Publications by authors named "Stéphanie Simon"

88 Publications

Small Hsps as Therapeutic Targets of Cystic Fibrosis Transmembrane Conductance Regulator Protein.

Int J Mol Sci 2021 Apr 20;22(8). Epub 2021 Apr 20.

Apoptosis, Cancer and Development Laboratory, Lyon Cancer Research Center, INSERM U1052-CNRS UMR5286, Claude Bernard University Lyon 1, Centre Léon Bérard, F-69008 Lyon, France.

Human small heat shock proteins are molecular chaperones that regulate fundamental cellular processes in normal and pathological cells. Here, we have reviewed the role played by HspB1, HspB4 and HspB5 in the context of Cystic Fibrosis (CF), a severe monogenic autosomal recessive disease linked to mutations in Cystic Fibrosis Transmembrane conductance Regulator protein (CFTR) some of which trigger its misfolding and rapid degradation, particularly the most frequent one, F508del-CFTR. While HspB1 and HspB4 favor the degradation of CFTR mutants, HspB5 and particularly one of its phosphorylated forms positively enhance the transport at the plasma membrane, stability and function of the CFTR mutant. Moreover, HspB5 molecules stimulate the cellular efficiency of currently used CF therapeutic molecules. Different strategies are suggested to modulate the level of expression or the activity of these small heat shock proteins in view of potential in vivo therapeutic approaches. We then conclude with other small heat shock proteins that should be tested or further studied to improve our knowledge of CFTR processing.
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http://dx.doi.org/10.3390/ijms22084252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8072646PMC
April 2021

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

Innovative and Highly Sensitive Detection of Enterotoxin Based on Receptor Interaction and Monoclonal Antibodies.

Toxins (Basel) 2021 04 8;13(4). Epub 2021 Apr 8.

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

enterotoxin (CPE) regularly causes food poisoning and antibiotic-associated diarrhea; therefore, reliable toxin detection is crucial. To this aim, we explored stationary and mobile strategies to detect CPE either exclusively by monoclonal antibodies (mAbs) or, alternatively, by toxin-enrichment via the cellular receptor of CPE, claudin-4, and mAb detection. Among the newly generated mAbs, we identified nine CPE-specific mAbs targeting five distinct epitopes, among them mAbs recognizing CPE bound to claudin-4 or neutralizing CPE activity in vitro. In surface plasmon resonance experiments, all mAbs and claudin-4 revealed excellent affinities towards CPE, ranging from 0.05 to 2.3 nM. Integrated into sandwich enzyme-linked immunosorbent assays (ELISAs), the most sensitive mAb/mAb and claudin-4/mAb combinations achieved similar detection limits of 0.3 pg/mL and 1.0 pg/mL, respectively, specifically detecting recombinant CPE from spiked feces and native CPE from 30 different culture supernatants. The implementation of mAb- and receptor-based ELISAs into a mobile detection platform enabled the fast detection of CPE, which will be helpful in clinical laboratories to diagnose diarrhea of assumed bacterial origin. In conclusion, we successfully employed an endogenous receptor and novel high affinity mAbs for highly sensitive and specific CPE-detection. These tools will be useful for both basic and applied research.
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http://dx.doi.org/10.3390/toxins13040266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8068247PMC
April 2021

Characterization of a highly neutralizing single monoclonal antibody to botulinum neurotoxin type A.

FASEB J 2021 May;35(5):e21540

Institut Pasteur, Unité des Toxines Bactériennes, UMR CNRS 2001, Paris, France.

