Classifications

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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
  • C12Q1/10—Enterobacteria
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/18—Testing for antimicrobial activity of a material
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
• • 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
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
  • G01N33/54386—Analytical elements
  • G01N33/54387—Immunochromatographic test strips
  • G01N33/54388—Immunochromatographic test strips based on lateral flow
• • 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
  • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
• • 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
  • G01N33/56911—Bacteria
  • G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
• • 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
  • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
  • C12Y305/02—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amides (3.5.2)
  • C12Y305/02006—Beta-lactamase (3.5.2.6)
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• This antibody can be used in kits and methods for detecting very quickly (in less than an hour), without using expensive equipment (a strip readable by the naked eye), the presence of bacteria producing enzymes of the type penicillinases, plasmid or hyper-produced AmpCs, ESBL or carbapenemases from colonies or in a sample.
• Description of the prior art Following the discovery of penicillin in 1928, its use only increased, making it possible to considerably reduce the mortality linked to infectious diseases.
• ESBL bacteria broad-spectrum ⁇ -lactamase bacteria
• ⁇ -lactamases cause the amide bond of the ⁇ -lactam ring to open, rendering the antibiotic agent ineffective and the bacteria resistant to its deleterious effect.
• the bacteria producing an ESBL are able to hydrolyze the ⁇ -lactam rings present in ⁇ -lactams such as penicillins, as well as the different classes of cephalosporins (C1G, C2G, C3G, C4G, C5G).
• antibiotics are used as a last resort, in order to limit the appearance of strains resistant to them.
• an antibiotic treatment based on cephalosporins is often prescribed, but it proves to be ineffective if the infecting bacteria produce an ESBL.
• it is necessary to adapt the antibiotic therapy to each type of infectious bacterium, according to its antibiogram, and this as soon as possible in order to have an optimal treatment and a high rate of therapeutic success (Maugat, Berger-Carbonne, and National Agency for Food, Environmental and Occupational Health Safety (ANSES), “Consumption of antibiotics and antibiotic resistance in France: an infection avoided is an antibiotic preserved!”).
• ESBL bacteria detection kit which is composed of a medium containing an antibiotic to kill Gram-positive bacteria, an antifungal compound, an antibiotic to kill Gram-negative bacteria not ESBL, and a colored indicator.
• the presence of ESBL bacteria is detected by incubating the sample to be tested on said support for 18-24 hours at 36-37°C and by detecting the change in color of the pH indicator, due to the acidification of the medium. related to the bacterial growth of ESBL bacteria.
• This colorimetric process is easy to use but the interpretation of the color change is subjective.
• this color change is induced by acidification of a medium buffered by bacterial growth.
• the hydrolysis of the substrate causes the appearance of a colored or fluorescent signal in the medium.
• the incubation time can be relatively long for enzymes with low enzymatic activity.
• some faint colorations may be difficult to interpret and a reading device is necessary for fluorogenic substrates.
• these substrates can be light sensitive, which poses a problem if the test is to be adapted in the field.
• WO 2013/72494 The method described in this application is aimed at the detection of bacteria producing broad-spectrum ⁇ -lactamases (broad-spectrum cephalosporin-hydrolyzing ⁇ -lactamase).
• This method comprises the following steps: a) carrying out cell lysis of the sample; b) reacting a fraction of the suspension obtained in step a) with a kit of reagents comprising: i) a broad-spectrum ⁇ -lactamase substrate chosen from the group consisting of cephalosporins, aztreonam and cephamycins, and ii) a pH indicator which will change color when the pH of the solution is between 6.4 and 8.4. This change in pH is induced by the hydrolysis of the cephalosporin, present in the medium, which causes a carboxylic acid function to appear.
• the color change in step b) indicates the presence of extended-spectrum ⁇ -lactamase producing bacteria in the sample.
