WO2022189555A1 - Méthode de détection d'agents biologiques infectieux par spectrométrie de masse - Google Patents

Méthode de détection d'agents biologiques infectieux par spectrométrie de masse Download PDF

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WO2022189555A1
WO2022189555A1 PCT/EP2022/056150 EP2022056150W WO2022189555A1 WO 2022189555 A1 WO2022189555 A1 WO 2022189555A1 EP 2022056150 W EP2022056150 W EP 2022056150W WO 2022189555 A1 WO2022189555 A1 WO 2022189555A1
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virus
protein
biological agent
infectious biological
targeted
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PCT/EP2022/056150
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English (en)
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Nathalie GILLARD
Patrice Filee
Maxime GAVAGE
François COTE
Anne-Catherine HUET
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Cer Groupe
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/18—Togaviridae; Flaviviridae
    • G01N2333/181—Alphaviruses or Group A arboviruses, e.g. sindbis, VEE, EEE, WEE or semliki forest virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/18—Togaviridae; Flaviviridae
    • G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
    • G01N2333/185—Flaviviruses or Group B arboviruses, e.g. yellow fever virus, japanese encephalitis, tick-borne encephalitis, dengue
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

  • the present invention is related to an improved detection method of an infectious biological agent, preferably a virus possibly present in a biological sample by mass spectrometry or by liquid chromatography followed by mass spectrometry.
  • the present invention is further related to the tools, such as the specific capture reagents and the infectious biological agent lysis solution, used for performing this method.
  • Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus that causes coronavirus disease 2019 (COVID-19), the respiratory illness responsible for the COVID-19 pandemic.
  • SARS-CoV-2 belongs to the broad family of viruses known as coronaviruses. It is a positive-sense single-stranded RNA virus, with a single linear RNA segment.
  • SARS-CoV-2 has four structural proteins, known as the S (spike), E (envelope), M (membrane), and N (nucleocapsid) proteins.
  • the N protein holds the RNA genome, and the S, E, and M proteins together create the viral envelope.
  • the spike protein is the protein responsible for allowing the virus to attach to (S1 subunit) and fuse (S2 subunit) with the membrane of a host cell.
  • Human ACE2 (angiotensin l-converting enzyme-2) is a type I surface transmembrane protein having a dipeptidyl carboxydipeptidase activity. This membrane protease is expressed in arteries, heart, kidneys, and epithelia of the lung and small intestine. Human ACE2 also plays a pivotal role in the human pathogenesis of SARS-CoV-2 by acting as the receptor for the spike (S) protein. The blockade of the ACE2 receptor and delivery of an excessive soluble form of ACE2 are among the investigated strategies to treat COVID-19.
  • SARS-CoV-2 There are many thousands of variants of SARS-CoV-2, which can be grouped into the much larger clades. Several notable variants of SARS-CoV-2, emerged in late 2020 and 2021 including:
  • the Alpha Variant (B.1 .1 .7) with the earliest documented sample origination from the United Kingdom; among the variants several mutations there is one in the receptor-binding domain of the spike protein that changes the asparagine at position 501 to tyrosine (N501 Y);
  • Beta Variant (B.1.351 ) with the earliest documented sample origination from South Africa, which has the same N501Y mutation, but also K417N and E484K mutations in the receptor-binding domain of the spike protein
  • Omicron Variant B.1 .1.529
  • EPE 3 amino acid insertion
  • Targeted analysis of proteins by mass spectrometry is classically based on tryptic proteolysis with subsequent characterization of the generated peptide products by liquid chromatography coupled to tandem mass spectrometry ((U)HPLC-MS/MS).
  • Copan Universal Transport Medium (UTM-RT), a conservation medium used for COVID-19 diagnostic nasopharyngeal swabs, consists, for instance, in a salt solution supplemented with proteins: bovine serum albumin and gelatine.