Compared to conventional antisera strategies, monoclonal antibodies (mAbs) represent an alternative and safer way to treat botulism, a fatal flaccid paralysis due to botulinum neurotoxins (BoNTs). In addition, mAbs offer the advantage to be produced in a reproducible manner. We previously identified a unique and potent mouse mAb (TA12) targeting BoNT/A1 with high affinity and neutralizing activity. In this study, we characterized the molecular basis of TA12 neutralization by combining Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS) with site-directed mutagenesis and functional studies. We found that TA12 recognizes a conformational epitope located at the interface between the H and H subdomains of the BoNT/A1 receptor-binding domain (H ). The TA12-binding interface shares common structural features with the ciA-C2 VHH epitope and lies on the face opposite recognized by ciA-C2- and the CR1/CR2-neutralizing mAbs. The single substitution of N1006 was sufficient to affect TA12 binding to H confirming the position of the epitope. We further uncovered that the TA12 epitope overlaps with the BoNT/A1-binding site for both the neuronal cell surface receptor synaptic vesicle glycoprotein 2 isoform C (SV2C) and the GT1b ganglioside. Hence, TA12 potently blocks the entry of BoNT/A1 into neurons by interfering simultaneously with the binding of SV2C and to a lower extent GT1b. Our study reveals the unique neutralization mechanism of TA12 and emphasizes on the potential of using single mAbs for the treatment of botulism type A.
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http://dx.doi.org/10.1096/fj.202002492RDOI Listing
May 2021

The Microbiome Meets Nanotechnology: Opportunities and Challenges in Developing New Diagnostic Devices.

Adv Mater 2021 May 14;33(18):e2006104. Epub 2021 Mar 14.

Nanobioelectronics and Biosensors Group, Institut Català de Nanociència i Nanotecnologia (ICN2), UAB Campus, Bellaterra, Barcelona, 08193, Spain.

Monitoring of the human microbiome is an emerging area of diagnostics for personalized medicine. Here, the potential of different nanomaterials and nanobiosensing technologies is reviewed for the development of novel diagnostic devices for the detection and measurement of microbiome-related biomarkers. Moreover, the current and future landscape of microbiome-based diagnostics is defined by exploring the advantages and disadvantages of current nanotechnology-based approaches, especially in the context of developing point-of-care (PoC) devices that would meet the international guidelines known as REASSURED (Real-time connectivity; Ease of specimen collection; Affordability; Sensitivity; Specificity; User-friendliness; Rapid & robust operation; Equipment-free; and Deliverability). Finally, the strategies of the latest international scientific consortia working in this field are analyzed, the current microbiome diagnostics market are reported and the principal ethical, legal, and societal issues related to microbiome R&D and innovation are discussed.
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http://dx.doi.org/10.1002/adma.202006104DOI Listing
May 2021

An antibody targeting type III secretion system induces broad protection against Salmonella and Shigella infections.

PLoS Negl Trop Dis 2021 03 12;15(3):e0009231. Epub 2021 Mar 12.

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

Salmonella and Shigella bacteria are food- and waterborne pathogens that are responsible for enteric infections in humans and are still the major cause of morbidity and mortality in the emerging countries. The existence of multiple Salmonella and Shigella serotypes as well as the emergence of strains resistant to antibiotics requires the development of broadly protective therapies. Recently, the needle tip proteins of the type III secretion system of these bacteria were successfully utilized (SipD for Salmonella and IpaD for Shigella) as vaccine immunogens to provide good prophylactic cross-protection in murine models of infections. From these experiments, we have isolated a cross-protective monoclonal antibody directed against a conserved region of both proteins. Its conformational epitope determined by Deep Mutational Scanning is conserved among needle tip proteins of all pathogenic Shigella species and Salmonella serovars, and are well recognized by this antibody. Our study provides the first in vivo experimental evidence of the importance of this common region in the mechanism of virulence of Salmonella and Shigella and opens the way to the development of cross-protective therapeutic agents.
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http://dx.doi.org/10.1371/journal.pntd.0009231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7990167PMC
March 2021

Quantitative Determination of Enterotoxins Types A to I and Variants in Dairy Food Products by Multiplex Immuno-LC-MS/MS.

J Agric Food Chem 2021 Mar 17;69(8):2603-2610. Epub 2021 Feb 17.