• This colorimetric method is easy to use but, as for WO2008/114001, the interpretation of the color change is subjective. In addition, this color change is induced by acidification of a medium buffered by bacterial growth. Therefore, a relatively long incubation time (16h-24h) is necessary to demonstrate the presence of the bacteria sought. - WO2016/156605
• This electrochemical method makes it possible to determine the presence of ESBL bacteria thanks to their electrochemical properties, visible using an apparatus. This technique has a high cost of use (expensive equipment and qualified personnel).
• the labeled antibody is saturated with the hydrolyzed antibiotic present in the sample in large quantity, and will therefore not bind to the immobilized antibiotic of the strip: there is then no signal on the test area of the strip. Otherwise, ie, if the sample contains non-ESBL bacteria (“negative” result), then the antibody introduced into the sample remains free to bind to the hydrolyzed antibiotics immobilized on the strip, and the signal becomes strong on the test area.
• the presence of the active enzyme in the sample therefore results in a reduction of the signal.
• This antibody is then used in kits and immunochromatographic tests to obtain a result very quickly (in less than an hour), without using expensive equipment (a strip readable by the naked eye).
• the use of such an antibody makes it possible to link the presence of active enzymes / ESBL bacteria in a sample (“positive” result) to the appearance of a signal, and not the reverse as proposed by many documents. of the prior art. Indeed, thanks to the antibody of the invention (recognizing the antibiotic with the intact ⁇ -lactam ring), it is possible to measure the disappearance of the substrate (intact antibiotic which is introduced into the sample) in a reliable, specific and reproducible, by measuring the appearance of a signal (easier to observe than the decrease in a signal).
• the test of the invention is extremely reliable: a sensitivity of 100% and a specificity of 100% were obtained for the detection of cephalosporinase activity (enzymatic activity of hydrolysis of cefotaxime) in many bacteria tested , in 40 minutes (see examples below).
• the detection kits and methods of the present invention make it possible to: - Facilitate the detection of bacteria capable of hydrolyzing ⁇ -lactams (a colored signal is synonymous with positivity) - possibly, identify which type of enzymes is expressed by the bacteria detected, - Reduce the detection time of bacteria capable of hydrolysing ⁇ -lactams (it only takes 40 minutes to obtain a result), - Detect all bacteria producing ⁇ - lactamases whether known or unknown (they allow to conclude even in case of appearance of new mutations) - Use immunochromatography, which has many advantages in terms of speed, cost, simplicity, and sensitivity.
• the present invention relates to new means for detecting the hydrolysis of ⁇ -lactam, and therefore for revealing the presence of bacteria producing a ⁇ -lactamase in a sample.
• These new means are based on the use of an antibody specifically developed by the inventors, to recognize an antibiotic with an intact ⁇ -lactam ring (and not its hydrolysis product).
• This antibody can be advantageously used in the detection kits and detection methods described below.
• these kits contain a strip (which is also an aspect of the invention in itself), on which the antibody of the invention has been deposited and dried.
• monoclonal antibodies were produced and selected to specifically recognize the intact form of ⁇ -lactam type antibiotics (ie, comprising a ⁇ -lactam nucleus) (cf. examples 1 and 2).
• immunogens containing an intact ⁇ lactam ring
• selection assays have been designed and implemented. Examples 1 and 2 of the application, presented below, describe how these immunogens were designed, then produced, and how highly discriminating antibodies could then be selected.
• antibiotics which were used in these examples are cefotaxime, a third generation cephalosporin (example 1), and meropenem, a new generation antibiotic which is hydrolyzed by certain ESBL enzymes called “carbapenemases" (example 2).
• these monoclonal antibodies were obtained by carrying out the following steps: A) Coupling of an antibiotic containing an intact ⁇ -lactam ring, with a large immunogenic molecule (BSA or hemocyanin type), away from the ⁇ -lactam core.
• BSA immunogenic molecule
• This coupling was achieved by activating the antibiotic with acetyl chloride, then by grafting thiol functions onto the large molecule, then by bringing the two activated molecules (this coupling step can also be carried out by various conventional chemical means known to those skilled in the art).
