.3390/toxins7124858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4690108PMC
November 2015

Mediator independently orchestrates multiple steps of preinitiation complex assembly in vivo.

Nucleic Acids Res 2015 Oct 3;43(19):9214-31. Epub 2015 Aug 3.

Institute for Integrative Biology of the Cell (I2BC), Institut de Biologie et de Technologies de Saclay (iBiTec-S), CEA, CNRS, Université Paris Sud, F-91191 Gif-sur-Yvette cedex, France

Mediator is a large multiprotein complex conserved in all eukaryotes, which has a crucial coregulator function in transcription by RNA polymerase II (Pol II). However, the molecular mechanisms of its action in vivo remain to be understood. Med17 is an essential and central component of the Mediator head module. In this work, we utilised our large collection of conditional temperature-sensitive med17 mutants to investigate Mediator's role in coordinating preinitiation complex (PIC) formation in vivo at the genome level after a transfer to a non-permissive temperature for 45 minutes. The effect of a yeast mutation proposed to be equivalent to the human Med17-L371P responsible for infantile cerebral atrophy was also analyzed. The ChIP-seq results demonstrate that med17 mutations differentially affected the global presence of several PIC components including Mediator, TBP, TFIIH modules and Pol II. Our data show that Mediator stabilizes TFIIK kinase and TFIIH core modules independently, suggesting that the recruitment or the stability of TFIIH modules is regulated independently on yeast genome. We demonstrate that Mediator selectively contributes to TBP recruitment or stabilization to chromatin. This study provides an extensive genome-wide view of Mediator's role in PIC formation, suggesting that Mediator coordinates multiple steps of a PIC assembly pathway.
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http://dx.doi.org/10.1093/nar/gkv782DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4627066PMC
October 2015

Phage amplification and immunomagnetic separation combined with targeted mass spectrometry for sensitive detection of viable bacteria in complex food matrices.

Anal Chem 2015 Jun 14;87(11):5553-60. Epub 2015 May 14.

†bioMérieux S.A., chemin de l'orme, 69280 Marcy-l'Etoile, France.

We have developed and describe here for the first time a highly sensitive method for the fast and unambiguous detection of viable Escherichia coli in food matrices. The new approach is based on using label-free phages (T4), obligate parasites of bacteria, which are attractive for pathogen detection because of their inherent natural specificity and ease of use. A specific immunomagnetic separation was used to capture the progeny phages produced. Subsequently, T4 phage markers were detected by liquid chromatography coupled to targeted mass spectrometry. Combining the specificity of these three methodologies is of great interest in developing an alternative to conventional time-consuming culture-based technologies for the detection of viable bacteria for industrial applications. First, optimization experiments with phage T4 spiked in complex matrices (without a phage amplification event) were performed and demonstrated specific, sensitive, and reproducible phage capture and detection in complex matrices including Luria-Bertani broth, orange juice, and skimmed milk. The method developed was then applied to the detection of E. coli spiked in foodstuffs (with a phage amplification event). After having evaluated the impact of infection duration on assay sensitivity, we showed that our assay specifically detects viable E. coli in milk at an initial count of ≥1 colony-forming unit (cfu)/mL after an 8-h infection. This excellent detection limit makes our new approach an alternative to PCR-based assays for rapid bacterial detection.
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http://dx.doi.org/10.1021/ac504508aDOI Listing
June 2015

Photolinker-free photoimmobilization of antibodies onto cellulose for the preparation of immunoassay membranes.

J Mater Chem B 2015 Feb 18;3(6):1079-1088. Epub 2014 Dec 18.

CEA Saclay, IRAMIS, NIMBE, LICSEN (Laboratory of Innovation in Surface Chemistry and Nanosciences), F-91191 Gif sur Yvette, France.