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

Staphylococcal enterotoxins (SEs) are responsible for frequent food poisoning outbreaks worldwide. Specific identification of SEs is crucial for confirmation of food poisoning, tracking of the incriminated foods or food ingredients, and removal from the food chain. Here, we report on a new food testing protocol addressing the challenge of low abundance of SEs in contaminated food and high sequence heterogeneity. Multiplex ability of targeted high-resolution mass spectrometry was succesfully applied to the simultaneous and quantitative determination of the eight most frequent SEs including sequence variants. In this aim, between three and eight proteotypic peptides of each SE were selected by carefully considering amino acid variations within each type, and sequence homology between types. Quantification of trace levels of SEs directly in food samples was reached by immunoaffinity enrichment and optimized analytical conditions. The assay was validated in dairy food products with a lower limit of quantification down to 0.1 ng/g (in milk), and quantification of SEs was successfully demonstrated in real-life samples collected during staphylococcal food poisoning outbreaks. Importantly, the ability of the method to detect diverse sequence variants was also illustrated. By enabling for the first time the simultaneous quantification of the eight most frequent SEs, the new mass spectrometry-based assay would facilitate the laboratory confirmation of positive samples in situation of food poisoning outbreaks.
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http://dx.doi.org/10.1021/acs.jafc.0c07545DOI Listing
March 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

Highly Sensitive and Specific Detection of Staphylococcal Enterotoxins SEA, SEG, SEH, and SEI by Immunoassay.

Toxins (Basel) 2021 02 9;13(2). Epub 2021 Feb 9.

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

Staphylococcal food poisoning (SFP) is one of the most common foodborne diseases worldwide, resulting from the ingestion of staphylococcal enterotoxins (SEs), primarily SE type A (SEA), which is produced in food by enterotoxigenic strains of staphylococci, mainly . Since newly identified SEs have been shown to have emetic properties and the genes encoding them have been found in food involved in poisoning outbreaks, it is necessary to have reliable tools to prove the presence of the toxins themselves, to clarify the role played by these non-classical SEs, and to precisely document SFP outbreaks. We have produced and characterized monoclonal antibodies directed specifically against SE type G, H or I (SEG, SEH or SEI respectively) or SEA. With these antibodies, we have developed, for each of these four targets, highly sensitive, specific, and reliable 3-h sandwich enzyme immunoassays that we evaluated for their suitability for SE detection in different matrices (bacterial cultures of , contaminated food, human samples) for different purposes (strain characterization, food safety, biological threat detection, diagnosis). We also initiated and described for the first time the development of monoplex and quintuplex (SEA, SE type B (SEB), SEG, SEH, and SEI) lateral flow immunoassays for these new staphylococcal enterotoxins. The detection limits in buffer were under 10 pg/mL (0.4 pM) by enzyme immunoassays and at least 300 pg/mL (11 pM) by immunochromatography for all target toxins with no cross-reactivity observed. Spiking studies and/or bacterial supernatant analysis demonstrated the applicability of the developed methods, which could become reliable detection tools for the routine investigation of SEG, SEH, and SEI.
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http://dx.doi.org/10.3390/toxins13020130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7916246PMC
February 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

Implementing a standardized screening protocol for parental depression, anxiety, and PTSD symptoms in the Neonatal Intensive Care Unit.

Early Hum Dev 2021 Mar 16;154:105279. Epub 2020 Nov 16.

Stanford University School of Medicine, Palo Alto, CA, USA. Electronic address:

The aim of this paper is to describe the development of a standardized screening program for parents of infants in the Neonatal Intensive Care Unit (NICU) and to assess its implementation. The standardized screening protocol assessed parental mental health symptoms including depression, anxiety and trauma. Screening began at 14 days post NICU admission and was implemented as part of routine medical care for all caregivers with infants admitted to the NICU at two weeks of age. Screenings were facilitated by pediatric social workers and psychology postdoctoral fellows and included review of critical self-harm items. A total of 158 parents ages 18-42 years (mean = 31.04) were eligible for screening, with 150 completed screenings. Positive screens on any of the three measures resulted in a mental health referral. Approximately 27% of parents had a positive screen that resulted in a mental health referral. The standardized screening protocol was found to be feasible, widely accepted, and effective in establishing referrals for in house mental health services. This model can be used as an example to help other NICUs implement their own universal screening protocols.
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http://dx.doi.org/10.1016/j.earlhumdev.2020.105279DOI Listing
March 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

Prevention of posttraumatic stress disorder in mothers of preterm infants using trauma-focused group therapy: Manual development and evaluation.