• D) Production of hybridomas producing the selected antibodies from splenocytes of immunized mice which have been fused with murine myeloma cells).
• E Isolation of each hybridoma produced in a well and confirmation that the monoclonal antibodies produced recognize the antibiotic whose ⁇ -lactam ring is intact (cf. test of FIG. 1 and test 1 of FIG. 9).
• F Identification and selection of hybridomas producing antibodies which do not recognize at all the antibiotic whose ⁇ -lactam ring is hydrolyzed (negative antibodies in test 2 of FIG. 2 and FIG. 9).
• G Determination of the specificity of each selected antibody towards the antibiotic with an intact ⁇ -lactam ring (tests 3 and 4 of FIG. 2 and FIG. 9) with different concentrations of the intact and hydrolyzed antibiotic).
• the present invention therefore relates to a monoclonal antibody specifically recognizing an antibiotic molecule containing an intact ⁇ -lactam ring, this antibody not recognizing the same hydrolyzed antibiotic molecule, ie, when its ⁇ -lactam ring has been hydrolysed, for example by a ⁇ -lactamase.
• the antibody of the invention is capable of binding only to antibiotics whose ⁇ -lactam cycle is intact. It therefore makes it possible to discriminate between the two forms of the antibiotic, since it will be complexed when it is brought into contact with the intact form of the antibiotic, but will remain free in the presence of the hydrolyzed form of the latter.
• an “intact” is synonymous with “non-hydrolyzed”.
• an “intact” ⁇ -lactam ring is a ⁇ -lactam ring which is closed, because it has not undergone hydrolysis by a ⁇ -lactamase.
• an “intact” or “non-hydrolyzed” antibiotic will be called an antibiotic whose ⁇ -lactam cycle is closed and therefore functional (this cycle in fact provides the antimicrobial effect of the antibiotic).
• the term “hydrolyzed” will designate an antibiotic whose ⁇ -lactam ring is open, because it has been hydrolyzed by a ⁇ -lactamase, naturally (in a natural sample) or artificially (for example by an enzyme of synthesis).
• a “hydrolyzed” antibiotic is generally non-functional, ie, it has little or no antimicrobial effect.
• the term “monoclonal antibody” refers to an antibody derived from a homogeneous antibody population. More particularly, the individual antibodies of a population of monoclonal antibodies are identical. In other words, a monoclonal antibody consists of a homogeneous antibody population derived from the growth of a single cell clone (e.g.
• a hybridoma a eukaryotic host cell transfected with a DNA molecule encoding the antibody homogeneous, a prokaryotic host cell transfected with a DNA molecule coding for the antibody, etc.
• It is generally characterized by heavy chains and light chains.
• Monoclonal antibodies are highly specific and are directed against a single antigen.
• An "antigen" is a predetermined molecule to which an antibody can bind selectively at a region called an epitope.
• the target epitope includes the ⁇ -lactam cycle of an antibiotic.
• the monoclonal antibody of the invention has a very strong affinity towards the target intact antibiotic and a very weak affinity towards the antibiotic which has been hydrolyzed, for example by a ⁇ -lactamase .
• its dissociation constant K d for the target antibiotic is between approximately 10 nM and approximately 1 pM. More preferably, said K d is between about 10 pM and about 40 pM.
• K d refers to the dissociation constant of a given antibody-antigen complex.
• K d k off / k on with k off consisting of the “off rate” constant for the dissociation of the antibody from the antibody-antigen complex and kon being the level at which the antibody associates with the antigen (Chen Y et al., 1999, J. Mol. Biol., 293:865-881). It is also possible to measure the affinity of the antibody of the invention towards its target by measuring its affinity constant, K a , which corresponds to the inverse of K d .
• the affinity constant K a of the antibody of the invention for the target antibiotic is greater than approximately 10 9 M -1 , more preferentially greater than 1011 M -1 and even more preferably, greater than 10 12 M ⁇ 1 .