  • non-targeted proteins interfere with downstream sample preparation and LC-MS/MS analysis and thus with COVID-19 diagnosis.
  • the presence of abundant proteins can interfere with viral proteins proteolysis yield.
  • the presence of these peptides generated from these non-targeted proteins can induce ion suppression effect (competition for ionisation efficiency in the ionisation source, between the analyte(s) of interest and other endogenous or exogenous species) and affect viral peptides detection.
  • a time-saving analytical method is also a major concern in epidemiological survey. This requires to transpose the method on an automated and a high-throughput platform and to simplify the test protocol by bypassing some steps such as buffer substitution and elution.
  • the present invention aims to provide a detection method and tools for performing this method, that do not present the drawbacks of the methods of the state of the art.
  • the present invention aims to provide a method and tools for preforming this method, which reduce or avoid possible detection problems, such as background detection noise due to interferences generated by the presence of numerous unspecific proteins or peptides, such as albumin, possibly present in the composition submitted to LC-MS/MS detection.
  • the aim of the present invention is to obtain a simplified method and tools for performing this method that will reduce or avoid possible false positive or false negative detection of the tested infectious biological agents, especially when several infectious biological agents possibly present simultaneously in the same biological sample to be tested.
  • a further aim of the invention is to obtain such method and tools for performing this method that could be applied upon different types of samples, especially upon nasopharyngial fluid, saliva, urine, plasma or whole blood, possibly containing the target infectious biological agent(s).
  • the present invention is related to an infectious biological agent, preferably a virus, detection method, comprising the steps of:
  • a biological sample comprising or not an infectious biological agent, preferably a virus
  • a lysis buffer preferably comprising a sufficient amount of n-Octyl ⁇ -D- glucoside, able to inactivate the infectious biological agent and disassemble the one or more targeted infectious biological agent protein(s) from the infectious biological agent, but keeping proteins antigenicity;
  • the released targeted infectious biological agent protein(s) by means of protein-protein interactions between the released targeted infectious biological agent protein(s) and the one or more capture reagent(s), preferably by one or more immuno/receptor reagent(s), that can be selected from the group consisting of virus receptors, antibodies or hypervariable portions thereof, preferably monoclonal antibodies and possibly different monoclonal antibodies (more preferably the antibodies produced by the cell lines LMBP 14246CB or LMBP 14247CB) , nanobodies, alphabodies, microbodies, affytins, fymomers, affilines, affimers, or a mixture of two or more thereof, wherein antigenicity and conformational properties of the targeted infectious biological agent protein(s) are maintained;
  • the infectious biological agent is preferably a virus, more preferably, a corona virus, such as the SARS-CoV- 2 and the targeted virus proteins are the spike protein (S protein) and/or the nucleoprotein (N protein) of SARS-CoV-2, a respiratory virus infecting humans, preferably selected from the group consisting of an adenovirus, an influenza virus, a rhinovirus, a parainfluenza or a Respiratory Syncytial virus ,
  • a corona virus such as the SARS-CoV- 2 and the targeted virus proteins are the spike protein (S protein) and/or the nucleoprotein (N protein) of SARS-CoV-2
  • a respiratory virus infecting humans preferably selected from the group consisting of an adenovirus, an influenza virus, a rhinovirus, a parainfluenza or a Respiratory Syncytial virus ,
  • a virus infecting animals preferably selected from the group consisting of an Avian Influenza, the African horse sickness virus or the African swine fever (ASF) virus, or,
  • a virus infecting humans preferably the Zika virus, the dengue virus or the chikungunya virus.
  • a multiplexed detection of different infectious biological agents preferably different viruses, is obtained from the same biological sample.
  • the sufficient amount of n-Octyl ⁇ -D- glucoside is present in the lysis buffer at a concentration comprised between 0.01% and 5% by weight, preferably between 0,1% and 2% by weight, based on the total weight of the lysis buffer and the invention is also related to a virus lysis buffer comprising n-Octyl ⁇ -D-glucoside, preferably at a concentration comprised between 0.1% and 5% by weight, more preferably between 0.1% and 2% by weight, based on the total weight of the lysis buffer.