Early Hum Dev 2021 Mar 16;154:105282. Epub 2020 Nov 16.

Division of Child and Adolescent Psychiatry, Stanford University School of Medicine, 401 Quarry Road, Palo Alto, CA 94305, USA. Electronic address:

Background: Preterm birth has been associated with a number of adverse maternal psychological outcomes.

Aims: The current study aims to develop and evaluate the feasibility of a trauma-focused group intervention that is designed to reduce maternal symptoms of anxiety, depression, and posttraumatic stress in a sample of mothers of preterm infants hospitalized in a neonatal intensive care unit (NICU).

Study Design: The study was a one-group pre-/post quasi-experimental design. Participants received a 6-session intervention targeting parental trauma.

Subjects: English-speaking mothers (N = 19) greater than 18 years of age of infants 23-34 weeks gestational age hospitalized in the NICU at Lucile Packard Children's Hospital Stanford.

Outcome Measures: Beck Anxiety Inventory (BAI), Beck Depression Inventory, Second Edition (BDI-II), Davidson Trauma Scale (DTS).

Results: Results from the study indicate that the intervention is feasible, able to be implemented with a high degree of fidelity, is rated as highly satisfactory by participants, and leads to statistically significant reductions in symptoms of anxiety, depression, and posttraumatic stress at 6-week and 6-month follow-ups.

Conclusions: Though encouraging, these findings are preliminary, and future studies should strive to reproduce these findings with a larger sample size and a comparison group.
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http://dx.doi.org/10.1016/j.earlhumdev.2020.105282DOI Listing
March 2021

Methylprednisolone pulse treatment improves ProSP-C trafficking in twins with SFTPC mutation: An isoform story?

Br J Clin Pharmacol 2021 May 4;87(5):2361-2373. Epub 2021 Jan 4.

Université Paris Est Creteil, INSERM, IMRB, F-94010 Creteil, France.

Mutations in the gene encoding surfactant protein C (SP-C) cause interstitial lung disease (ILD), and glucocorticosteroid (GC) treatment is the most recognized therapy in children. We aimed to decipher the mechanisms behind successful GC treatment in twins carrying a BRICHOS c.566G > A (p.Cys189Tyr) mutation in the SP-C gene (SFTPC). METHODS: The twins underwent bronchoscopy before and after GC treatment and immunoblotting analysis of SP-C proprotein (proSP-C) and SP-C mature in bronchoalveolar fluid (BALF). Total RNA was extracted and analysed using quantitative real-time PCR assays. In A549 cells, the processing of mutated protein C189Y was studied by immunofluorescence and immunoblotting after heterologous expression of eukaryotic vectors containing wild type or C189Y mutant cDNA. RESULTS: Before treatment, BALF analysis identified an alteration of the proSP-C maturation process. Functional study of C189Y mutation in alveolar A549 cells showed that pro-SP-C was retained within the endoplasmic reticulum together with ABCA3. After 5 months of GC treatment with clinical benefit, the BALF analysis showed an improvement of proSP-C processing. SFTPC mRNA analysis in twins revealed a decrease in the expression of total SFTPC mRNA and a change in its splicing, leading to the expression of a second shorter proSP-C isoform. In A549 cells, the processing and the stability of this shorter wild-type proSP-C isoform was similar to that of the longer isoform, but the half-life of the mutated shorter isoform was decreased. These results suggest a direct effect of GC on proSP-C metabolism through reducing the SFTPC mRNA level and favouring the expression of a less stable protein isoform.
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http://dx.doi.org/10.1111/bcp.14645DOI Listing
May 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

Phosphorylation of the Chaperone-Like HspB5 Rescues Trafficking and Function of F508del-CFTR.

Int J Mol Sci 2020 Jul 8;21(14). Epub 2020 Jul 8.

Univ Paris Est Creteil, INSERM, IMRB, F-94010 Creteil, France.