• Antibodies with low affinity for a target generally bind slowly to that target and tend to dissociate from it easily, while antibodies with high affinity for a target generally bind the target quickly, and tend to stay bound with it. Longer.
• a variety of methods for measuring binding affinity are known in the art (e.g., by equilibrium dialysis, or by fluorescence, or even with Biacore assays), any of which can be used for purposes of the present invention.
• the present invention also relates to fragments of monoclonal antibodies which are functional (ie, which specifically recognize an antibiotic molecule whose ⁇ -lactam ring is intact, but not when hydrolyzed).
• This fragment can be, for example, chosen from Fv, Fab, (Fab′) 2 , Fab′, scFv, scFv-Fc fragments and diabodies.
• the monoclonal antibody of the invention can be produced and isolated by conventional means using any known technique allowing the production of antibody molecules by cell lines in culture.
• Target antibiotic designates any antibiotic known to contain, in its chemical formula, a ⁇ -lactam ring. Otherwise called “ ⁇ -lactam antibiotic” or “ ⁇ -lactam antibiotic”, it can be chosen from penicillins, cephalosporins, monobactams, and carbapenems, which all contain a ⁇ -lactam nucleus in their molecular structure.
• the target antibiotic can in particular be a penicillin chosen from benzylpenicillin (penicillin G), phenoxymethylpenicillin (penicillin V), methicillin, dicloxacillin, fucloxacillin, amoxyicillin, ampicillin, piperacillin, ticarcillin, azlocillin, and carbenicillin.
• the target antibiotic can in particular be a cephalosporin chosen from cephalexin, cephalotin, cephazolin, cefaclor, cefuroxime, cefamandole, cefotetan, cefoxitin, ceftriaxone, cefixin, cefotaxime, ceftazidime, cefepime, cefpirome, ceftaroline, and ceftobiprole.
• the target antibiotic can in particular be a carbapenem chosen from thienamycin, imipenem, meropenem, ertapenem, biapenem, tebipenem and doripenem.
• the target antibiotic can in particular be a monobactam such as aztreonam.
• the target antibiotic can in particular be a cephamycin chosen from cefmetazole and latamoxef.
• the discriminating antibody of the invention is characterized in that it specifically recognizes an antibiotic molecule chosen from penicillins, cephalosporins, monobactams, carbapenems, and cephamycins, whose ⁇ - ring lactam is intact.
• the antibody of the invention is characterized in that it specifically recognizes an antibiotic molecule chosen from the group consisting of: benzylpenicillin (penicillin G), phenoxymethylpenicillin (penicillin V), methicillin , dicloxacillin, fucloxacillin, amoxyicillin, ampicillin, piperacillin, ticarcillin, azlocillin, carbenicillin, cephalexin, cephalotin, cephazolin, cefaclor, cefuroxime, cefamandole, cefotetan, cefoxitin, ceftriaxone, cefixin, cefotaxime, ceftazidime, cefepime, cefpirome, ceftaroline, ceftobirpole, thienamycin, imipenem, meropenem, ertapenem, biapenem, tebipenem, doripenem, l aztreonam, cefmetazole, and latam
• an antibiotic molecule
• the antibody of the invention is characterized in that it specifically recognizes an antibiotic molecule chosen from the group consisting of thienamycin, imipenem, meropenem, ertapenem, and doripenem. Conversely, said antibody does not recognize (or has a low affinity for) these antibiotic molecules when the ⁇ -lactam ring has been hydrolyzed, for example by the effect of a ⁇ -lactamase enzyme.
• ⁇ -lactamase designates, within the framework of the invention, the following enzymes: penicillinases (eg: TEM-1, SHV-1, etc.), cephalosporinases (eg: AmpC), ⁇ -lactamases broad spectrum (eg derivatives of TEM, SHV, CTX-M, etc.), as well as carbapenemases.