  • Another aspect of the invention is related to (hybridoma) cells having the deposit number LMBP14246CB and LMBP14247CB and to monoclonal antibodies directed against viral SARS-CoV-2 ( linear) epitopes and produced by these (hybridoma) cells.
  • a last aspect of the invention is related to beads having attached thereto one or more capture reagent(s) directed against SARS-CoV-2 virus protein(s), preferably beads having attached thereto one or more capture reagent(s) directed against an infectious biological agent, preferably a virus; more preferably a virus selected from the group consisting of an adenovirus, an influenza virus, a rhinovirus, a parainfluenza virus, the Respiratory Syncytial virus, an avian virus, an Influenza virus, the African horse sickness virus or the African swine fever (ASF) virus, the zika virus, the dengue virus or the chikungunya virus, wherein the capture reagent is selected from the group consisting of virus receptors, (preferably monoclonal) antibodies (more preferably the monoclonal antibodies produced by the cells having the deposit numbers LMBP 14246CB and/or LMBP 14247CB) or hypervariable fragments thereof, nanobodies, alphabodies, microbodies,
  • the above described tools for performing the method of the invention i.e. the lysis buffer, the capture reagents , specifically the antibodies (more preferably the antibodies produced by the cells having the deposit number LMBP 14246CB and/or LMBP 14247CB) and the beads above described are included together in a kit, with instructions to perform the method steps of the invention.
  • the figures 1 to 3 represent the detection LC-MS/MS signal results obtained from examples 1 to 3.
  • This methodology could comprise the use of the proven power of a detergent-based buffer for extraction of a target infectious biological agent (viral) protein(s) or fragments of protein(s) in combination with, and compatible with, rapid purification, such as immuno/receptor-purification, at protein level, on-beads protein and/or protein fragments, proteolytic digestion and final release of targeted peptides thereof.
  • a target infectious biological agent viral
  • rapid purification such as immuno/receptor-purification
  • the inventors have found that one or more, in particular a combination, of the problems of the state of the art can be solved by using the power of a detergent-based buffer for an infectious biological agent, preferably virus protein(s) or virus protein(s) fragment(s) extraction in combination with, and compatible with, rapid purification, such as immuno/receptor-purification, at protein level, on-beads protein proteolytic digestion and final release of targeted infectious biological agent (virus) peptides.
  • an infectious biological agent preferably virus protein(s) or virus protein(s) fragment(s) extraction in combination with, and compatible with, rapid purification, such as immuno/receptor-purification, at protein level, on-beads protein proteolytic digestion and final release of targeted infectious biological agent (virus) peptides.
  • the present invention concerns the method and the tools as described in the enclosed claims, in particular an infectious biological agent (virus) detection method, comprising the steps of:
  • a lysis buffer able to inactivate the infectious biological agent (virus) and disassemble the one or more targeted infectious biological agent (virus) protein(s) from the infectious biological agent, preferably the virus;
  • the lysis buffer preferably comprising a sufficient amount of n-Octyl ⁇ -D-glucoside, to release targeted infectious biological agent (virus) protein(s) from the biological sample, wherein the released targeted infectious biological agent (virus) protein(s) is or are soluble in the buffer and wherein the released targeted proteins keep advantageously their antigenicity ;
  • any (infectious agent, preferably any virus or other) interfering compound(s) such as co-extracted human or animal compounds or added sample conservation or transport molecule(s) (i.e. Bovine Serum Albumin (BSA)) from the sample and not bound to the beads;
  • interfering compound(s) such as co-extracted human or animal compounds or added sample conservation or transport molecule(s) (i.e. Bovine Serum Albumin (BSA)) from the sample and not bound to the beads;
  • BSA Bovine Serum Albumin
  • the method according to the invention is related to the preparation and the analysis of an infectious biological agent (virus(es)) contaminated biological sample.