Paper-based detection devices such as lateral flow immunoassays (LFIAs) are inexpensive, rapid, user-friendly and therefore highly promising for providing resource-limited settings with point-of-care diagnostics. Recently, this biosensing field has trended towards three-dimensional microfluidic devices and multiplexed assay platforms. However, many multiplexed paper-based biosensors implement methods incompatible with the conventional LFIA carrier material: nitrocellulose. It thus tends to be replaced by cellulose. This major material change implies to undertake a covalent immobilization of biomolecules onto cellulose which preserves their biological activity. In this perspective, the immobilization process elaborated in this study is entirely biocompatible. While antibody immobilization onto cellulose usually requires chemical modifications of either the biomolecule and/or the membrane, the light-based procedure presented here was performed without any chemical photolinker. Native biomolecules have been successfully immobilized onto paper sheets which therefore enable to perform LFIAs. More generally, the process expounded herein is fast, simple, cost-saving, environmentally-friendly and would be helpful to immobilize chemical-sensitive biomolecules onto cellulose sheets.
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http://dx.doi.org/10.1039/c4tb01138dDOI Listing
February 2015

Fast and sensitive detection of enteropathogenic Yersinia by immunoassays.

J Clin Microbiol 2015 Jan 29;53(1):146-59. Epub 2014 Oct 29.

CEA Saclay, iBiTec-S, Service de Pharmacologie et d'Immunoanalyse, Laboratoire d'Etudes et de Recherches en Immunoanalyse, Gif sur Yvette, France

Yersinia enterocolitica and Yersinia pseudotuberculosis, the two Yersinia species that are enteropathogenic for humans, are distributed worldwide and frequently cause diarrhea in inhabitants of temperate and cold countries. Y. enterocolitica is a major cause of foodborne disease resulting from consumption of contaminated pork meat and is further associated with substantial economic cost. However, investigation of enteropathogenic Yersinia species is infrequently performed routinely in clinical laboratories because of their specific growth characteristics, which make difficult their isolation from stool samples. Moreover, current isolation procedures are time-consuming and expensive, thus leading to underestimates of the incidence of enteric yersiniosis, inappropriate prescriptions of antibiotic treatments, and unnecessary appendectomies. The main objective of the study was to develop fast, sensitive, specific, and easy-to-use immunoassays, useful for both human and veterinary diagnosis. Monoclonal antibodies (MAbs) directed against Y. enterocolitica bioserotypes 2/O:9 and 4/O:3 and Y. pseudotuberculosis serotypes I and III were produced. Pairs of MAbs were selected by testing their specificity and affinity for enteropathogenic Yersinia and other commonly found enterobacteria. Pairs of MAbs were selected to develop highly sensitive enzyme immunoassays (EIAs) and lateral flow immunoassays (LFIs or dipsticks) convenient for the purpose of rapid diagnosis. The limit of detection of the EIAs ranged from 3.2 × 10(3) CFU/ml to 8.8 × 10(4) CFU/ml for pathogenic serotypes I and III of Y. pseudotuberculosis and pathogenic bioserotypes 2/O:9 and 4/O:3 of Y. enterocolitica and for the LFIs ranged from 10(5) CFU/ml to 10(6) CFU/ml. A similar limit of detection was observed for artificially contaminated human feces.
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http://dx.doi.org/10.1128/JCM.02137-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4290913PMC
January 2015

A rapid lateral flow immunoassay for the detection of tyrosine phosphatase-like protein IA-2 autoantibodies in human serum.

PLoS One 2014 21;9(7):e103088. Epub 2014 Jul 21.

CEA, iBiTec-S, Service de Pharmacologie et d'Immunoanalyse, Laboratoire d'Etudes et de Recherches en Immunoanalyse, Gif sur Yvette, France.