• penicillinases eg: TEM-1, SHV-1, etc.
• cephalosporinases eg: AmpC
• ⁇ -lactamases broad spectrum eg derivatives of TEM, SHV, CTX-M, etc.
• ESBLs Broad-spectrum ⁇ -lactamases
• ESBLs are a large, very heterogeneous family of bacterial enzymes discovered in the 1980s in France, then in Germany. They are induced either by plasmids (frequent) or by mutation of the natural genome in Klebsiella spp, coding for a ⁇ -lactamase SHV. Both mechanisms provide affected bacteria with the ability to hydrolyze a wide variety of penicillins and cephalosporins.
• the majority of ESBLs are the result of genetic mutations of natural ⁇ -lactamases, in particular TEM-1, TEM-2 and SHV-1. They are very active against penicillins and moderately active against first-generation cephalosporins.
• the antibody of the invention is preferably labeled in a detectable manner, for example in that it is coupled to a fluorochrome, a radioactive ion, a contrast agent, a metal ion, to a chromophore, an enzyme or any other marker visible to the naked eye or detectable by imaging.
• the invention also relates to the use of a discriminating antibody according to the invention (as described above) in tests for rapidly detecting and very reliably, in any sample, the enzymatic activity of a ⁇ -lactamase enzyme and therefore the presence of bacteria having ⁇ -lactamase activity (otherwise called "bacteria resistant to ⁇ -lactam antibiotics"). More specifically, the antibody of the invention can be used to quickly and reliably detect the presence of bacteria having broad-spectrum ⁇ -lactamase (ESBL) activity in a sample.
• ESBL broad-spectrum ⁇ -lactamase
• the present invention therefore covers the use of the discriminating antibody of the invention, as described above, for detecting the presence of a functional ⁇ -lactamase enzyme, preferably with a broad spectrum, in a sample.
• sample means any biological or environmental fraction, solid or liquid, capable of containing a ⁇ -lactamase enzyme as described above, or bacteria expressing such an enzyme, said enzyme being likely to be functional. It may be, for example, an environmental sample, or even a human, animal or plant biological fluid.
• the sample used in the methods of the invention contains bacteria.
• biological fluid refers to any sample which has been obtained from an individual human, animal, or plant and which is fluid or viscous. It may be, for example, a biological fluid produced by a human or an animal, such as urine, cerebrospinal fluid, pleural fluid, synovial fluid, peritoneal fluid, amniotic fluid, stomach, blood, serum, plasma, lymph fluid, interstitial fluid, saliva, physiological secretions, tears, mucus, sweat, milk, semen, seminal fluid, vaginal secretions, a fluid from ulcers and other surface rashes, blisters, faeces or abscesses.
• any said sample contains bacteria.
• the antibody of the invention can be used in any immunological test making it possible to unequivocally detect the reduction in the quantity of intact antibiotic when it is brought into contact with the sample. This decrease being due to the action of the ⁇ -lactamase enzymes present in the sample, the antibody of the invention will thus be able to reveal their presence.
• the antibody of the invention is preferably used in a biological test by competition, ie, in a test in which the presence of a ⁇ -lactamase will be demonstrated by the appearance (and not the reduction) of a detectable signal.
• the antibody of the invention will be either labeled or immobilized, and will be brought into contact with the cycle-intact antibiotic that it specifically recognizes, which will itself be either labeled or immobilized.
• said test involves a labeled (but mobile) antibody of the invention, and an immobilized intact antibiotic.
• said assay involves immobilized antibody of the invention, and unlabeled and labeled (but mobile) intact antibiotic.
• said test is carried out entirely in the liquid phase, and involves using antibodies of the invention and associated antibiotics which will have been labeled with different detectable markers. If different fluorophores are used, it will be possible to measure the co-localization of antibodies / antibiotics by the FRET technique.