  • the method comprises deactivation/disassembling steps of the infectious biological agent, preferably viral particles using a (preferably mass spectroscopy (MS)-compatible) buffer in such a way that the antigenicity of the infectious biological agent (virus) protein(s) or infectious biological agent (virus) protein(s) fragment(s) to be detected (the so-called targeted infectious biological agent (virus) protein(s) or targeted infectious biological agent(virus) protein(s) fragment(s)) is preserved.
  • the present method is further based on a selective enrichment of targeted protein(s) or fragments of this targeted protein or these targeted proteins from a large sample volume by a specific mixture of capture reagents specifically directed against these proteins, for example monoclonal antibodies, antigen-purified polyclonal antibodies or infectious biological agent (virus) receptor(s), attached or fixed to beads.
  • these beads are magnetic beads.
  • the method may further comprise the step of eliminating any infectious biological agent (virus) or another interfering compound(s) from the sample and being not specifically bound to the beads.
  • infectious biological agent virus
  • other interfering compound(s) are proteins from the sample transport medium or inorganic and organic elements possibly present into the sample.
  • the present method is further based on-beads proteolysis of the targeted protein(s) or fragments of this (these) protein(s), resulting in targeted infectious biological agent (virus) peptides.
  • the method is further based on the detection of the obtained targeted infectious biological agent(virus) peptides by means of mass spectroscopy, preferably by means of a liquid chromatography step followed(coupled) by mass spectrometry.
  • the method allows one or more of:
  • infectious biological agent (virus) variants This can be a powerful asset in epidemio-surveillance; and simultaneous detection of different infectious biological agent (virus(es) and/or infectious biological agent (virus) proteins and their variants, in the same, single, experiment or measurement; this aspect of the invention can be advantageous, when different infectious agents lead to similar disease symptoms or even can be associated in some co-infection cases (Recent published data related that influenza A virus (IAV) elevated its expression and that co-infection with IAV enhances SARS-CoV-2 infectivity).
  • IAV influenza A virus
  • the tested samples can be obtained from any mammal subject possibly suffering from a disease or syndrome, but preferably from a human patient and is preferably selected from the group consisting of saliva, urine, plasma, whole blood or a naso- pharyngial sample.
  • the infectious biological agent to be detected could be any microorganism involved in animals diseases, especially in human diseases and which is preferably selected from the group consisting of bacteria, parasites (such as plasmodium or a tryponozoma species) and/or viruses.
  • the virus can be a respiratory virus infecting humans or animals and can be selected from the group consisting of a, an adenovirus, an influenza virus, a rhinovirus, a parainfluenza or a Respiratory Syncytial Virus, can be selected from the group consisting of Avian Influenza, the African horse sickness or the African swine fever (ASF) or can be selected from the group consisting the zika virus, the dengue virus or the chikungunya virus.
  • a respiratory virus infecting humans or animals can be selected from the group consisting of a, an adenovirus, an influenza virus, a rhinovirus, a parainfluenza or a Respiratory Syncytial Virus
  • ASF African swine fever
  • the present method relates to the detection of the SARS-CoV-2 virus from (swab) samples and to the complexity of its epidemiological context (antigenic drift and co-infection).
  • a purification step is advantageously applied during sample preparation to eliminate any interference from the biological sample(s) prior to MS analysis.
  • Specific purification steps are preferred, such as immuno/receptor-capture.
  • the specificity of the antibodies allows to retain proteins bearing the targeted epitopes (so-called targeted virus proteins of the invention), while interferences are eliminated from the biological sample(s).
  • targeted virus proteins of the invention proteins bearing the targeted epitopes
  • interferences are eliminated from the biological sample(s).
  • the emergency of variants can lead to some antigenic drifts, which can interfere with the efficiency of immuno/receptor-capture and thus false negative sample(s).