Type 1 diabetes (T1D) results from the destruction of pancreatic insulin-producing beta cells and is strongly associated with the presence of islet autoantibodies. Autoantibodies to tyrosine phosphatase-like protein IA-2 (IA-2As) are considered to be highly predictive markers of T1D. We developed a novel lateral flow immunoassay (LFIA) based on a bridging format for the rapid detection of IA-2As in human serum samples. In this assay, one site of the IA-2As is bound to HA-tagged-IA-2, which is subsequently captured on the anti-HA-Tag antibody-coated test line on the strip. The other site of the IA-2As is bound to biotinylated IA-2, allowing the complex to be visualized using colloidal gold nanoparticle-conjugated streptavidin. For this study, 35 serum samples from T1D patients and 44 control sera from non-diabetic individuals were analyzed with our novel assay and the results were correlated with two IA-2A ELISAs. Among the 35 serum samples from T1D patients, the IA-2A LFIA, the in-house IA-2A ELISA and the commercial IA-2A ELISA identified as positive 21, 29 and 30 IA-2A-positive sera, respectively. The major advantages of the IA-2A LFIA are its rapidity and simplicity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0103088PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4105419PMC
November 2015

Fast and direct extraction of cell-associated hepatotoxins from toxic cyanobacteria.

Water Environ Res 2014 May;86(5):470-7

Microcystins are an important group of toxins produced by cyanobacteria of different genera. An increasing number of water contaminations by this class of toxins have been reported that are susceptible to generate important public health problems. We designed an efficient method for extracting these toxins on-site for a rapid testing of potentially contaminated water. The extraction parameters have been optimized using Microcystis aeruginosa and the technique successfully applied to different laboratory cultures and field samples. The procedure employs a simple and stable reagent mix of propanol, Tween 20, and trifluoroacetic acid. It is directly active on crude cell suspensions without any pre-treatment. Extraction yields measured by immunological quantification were at least equal to the best values obtained with the most commonly used laboratory techniques. An additional simple concentration/extraction step is also described that allows measurements on samples too dilute for direct detection by immunochromatographic techniques.
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http://dx.doi.org/10.2175/106143013x13807328849891DOI Listing
May 2014

Detection of Yersinia pestis in environmental and food samples by intact cell immunocapture and liquid chromatography-tandem mass spectrometry.

Anal Chem 2014 Jun 29;86(12):6144-52. Epub 2014 May 29.

Service de Pharmacologie et d'Immunoanalyse, Institut de Biologie et de Technologies de Saclay (iBiTec-S), Commissariat à l'Énergie Atomique et aux Énergies Alternatives (CEA) , 91191 Gif-sur-Yvette, France.

Yersinia pestis is the causative agent of bubonic and pneumonic plague, an acute and often fatal disease in humans. In addition to the risk of natural exposure to plague, there is also the threat of a bioterrorist act, leading to the deliberate spread of the bacteria in the environment or food. We report here an immuno-liquid chromatography-tandem mass spectrometry (immuno-LC-MS/MS) method for the direct (i.e., without prior culture), sensitive, and specific detection of Y. pestis in such complex samples. In the first step, a bottom-up proteomics approach highlighted three relevant protein markers encoded by the Y. pestis-specific plasmids pFra (murine toxin) and pPla (plasminogen activator and pesticin). Suitable proteotypic peptides were thoroughly selected to monitor the three protein markers by targeted MS using the selected reaction monitoring (SRM) mode. Immunocapture conditions were optimized for the isolation and concentration of intact bacterial cells from complex samples. The immuno-LC-SRM assay has a limit of detection of 2 × 10(4) CFU/mL in milk or tap water, which compares well with those of state-of-the-art immunoassays. Moreover, we report the first direct detection of Y. pestis in soil, which could be extremely useful in confirming Y. pestis persistence in the ground.
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http://dx.doi.org/10.1021/ac501371rDOI Listing
June 2014

A simple and fast non-radioactive bridging immunoassay for insulin autoantibodies.

PLoS One 2013 29;8(7):e69021. Epub 2013 Jul 29.

Commisariat à l'Energie Atomique, iBiTec-S, Service de Pharmacologie et d'Immunoanalyse, Commisariat à l'Energie Atomique Saclay, Gif sur Yvette, France.