• ⁇ -lactamase activity it is for example possible to detect the presence of ⁇ -lactamase activity by comparing the signal obtained under the following conditions: - When the sample is incubated in the presence of the intact antibiotic before being brought into contact with the antibodies of the invention immobilized in well or on a support, in the presence of an intact antibiotic labeled (with biotin or with another label). - When the antibody of the invention is brought into contact with the intact antibiotic without having been brought into contact with the sample or having been brought into contact with a sample not containing any enzyme hydrolyzing the antibiotic, before to be brought into contact with the labeled intact antibiotic (the signal is then minimal).
• the antibody of the invention is brought into contact with the intact antibiotic after its incubation with a purified ⁇ -lactamase, before being brought into contact with the labeled intact antibiotic (the signal is then maximal).
• the antibiotic used in intact form
• the invention relates to methods making it possible to detect bacteria producing functional ⁇ -lactamase enzymes, using antibodies according to the invention and the antibiotics which they specifically recognize.
• an antibiotic possessing a ⁇ -lactam ring, as described above is available in intact form, and possibly labeled, and an antibody, optionally labeled, allowing it to be detected specifically (because produced as described above using said antibiotic as an immunogen)
• said method may advantageously comprise the following steps, in this order: a) Bringing the sample to be tested into contact with said intact antibiotic, b) Adding said antibody to the sample, c) Detecting whether said antibodies are complexed with the intact antibiotic.
• the signal obtained can then be compared with that which is generated when the antibody and the intact antibiotic have not been brought into contact with the sample (or when they have been brought into contact with a sample not containing of enzyme hydrolyzing the antibiotic), or when the intact antibiotic has been hydrolyzed by a ⁇ -lactamase before being brought into contact with the antibody.
• step d) is proportional to the quantity of ⁇ -lactamase enzymes present in the sample.
• the more the enzyme is concentrated in the sample tested the faster the hydrolysis of the intact antibiotic will be, the more the antibody added to the sample will remain free to complex with the intact antibiotic which will be brought into contact later.
• the intensity of the signal will be all the more intense as the concentration of enzyme initially present in the sample is high.
• the invention relates to a method making it possible to detect the presence of bacteria producing a functional ⁇ -lactamase enzyme, preferably with an extended spectrum, said method using at least one antibody as defined in the invention and the antibiotic molecule containing an intact ⁇ -lactam ring specifically recognized by said antibody. More specifically, said method uses i) an antibiotic possessing an intact ⁇ -lactam ring, as described above, in a labeled form and/or in an unlabeled form, and ii) at least one antibody making it possible to specifically detect this an intact antibiotic produced as described above using said antibiotic or an intact analog as an immunogen, said antibody being immobilized on a solid or readily detectable support.
• the method can also use, in addition to the antibody specific for the intact form, capture antibodies known to recognize the hydrolytic enzymes expressed by bacteria.
• This step makes it possible, if the bacteria in the sample have an ESBL activity, to determine which enzyme is responsible for this activity and to classify the bacteria according to this parameter (or to detect an activity due to a type of enzyme not known). It is in particular possible, to complete the information that the sample studied contains ESBL bacteria, to use anti-CTX-M or anti-carbapenemase antibodies known in the art (for example those described in Bernabeu et al. , 2020; Boutal et al., 2018).
• the present invention also relates to a kit making it possible to implement such methods.
• This kit contains at least the antibody of the invention and potentially the antibiotic which was used to produce it. It also contains, optionally, means for labeling the antibiotic and/or the antibody so as to be able to detect it(these).
• the antibody and/or the antibiotic is (are) immobilized on a solid support (for example a strip), or already labelled.
• a method according to the invention comprises, for example, the following steps, in this order: a) bringing the sample to be tested into contact with said antibiotic containing an intact, unlabeled ⁇ -lactam ring, b) Bringing the sample into contact with said antibody which has been immobilized on a solid support (or which is detectable), c) Bringing the antibiotic containing an intact, labeled ⁇ -lactam ring into contact with said antibody, or with the sample from step b), containing said antibody, d) Detecting whether said antibody is complexed with the labeled intact antibiotic brought into contact in step c).