Cystic Fibrosis is a lethal monogenic autosomal recessive disease linked to mutations in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein. The most frequent mutation is the deletion of phenylalanine at position 508 of the protein. This F508del-CFTR mutation leads to misfolded protein that is detected by the quality control machinery within the endoplasmic reticulum and targeted for destruction by the proteasome. Modulating quality control proteins as molecular chaperones is a promising strategy for attenuating the degradation and stabilizing the mutant CFTR at the plasma membrane. Among the molecular chaperones, the small heat shock protein HspB1 and HspB4 were shown to promote degradation of F508del-CFTR. Here, we investigated the impact of HspB5 expression and phosphorylation on transport to the plasma membrane, function and stability of F508del-CFTR. We show that a phosphomimetic form of HspB5 increases the transport to the plasma membrane, function and stability of F508del-CFTR. These activities are further enhanced in presence of therapeutic drugs currently used for the treatment of cystic fibrosis (VX-770/Ivacaftor, VX-770+VX-809/Orkambi). Overall, this study highlights the beneficial effects of a phosphorylated form of HspB5 on F508del-CFTR rescue and its therapeutic potential in cystic fibrosis.
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http://dx.doi.org/10.3390/ijms21144844DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7402320PMC
July 2020

Altered age-linked regulation of plasma DYRK1A in elderly cognitive complainers (INSIGHT-preAD study) with high brain amyloid load.

Alzheimers Dement (N Y) 2020 2;6(1):e12046. Epub 2020 Jul 2.

INSERM U 1127, CNRS UMR 7225 UPMC Univ Paris 06 UMR S 1127, Institut du Cerveau et la Moelle épinière, ICM Sorbonne Universités Paris France.

Introduction: An effective therapy has not yet been developed for Alzheimer's disease (AD), in part because pathological changes occur years before clinical symptoms manifest. We recently showed that decreased plasma DYRK1A identifies individuals with mild cognitive impairment (MCI) or AD, and that aged mice have higher DYRK1A levels.

Methods: We assessed DYRK1A in plasma in young/aged controls and in elderly cognitive complainers with low (L) and high (H) brain amyloid load.

Results: DYRK1A level increases with age in humans. However, plasma from elderly individuals reporting cognitive complaints showed that the H group had the same DYRK1A level as young adults, suggesting that the age-associated DYRK1A increase is blocked in this group. L and H groups had similar levels of clusterin.

Discussion: These results are reflective of early changes in the brain. These observations suggest that plasma DYRK1A and not clusterin could be used to classify elderly memory complainers for risk for amyloid beta pathology.
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http://dx.doi.org/10.1002/trc2.12046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7331462PMC
July 2020

SipD and IpaD induce a cross-protection against Shigella and Salmonella infections.

PLoS Negl Trop Dis 2020 05 28;14(5):e0008326. Epub 2020 May 28.

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

Salmonella and Shigella species are food- and water-borne pathogens that are responsible for enteric infections in both humans and animals and are still the major cause of morbidity and mortality in the emerging countries. The existence of multiple Salmonella and Shigella serotypes as well as the emergence of strains resistant to antibiotics require the development of broadly protective therapies. Those bacteria utilize a Type III Secretion System (T3SS), necessary for their pathogenicity. The structural proteins composing the T3SS are common to all virulent Salmonella and Shigella spp., particularly the needle-tip proteins SipD (Salmonella) and IpaD (Shigella). We investigated the immunogenicity and protective efficacy of SipD and IpaD administered by intranasal and intragastric routes, in a mouse model of Salmonella enterica serotype Typhimurium (S. Typhimurium) intestinal challenge. Robust IgG (in all immunization routes) and IgA (in intranasal and oral immunization routes) antibody responses were induced against both proteins. Mice immunized with SipD or IpaD were protected against lethal intestinal challenge with S. Typhimurium or Shigella flexneri (100 Lethal Dose 50%). We have shown that SipD and IpaD are able to induce a cross-protection in a murine model of infection by Salmonella and Shigella. We provide the first demonstration that Salmonella and Shigella T3SS SipD and IpaD are promising antigens for the development of a cross-protective Salmonella-Shigella vaccine. These results open the way to the development of cross-protective therapeutic molecules.
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http://dx.doi.org/10.1371/journal.pntd.0008326DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7282677PMC
May 2020

Revisiting the bacterial mutagenicity assays: Report by a workgroup of the International Workshops on Genotoxicity Testing (IWGT).