  • the resolution of this problem requires specific selection of capture reagents, in particular specific mixtures of monoclonal antibodies or antigen-purified polyclonal antibodies or infectious biological agent (viral) receptor(s).
  • the antigenic drift observed in a virus or another infectious biological agent is considered as a way to escape the immune system or to strengthen the virulence by increasing the affinity to the infectious biological agent (viral) mammal receptor.
  • the fact that variants conserve their affinity for their infectious biological agent (viral) mammal receptor allows to secure the purification step, allowing to conduct a mass spectrometry detection of infectious biological agent (viral) protein(s).
  • the invention provides a solution to bottlenecks in mass spectrometry in a fully integrated approach.
  • the invention provides means for the use of mass spectrometry to bring analytical support in epidemiological survey, of an infectious biological agent, such as SARS- CoV-2 pandemic.
  • infectious biological agent virus(es)
  • infectious biological agent virus
  • virus protein(s) or protein(s) fragments disassembly (from viral or other infectious biological agents particles)
  • one or more specific capture reagent(s) preferably monoclonal antibodies, a mixture of monoclonal antibodies, polyclonal antibodies, Ace2 or other mammal receptor(s) or any engineered (hypervariable) fragment thereof, to capture specific, targeted infectious biological agent (virus(es) protein(s) or fragments thereof;
  • the ability to isolate highly purified infectious biological agent, especially the SARS-CoV-2 proteins for such uses can be achieved through the development of new tools such as new highly specific mixture of monoclonal antibodies, antigen-purified polyclonal antibodies or human Ace2 receptor recombinant forms, which are capable of interacting with infectious biological agent, especially the SARS-CoV-2 protein(s) or SARS-CoV-2 protein fragments.
  • the present invention provides a detection or analysis method of infectious biological agent (virus) protein(s) or protein(s) fragments(s)by means of (U)HPLC- MS/MS, coupled to the development of a deactivation/disassembly buffer, the production of specific capture reagent(s), such as monoclonal/polyclonal antibodies and infectious biological agent (viral) mammal receptors, and the use of a solid phase for automation and simplification of the overall process.
  • infectious biological agent virus
  • the applied method was developed and proven on the SARS-CoV-2 case and extended to the detection of others viral or non-viral infectious biological agents, such as the influenza virus (IAV H1 N1 ) and to the multiplexed detection of the SARS-CoV-2 and of the IAV H1 N1 ).
  • Various recombinant proteins were first biologically produced in bacteria (nucleoprotein, N protein) and in mammal cells (spike proteins, S protein) and these antigens were used for immunization of mouse and rabbit.
  • Monoclonal antibodies from mouse and antigen-purified polyclonal antibodies from rabbit having the capability of specifically identifying and binding to SARS-CoV-2 proteins (nucleoprotein or spike proteins) were developed and characterized.
  • Recombinant Human Ace2 Fc fusion retaining SARS-CoV-2 spike protein binding was produced in mammal cells and its spike protein binding activity was validated by ELISA using a recombinant spike protein.
  • the invention includes three major parts:
  • This method comprises:
  • This step includes the stock (magnetic) beads washing step, an incubation step with antibodies or Fc fusion, and an elimination step of unbound antibodies/Fc fusion, all steps advantageously being preferably performed in a 50 mM PBS buffer;
  • the beads are preferably magnetic beads;
  • infectious biological agent virus and other interfering
  • infectious biological agent such as co-extracted animal or human compounds or added sample carrier or conservation molecules (BSA) not bound to the beads
  • BSA sample carrier or conservation molecules
  • a proteolytic digestion step preferably by addition of trypsin, of the released, targeted infectious biological agent (virus) protein(s) or protein(s) fragment(s) bound to the, preferably magnetic, beads, thereby obtaining released targeted infectious biological agent (virus) peptide(s);
  • a lysis buffer preferably a lysis buffer comprising n- Octyl ⁇ -D-glucoside, more preferably a lysis buffer comprising n-Octyl ⁇ -D-glucoside at a concentration comprised between 0.01% and 5% by weight, more preferably at a concentration comprised between 0,1% and 2% by weight, based on the total weight of the lysis buffer, is adequate for performing the deactivation/disassembling step while not disturbing further analysis by means of (U)HPLC-MS/MS and immune-purification .