• the (unlabeled) antibiotic added to the sample (step a) will remain in intact form, the antibodies of the invention will bind to this intact antibiotic and will not will not be able to bind the intact antibiotic marked in step c). Conversely, if the enzyme is present in the sample, the antibiotic (unlabeled) will be hydrolyzed in step a), and the antibody of the invention will remain free to bind to the intact antibiotic labeled l step c).
• the signal detected in step d), when the labeled antibiotic binds to the antibody of the invention, is thus proportional to the presence of enzyme in the sample (the signal increases the more enzyme there is in the sample, since the imposed competition is to the detriment of the labeled antibiotic, which is added after the unlabeled antibiotic). It is advantageous to compare the signal obtained in the presence of the sample with that which is generated when the antibody and the intact antibiotic have not been brought into contact with the sample (or with a sample containing no enzyme hydrolyzing the antibiotic), or when the intact antibiotic has been hydrolyzed with a ⁇ -lactamase before being brought into contact with the antibody (negative and positive controls).
• kits making it possible to implement such methods.
• This kit contains at least the antibody of the invention and possibly the antibiotic which was used to produce it. It also contains, optionally, means for labeling the antibiotic so as to be able to detect it and/or antibodies known to recognize ⁇ -lactamase enzymes.
• the antibody is supplied already immobilized on a solid support (for example a strip), or already labeled.
• the invention relates to a method making it possible to detect bacteria producing functional ⁇ -lactamases, said method using at least i) an antibiotic possessing a ⁇ -lactam ring, as described above, under intact form, and ii) an antibody capable of specifically detecting this intact antibiotic, produced as described above using said antibiotic as an immunogen, said antibody being labeled so as to be detectable.
• a method according to the invention comprises for example the following steps, in this order: a) Bringing the sample to be tested into contact with an antibiotic containing an intact ⁇ -lactam ring, b) Bringing the sample into contact after step a) with the antibody, which has been labeled, c) bringing the antibody obtained after step b) into contact with the antibiotic containing an intact ⁇ -lactam ring, which has been immobilized on a solid support, or adding labeled intact antibiotic distinctly from the antibody, d) Detecting whether said labeled antibody is complexed with the intact antibiotic brought into contact in step c).
• the antibiotic (unlabeled added in step a) added in the sample will remain in intact form, the antibodies of the invention will bind to this intact antibiotic and will not be able to bind the intact antibiotic immobilized in step c).
• the enzyme is present in the sample, the antibiotic (unlabeled) added in step a) will be hydrolyzed, and the labeled antibody of the invention in step b) will remain free to attach to the immobilized or labeled intact antibiotic during step c).
• the signal detected in step d) when the immobilized antibiotic binds to the labeled antibody of the invention is thus proportional to the presence of enzyme in the sample (the signal increases the more enzyme there is in the sample, since the imposed competition is to the detriment of the immobilized antibiotic).
• the signal obtained in the presence of the sample can advantageously be compared with that which is generated when the antibody and the intact antibiotic have not been brought into contact with the sample (or with a sample not containing of enzyme hydrolyzing the antibiotic), or when the intact antibiotic has been hydrolyzed with a ⁇ -lactamase enzyme before being brought into contact with the antibody (negative and positive controls). If the antibiotic is immobilized, it will be possible to observe the appearance of the signal where the antibiotic is immobilized.
• FRET fluorescence resonance energy transfer
• the method of the invention comprises, for example, the following steps: a) bringing the sample to be tested into contact with the antibiotic molecule containing an intact, unlabeled ⁇ -lactam ring, b) Bringing the sample obtained after step a) into contact with the antibody of the invention (which specifically recognizes the intact form of the antibiotic added in step a) and with at least one antibody specifically recognizing a ⁇ -lactamase enzyme (for example CTX-M or carbapenemase), said antibodies having been labeled beforehand, c) bringing the solution of step b) into contact with said antibiotic molecule containing an intact ⁇ -lactam ring and with antibodies specifically recognizing a ⁇ -lactamase enzyme (e.g.