Mutat Res 2020 01 13;849:503137. Epub 2020 Jan 13.

Non-Clinical Safety Janssen R&D, a Division of Janssen Pharmaceutica N.V., Beerse, Belgium.

The International Workshop on Genotoxicity Testing (IWGT) meets every four years to obtain consensus on unresolved issues associated with genotoxicity testing. At the 2017 IWGT meeting in Tokyo, four sub-groups addressed issues associated with the Organization for Economic Cooperation and Development (OECD) Test Guideline TG471, which describes the use of bacterial reverse-mutation tests. The strains sub-group analyzed test data from >10,000 chemicals, tested additional chemicals, and concluded that some strains listed in TG471 are unnecessary because they detected fewer mutagens than other strains that the guideline describes as equivalent. Thus, they concluded that a smaller panel of strains would suffice to detect most mutagens. The laboratory proficiency sub-group recommended (a) establishing strain cell banks, (b) developing bacterial growth protocols that optimize assay sensitivity, and (c) testing "proficiency compounds" to gain assay experience and establish historical positive and control databases. The sub-group on criteria for assay evaluation recommended that laboratories (a) track positive and negative control data; (b) develop acceptability criteria for positive and negative controls; (c) optimize dose-spacing and the number of analyzable doses when there is evidence of toxicity; (d) use a combination of three criteria to evaluate results: a dose-related increase in revertants, a clear increase in revertants in at least one dose relative to the concurrent negative control, and at least one dose that produced an increase in revertants above control limits established by the laboratory from historical negative controls; and (e) establish experimental designs to resolve unclear results. The in silico sub-group summarized in silico utility as a tool in genotoxicity assessment but made no specific recommendations for TG471. Thus, the workgroup identified issues that could be addressed if TG471 is revised. The companion papers (a) provide evidence-based approaches, (b) recommend priorities, and (c) give examples of clearly defined terms to support revision of TG471.
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http://dx.doi.org/10.1016/j.mrgentox.2020.503137DOI Listing
January 2020

Top-Down and Bottom-Up Proteomics of Circulating S100A8/S100A9 in Plasma of Septic Shock Patients.

J Proteome Res 2020 02 22;19(2):914-925. Epub 2020 Jan 22.

Service de Pharmacologie et Immunoanalyse (SPI), CEA, INRA, Université Paris-Saclay , Gif-sur Yvette F-91191 , France.

Well-characterized prognostic biomarkers and reliable quantitative methods are key in sepsis management. Among damage-associated molecular patterns, S100A8/S100A9 complexes are reported to be markers for injured cells and to improve the prediction of death in septic shock patients. In view of the structural diversity observed for the intracellular forms, insight into circulating complexes and proteoforms is required to establish prognostic biomarkers. Here, we developed top-down and bottom-up proteomics to characterize the association of S100A8 and S100A9 in complexes and major circulating proteoforms. An antibody-free method was developed for absolute quantification of S100A8/S100A9 in a cohort of 49 patients to evaluate the prognostic value on the first day after admission for septic shock. The predominant circulating forms identified by top-down proteomics were S100A8, mono-oxidized S100A8, truncated acetylated S100A9, and S-nitrosylated S100A9. S100A8, truncated acetylated S100A9, and mono-oxidized S100A8 discriminated between survivors and nonsurvivors, along with total S100A8/S100A9 measured by the antibody-free bottom-up method. Overall, new insights into circulating S100A8/S100A9 and confirmation of its prognostic value in septic shock are crucial in qualification of this biomarker. Also, the simple antibody-free assay would support the harmonization of S100A8/S100A9 measurements.
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http://dx.doi.org/10.1021/acs.jproteome.9b00690DOI Listing
February 2020

Unsolved severe chronic rhinosinusitis elucidated by extensive genotyping.