Type 1 diabetes (T1D) is an autoimmune disease which results from the destruction of pancreatic beta cells. Autoantibodies directed against islet antigens are valuable diagnostic tools. Insulin autoantibodies (IAAs) are usually the first to appear and also the most difficult to detect amongst the four major islet autoantibodies. A non-radioactive IAA bridging ELISA was developed to this end. In this assay, one site of the IAAs from serum samples is bound to a hapten-labeled insulin (GC300-insulin), which is subsequently captured on anti-GC300 antibody-coated 96-well plates. The other site of the IAAs is bound to biotinylated insulin, allowing the complex to be detected by an enzyme-streptavidin conjugate. In the present study, 50 serum samples from patients with newly diagnosed T1D and 100 control sera from non-diabetic individuals were analyzed with our new assay and the results were correlated with an IAA radioimmunoassay (RIA). Using IAA bridging ELISA, IAAs were detected in 32 out of 50 T1D children, whereas with IAA RIA, 41 out of 50 children with newly diagnosed T1D were scored as positive. In conclusion, the IAA bridging ELISA could serve as an attractive approach for rapid and automated detection of IAAs in T1D patients for diagnostic purposes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0069021PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3726704PMC
February 2014

A one-step and biocompatible cellulose functionalization for covalent antibody immobilization on immunoassay membranes.

J Mater Chem B 2013 Jul 24;1(26):3277-3286. Epub 2013 May 24.

CEA/DSM/IRAMIS/SPCSI/Laboratoire de Chimie des Surfaces et Interfaces (LCSI), Gif sur Yvette, F-91191, France.

Among bioactive papers, many multiplexed assays implement methods incompatible with the conventional lateral flow immunoassay (LFIA) carrier material, nitrocellulose. Consequently, its replacement by cellulose has to be considered. This technological breakthrough requires a surface chemistry which ensures both the biomolecules covalent grafting to cellulose and the conservation of their biological activity. To comply with these requirements, the process elaborated in this study implements compounds and methods compatible with biological material. While cellulose chemical modification is usually operated under harsh conditions in organic solvents, the diazonium-based functionalization procedure presented here was performed onto cellulose sheets in water and at room temperature. Paper sheets have been successfully modified and bear different chemical functions which enable grafting of biomolecules by common bioconjugate techniques and to perform LFIAs. More generally, the chemical ways developed in this study are suitable for many biomolecules and would be helpful for any sensitive molecule immobilization onto cellulose sheets.
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http://dx.doi.org/10.1039/c3tb20380hDOI Listing
July 2013

Pathogen-free screening of bacteria-specific hybridomas for selecting high-quality monoclonal antibodies against pathogen bacteria as illustrated for Legionella pneumophila.

J Immunol Methods 2013 May 27;391(1-2):81-94. Epub 2013 Feb 27.

CEA, iBiTec-S, Service de Pharmacologie et d'Immunoanalyse, CEA Saclay, 91191 Gif-sur-Yvette, France.

Antibodies are potent biological tools increasingly used as detection, diagnostic and therapeutic reagents. Many technological advances have optimized and facilitated production and screening of monoclonal antibodies. We report here an original method to screen for antibodies targeting biosafety level 2 or 3 pathogens without the fastidious handling inherent to pathogen use. A double ELISA screening was performed using as coated antigen transformed Escherichia coli expressing at its surface a protein specific to the pathogenic bacteria versus control untransformed E. coli. This method was applied to Legionella, using the surface-exposed Mip protein (macrophage infectivity potentiator). This screening proved to be an excellent means of selecting mAbs that bind Legionella pneumophila 1 surface-exposed Mip protein. This method also appears more biologically relevant than screening using the recombinant Mip protein alone and less tedious than a test performed directly on Legionella bacteria. We obtained 21 mAbs that bind strongly to L. pneumophila serogroups 1 to 13, and we validated their use in a rapid ELISA (performed in 4.5 h) and an immunochromatographic test (20 min).
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http://dx.doi.org/10.1016/j.jim.2013.02.012DOI Listing
May 2013

Fast and simple detection of Yersinia pestis applicable to field investigation of plague foci.

PLoS One 2013 29;8(1):e54947. Epub 2013 Jan 29.