  • magnetic beads offer a series of advantages for the optimization of sample preparation steps, including immuno/receptor-capture, interference elimination and a proteolytic digestion, releasing targeted infectious biological agent (virus) peptide(s) in the biological sample and allowing a detection by UHPLC-MS/MS analysis of these released, targeted infectious biological agent (virus) peptide(s).
  • the invention method relies on a sample preparation comprising a deactivation/disassembling step of viral particles and a selective enrichment of targeted protein using specific capture reagents fixed to magnetic beads. Relevant examples of the application of this strategy on SARS-CoV-2 and Influenza positive samples are exposed here below.
  • a deposit of Biological material has been done according to the Budapest T reaty with the following deposit numbers LMBP 14246CB and LMBP 14247CB, both deposits been filed on February 08, 2022 at the Belgian Biological material collection BCCM/GeneCorner Plasmid collection Ghent University, Department of Biomedical Molecular Biology Technologiepark-Zwijnaarde 71 B-9052 Gent.
  • mice and rabbits Monoclonal antibodies from mouse and antigen-purified polyclonal antibodies from rabbit having the capability of specifically identifying and binding to SARS-CoV-2 proteins (N protein or S protein) were developed and characterized.
  • Recombinant Human Ace2 angiotensin l-converting enzyme-2
  • Fc fusion retaining SARS-CoV-2 S protein binding was produced in mammal cells and its S protein binding activity was validated by ELISA using a recombinant S protein.
  • Magnetic beads covalently coupled to protein G (DynabeadsTM Protein G for Immunoprecipitation) and n-Octyl ⁇ -D-glucoside detergent was also purchased from ThermoFisher scientific. MagneSphere® magnetic separation stand from Promega was used for magnetic beads isolation. Trypsin Gold from Promega was used for proteolysis.
  • Nasopharyngeal swabs collected from COVID-19 patients confirmed to be positive for the SARS-CoV-2 virus by RT-PCR testing were included. Swabs were conserved in various viral conservation media including Copan UTM-RT medium, Copan’s Liquid Amies and IMPROVIRALTM Viral Preservative Medium. Samples were conserved at -20°C until further processing. Influenza H1 N1 virus was obtained from an in-house virus culture with Madin- Darby canine kidney cells.
  • a volume of 50 pi of resuspended magnetic beads was considered for the immune- purification of each sample.
  • PBS phosphate saline buffer
  • antibodies or other immuno/receptor capture reagent
  • An amount of 5 pg of antibody was considered for 50 mI of resuspended magnetic beads.
  • Beads and antibody binding was obtained with 20 min incubation at 24°C under 1000 RPM orbital agitation. Beads were then washed with PBS to eliminate unbound antibodies.
  • Nasopharyngeal swab samples were transferred to micro-centrifuge tubes and viral inactivation and virus lysis was done by addition of 10 % n-Octyl ⁇ -D-glucoside prepared in 50 mM PBS (2% n-Octyl ⁇ -D-glucoside final concentration) and 20 min incubation at 24°C under 1000 RPM orbital agitation.
  • the inactivated samples were transferred to a micro centrifuge tube containing magnetic beads bound to antibodies (or other immuno/receptor capture reagent).
  • Protein of interest were immune-purified with a 60 min incubation at 24°C under 1000 RPM orbital agitation. Beads were then washed 50 mM triethylammonium bicarbonate (TEAB) solution.