Clin Case Rep 2019 Nov 27;7(11):2128-2134. Epub 2019 Sep 27.

INSERM U955 IMRB, Team 5 Créteil France.

Severe chronic rhinosinusitis in children should alert clinicians and extensive genotyping should be performed. We propose that thorough clinical and functional assessment in severe chronic rhinosinusitis is valuable to discover rare mutations which could be treated by CFTR correctors to postpone pulmonary infection.
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http://dx.doi.org/10.1002/ccr3.2443DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6878083PMC
November 2019

Four types of scrapie in goats differentiated from each other and bovine spongiform encephalopathy by biochemical methods.

Vet Res 2019 Nov 25;50(1):97. Epub 2019 Nov 25.

Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanita (ISS), 299-00161, Rome, Italy.

Scrapie in goats has been known since 1942, the archetype of prion diseases in which only prion protein (PrP) in misfolded state (PrP) acts as infectious agent with fatal consequence. Emergence of bovine spongiform encephalopathy (BSE) with its zoonotic behaviour and detection in goats enhanced fears that its source was located in small ruminants. However, in goats knowledge on prion strain typing is limited. A European-wide study is presented concerning the biochemical phenotypes of the protease resistant fraction of PrP (PrP) in over thirty brain isolates from transmissible spongiform encephalopathy (TSE) affected goats collected in seven countries. Three different scrapie forms were found: classical scrapie (CS), Nor98/atypical scrapie and one case of CH1641 scrapie. In addition, CS was found in two variants-CS-1 and CS-2 (mainly Italy)-which differed in proteolytic resistance of the PrP N-terminus. Suitable PrP markers for discriminating CH1641 from BSE (C-type) appeared to be glycoprofile pattern, presence of two triplets instead of one, and structural (in)stability of its core amino acid region. None of the samples exhibited BSE like features. BSE and these four scrapie types, of which CS-2 is new, can be recognized in goats with combinations of a set of nine biochemical parameters.
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http://dx.doi.org/10.1186/s13567-019-0718-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6878695PMC
November 2019

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

High plasma level of S100A8/S100A9 and S100A12 at admission indicates a higher risk of death in septic shock patients.

Sci Rep 2019 10 30;9(1):15660. Epub 2019 Oct 30.

Université Paris 7 Cité Sorbonne; UMR INSERM 1160, 110 Avenue de Verdun, Paris, 75010, France.

Biomarkers in sepsis for severity, prediction of outcome or reversibility of organ dysfunction are warranted. Measurements of plasma DAMP levels at admission can reflect the severity of cellular damage in septic shock, which might predict the prognosis and reduce the risk of overtreating patients with costly therapies. We measured plasma levels of two DAMPs, S100A8/S100A9 and S100A12 during the first 24 h of admission of septic shock patients. Forty-nine septic shock patients with a similar SOFA scores were selected from our sepsis database to compare a similar proportion of survivors and non-survivors. Plasma levels of S100A8/S100A9 and S100A12 were compared with healthy volunteers using in-house ELISA. Plasma levels of S100A8/S100A9 and S100A12 (5.71 [2.60-13.63] µg/mL and 0.48 [0.22-1.05] µg/mL) were higher in septic shock patients than in healthy volunteers (1.18 [0.74-1.93] µg/mL and 0.09 [0.02-0.39] µg/mL) (P < 0.0001 and P = 0.0030). Levels of S100A8/S100A9 and S100A12 in non-survivors at day 28 (11.70 [2.85-24.36] µg/mL and 0.62 [0.30-1.64] µg/mL) were significantly higher than in survivors (4.59 [2.16-7.47] µg/mL and 0.30 [0.20-0.49] µg/mL) (P = 0.0420 and P = 0.0248) and correlated well (Spearman r = 0.879, P < 0.0001). The high level of plasma calgranulins at admission in septic shock, were higher in non-survivors compared to survivors. These markers could indicate a higher risk of death when SOFA scores are similar and help the stratification of patients for improved care and therapy selection.
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http://dx.doi.org/10.1038/s41598-019-52184-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821805PMC
October 2019

Evaluation of In-Flow Magnetoresistive Chip Cell-Counter as a Diagnostic Tool.