CEA Saclay, iBiTec-S, Service de Pharmacologie et d'Immunoanalyse, Laboratoire d'Etudes et de Recherches en Immunoanalyse, Gif sur Yvette, France.

Yersinia pestis, the plague bacillus, has a rodent-flea-rodent life cycle but can also persist in the environment for various periods of time. There is now a convenient and effective test (F1-dipstick) for the rapid identification of Y. pestis from human patient or rodent samples, but this test cannot be applied to environmental or flea materials because the F1 capsule is mostly produced at 37°C. The plasminogen activator (PLA), a key virulence factor encoded by a Y. pestis-specific plasmid, is synthesized both at 20°C and 37°C, making it a good candidate antigen for environmental detection of Y. pestis by immunological methods. A recombinant PLA protein from Y. pestis synthesized by an Escherichia coli strain was used to produce monoclonal antibodies (mAbs). PLA-specific mAbs devoid of cross-reactions with other homologous proteins were further cloned. A pair of mAbs was selected based on its specificity, sensitivity, comprehensiveness, and ability to react with Y. pestis strains grown at different temperatures. These antibodies were used to develop a highly sensitive one-step PLA-enzyme immunoassay (PLA-EIA) and an immunostrip (PLA-dipstick), usable as a rapid test under field conditions. These two PLA-immunometric tests could be valuable, in addition to the F1-disptick, to confirm human plague diagnosis in non-endemic areas (WHO standard case definition). They have the supplementary advantage of allowing a rapid and easy detection of Y. pestis in environmental and flea samples, and would therefore be of great value for surveillance and epidemiological investigations of plague foci. Finally, they will be able to detect natural or genetically engineered F1-negative Y. pestis strains in human patients and environmental samples.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0054947PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3558477PMC
July 2013

Toxin detection in patients' sera by mass spectrometry during two outbreaks of type A Botulism in France.

J Clin Microbiol 2012 Dec 19;50(12):4091-4. Epub 2012 Sep 19.

Institut Pasteur, Unité des Bactéries Anaérobies et Toxines, Paris, France.

In two outbreaks of food-borne botulism in France, Clostridium botulinum type A was isolated and characterized from incriminated foods. Botulinum neurotoxin type A was detected in the patients' sera by mouse bioassay and in vitro endopeptidase assay with an immunocapture step and identification of the cleavage products by mass spectrometry.
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http://dx.doi.org/10.1128/JCM.02392-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3502950PMC
December 2012

Fast and sensitive detection of Bacillus anthracis spores by immunoassay.

Appl Environ Microbiol 2012 Sep 6;78(18):6491-8. Epub 2012 Jul 6.

CEA Service de Pharmacologie et d'Immunoanalyse, CEA/Saclay, Gif sur Yvette, France.

Bacillus anthracis is one of the most dangerous potential biological weapons, and it is essential to develop a rapid and simple method to detect B. anthracis spores in environmental samples. The immunoassay is a rapid and easy-to-use method for the detection of B. anthracis by means of antibodies directed against surface spore antigens. With this objective in view, we have produced a panel of monoclonal antibodies against B. anthracis and developed colorimetric and electrochemiluminescence (ECL) immunoassays. Using Meso Scale Discovery ECL technology, which is based on electrochemiluminescence (ECL) detection utilizing a sulfo-Tag label that emits light upon electrochemical stimulation (using a dedicated ECL plate reader, an electrical current is placed across the microplate with electrodes integrated into the bottom of the plate, resulting in a series of electrically induced reactions leading to a luminescent signal), a detection limit ranging between 0.3 × 10(3) and 10(3) CFU/ml (i.e., 30 to 100 spores per test), depending on the B. anthracis strain assayed, was achieved. In complex matrices (5 mg/ml of soil or simulated powder), the detection level (without any sample purification or concentration) was never altered more than 3-fold compared with the results obtained in phosphate-buffered saline.
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http://dx.doi.org/10.1128/AEM.01282-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3426714PMC
September 2012
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