  • TEAB triethylammonium bicarbonate
  • Immuno-purified proteins were then digested “on beads” with the addition of 60 pi of digestion solution composed 50 mM TEAB containing 5% acetonitrile and trypsin. The samples were incubated during 60 min at 37°C under 1000 RPM orbital agitation.
  • Liquid chromatographic parameters are the following : Chromatography column: ACQUITY UPLC BEH C18, 130A, 1.7 pm, 2.1 mm X 100 mm, UHPLC mobile phase A: water + 0.1 % formic acid, UHPLC mobile phase B: acetonitrile + 0.1 % formic acid, Flow rate: 0.2 ml/min, Oven temperature: 50°C, Injection volume: 10 pi.
  • the parameters are the following: Positive ESI with desolvation temperature: 500 °C, Source temperature: 150 °C, Cone gas: 150 L/h and Desolvatation gas: 700 L/h.
  • Example 1 Immuno-purification of SARS-CoV-2 N protein with monoclonal antibodies
  • This first example compares the LC-MS/MS signal obtained for the analysis of a SARS-CoV-2 positive clinical sample (conserved in UTM-RT transport medium) separately treated with two different sample preparation strategies.
  • the first strategy corresponds to protein precipitation with cold acetone followed by protein pellet resuspension and enzymatic digestion with trypsin.
  • the second strategy according to the invention is based on virus lysis, SARS-CoV- 2 N-protein immune-purification with monoclonal antibodies (preferably the antibodies produces by the cell lines LMBP 14246CB or LMBP 14247CB of the invention) linked to magnetic beads followed by “on-beads” protein enzymatic digestion with trypsin.
  • the LC- MS/MS signal obtained for 3 tryptic peptides of SARS-CoV-2 N protein are compared in the figure 1.
  • Example 2 Immuno-purification of SARS-CoV-2 S protein with recombinant human Ace2 Fc fusion
  • This second example compares the LC-MS/MS signal obtained for the analysis of a SARS-CoV-2 positive clinical sample (conserved in UTM-RT transport medium) separately treated with two different sample preparation strategies.
  • the first strategy corresponds to protein precipitation with cold acetone followed by protein pellet resuspension and enzymatic digestion with trypsin.
  • the second applied strategy is based on virus lysis, SARS-CoV-2 S protein immune-purification with recombinant human Ace2 Fc fusion linked to magnetic beads followed by “on-beads” protein enzymatic digestion with trypsin.
  • the LC-MS/MS signal obtained for 3 tryptic peptides of SARS-CoV-2 S protein are compared in the figure 2.
  • Example 3 Simultaneous immune-purification of N protein from SARS-CoV-2 and N protein from Influenza H1N1
  • This third example corresponds to the LC-MS/MS analysis of a SARS-CoV-2 positive clinical sample manually contaminated with Influenza H1N1 virus from in-house culture.
  • virus lysis the N protein of SARS-CoV-2 and the N protein from Influenza H1 N1 were simultaneously immune-purified with a mix of monoclonal antibodies (specific to SARS- CoV-2 N protein and Influenza H1N1 N protein) linked to magnetic beads followed by “on- beads” protein enzymatic digestion with trypsin.
  • the invention method advantageously improves detection sensitivity and specificity, but can also be simultaneously applied to different targets thanks to the combination of multiple capture agents.

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Abstract

La présente invention concerne une méthode relative à un ou à plusieurs agents biologiques infectieux, de préférence à la détection de virus éventuellement présents dans un échantillon biologique par spectrométrie de masse ou par chromatographie liquide couplée à une spectrométrie de masse. La présente invention concerne en outre des outils, en particulier des réactifs de capture d'immunorécepteurs sélectifs, tels que des anticorps et un tampon de lyse, pour la réalisation de cette méthode.
PCT/EP2022/056150 2021-03-10 2022-03-10 Méthode de détection d'agents biologiques infectieux par spectrométrie de masse WO2022189555A1 (fr)

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