Biosensors (Basel) 2019 Aug 31;9(3). Epub 2019 Aug 31.

SPEC, CEA, CNRS, Université Paris-Saclay, CEA Saclay, CEDEX, 91191 Gif-sur-Yvette, France.

Inexpensive simple medical devices allowing fast and reliable counting of whole cells are of interest for diagnosis and treatment monitoring. Magnetic-based labs on a chip are one of the possibilities currently studied to address this issue. Giant magnetoresistance (GMR) sensors offer both great sensitivity and device integrability with microfluidics and electronics. When used on a dynamic system, GMR-based biochips are able to detect magnetically labeled individual cells. In this article, a rigorous evaluation of the main characteristics of this magnetic medical device (specificity, sensitivity, time of use and variability) are presented and compared to those of both an ELISA test and a conventional flow cytometer, using an eukaryotic malignant cell line model in physiological conditions (NS1 murine cells in phosphate buffer saline). We describe a proof of specificity of a GMR sensor detection of magnetically labeled cells. The limit of detection of the actual system was shown to be similar to the ELISA one and 10 times higher than the cytometer one.
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http://dx.doi.org/10.3390/bios9030105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6784370PMC
August 2019

Quantification of low abundance Yersinia pestis markers in dried blood spots by immuno-capture and quantitative high-resolution targeted mass spectrometry.

Eur J Mass Spectrom (Chichester) 2019 Jun;25(3):268-277

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

Plague, caused by the bacterium Yersinia pestis, is still present in several countries worldwide. Besides, Y. pestis has been designated as Tier 1 agent, the highest rank of bioterrorism agents. In this context, reliable diagnostic methods are of great importance. Here, we have developed an original workflow based upon dried blood spot for simplified sampling of clinical specimens, and specific immuno-mass spectrometry monitoring of Y. pestis biomarkers. Targeted proteins were selectively enriched from dried blood spot extracts by multiplex immunocapture using antibody-coated magnetic beads. After accelerated on-beads digestion, proteotypic peptides were monitored by multiplex LC-MS/MS through the parallel reaction monitoring mode. The DBS-IC-MS assay was designed to quantify both F1 and LcrV antigens, although 10-fold lower sensitivity was observed with LcrV. The assay was successfully validated for F1 with a lower limit of quantification at 5 ng·mL in spiked blood, corresponding to only 0.1 ng on spots. In vivo quantification of F1 in blood and organ samples was demonstrated in the mouse model of pneumonic plague. The new assay could help to simplify the laboratory confirmation of positive point of care F1 dipstick.
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http://dx.doi.org/10.1177/1469066718795978DOI Listing
June 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

Fab is the most efficient format to express functional antibodies by yeast surface display.

MAbs 2018 07 24;10(5):720-729. Epub 2018 May 24.

a Service d'Ingénierie Moléculaire des Protéines (SIMOPRO), CEA, Université Paris-Saclay , Gif/Yvette , France.

Multiple formats are available for engineering of monoclonal antibodies (mAbs) by yeast surface display, but they do not all lead to efficient expression of functional molecules. We therefore expressed four anti-tumor necrosis factor and two anti-IpaD mAbs as single-chain variable fragment (scFv), antigen-binding fragment (Fab) or single-chain Fabs and compared their expression levels and antigen-binding efficiency. Although the scFv and scFab formats are widely used in the literature, 2 of 6 antibodies were either not or weakly expressed. In contrast, all 6 antibodies expressed as Fab revealed strong binding and high affinity, comparable to that of the soluble form. We also demonstrated that the variations in expression did not affect Fab functionality and were due to variations in light chain display and not to misfolded dimers. Our results suggest that Fab is the most versatile format for the engineering of mAbs.
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http://dx.doi.org/10.1080/19420862.2018.1468952DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6150635PMC
July 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

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