• CTX-M or carbapenemase which have been immobilized on a solid support, d) Detecting whether said labeled antibodies are complexed to the intact antibiotic and/or to the anti- ⁇ -lactamase antibodies complexed with the ⁇ -lactamase enzyme, brought into contact in step c).
• the present invention also relates to a kit making it possible to implement such methods.
• This kit contains at least the antibody of the invention and possibly the antibiotic which was used to produce it. It also contains, optionally, means for labeling the antibody so as to be able to detect it and/or antibodies known to recognize ⁇ -lactamase enzymes.
• kits of the invention are supplied in free form (for step a)) and also in immobilized form on a solid support (for example a strip), or already labeled. Other tests, more or less complex, can be developed by using the discriminating antibody of the invention, as well as the antibiotic used to obtain it.
• All the kits of the invention can also contain a ⁇ -lactamase enzyme which can be used to verify the specificity of the antibody of the invention.
• All the kits of the invention can also contain a control sample not containing ⁇ -lactamase.
• All the kits of the invention can also contain the means for detecting the labeled antibody of the invention (for example, antibodies recognizing the constant part of a mouse immunoglobulin).
• kits of the invention may finally contain instructions making it possible to explain to users the details of the experiments to be carried out in order to detect bacteria producing functional ⁇ -lactamases in a rapid and efficient manner.
• Strip of the Invention the immunological test of the invention is an immunochromatographic test supported by a detection “strip”.
• the test of the invention is called “test strip” and the solid support used in the method of the invention is a strip.
• This strip constitutes a particularly important aspect of the present invention. Indeed, the present inventors have demonstrated that the antibody of the invention can advantageously be used in a strip test.
• the antibody of the invention is then preferably coupled to a fluorochrome or to a chromophore, or to any other marker visible to the naked eye, for example colloidal gold.
• the “strip test” is a simple, fast, inexpensive detection system that can be used by non-specialists in the field.
• the strips are generally formed of three distinct zones, which are fixed on a support, most often plastic: 1) an absorption zone promoting migration, 2) a reaction zone (generally formed of a nitrocellulose membrane) and 3) a deposit zone where the sample to be tested is deposited, located at the opposite end of the absorption zone (cf. figure 5).
• said “tracer” antibody will be the discriminating antibody of the invention, described above.
• two lines are usually present. The first of these lines, called the “test” (LT) line, will here be composed of antibiotics with a ⁇ -lactam cycle. The signal obtained at this test line will indicate the presence or absence of ⁇ -lactamase in the sample.
• a second line, called the “control” (LC) line will consist of antibodies directed against the antibody of the invention.
• the strip according to the invention can contain several test lines and a control line.
• One test line will correspond to an area where the intact antibiotic coupled to BSA or another carrier structure (e.g. casein, dextran or polylysine), has been immobilized, and the other test lines will correspond to areas where antibodies anti- ⁇ -lactamase were immobilized.
• the deposit zone will advantageously contain the antibodies of the invention as well as the anti- ⁇ -lactamase antibodies, all labeled in a detectable manner (the labels used being distinct or identical).
• the detection of the presence of the target enzyme will be much more reliable.
• the bacteria in the sample express a ⁇ -lactamase enzyme recognized by the labeled antibodies present on the deposit zone, these will bind to the ⁇ -lactamase enzyme, and the enzyme will also be able to bind to the anti- ⁇ -lactamase antibodies immobilized on the test area. A marking will therefore be visible on the test line corresponding to these antibodies, and it will be possible to deduce whether the bacteria carrying the ESBL activity express the enzyme recognized by the antibody immobilized on this line.
• the reaction zone can also contain several test lines on which different antibiotics are immobilized. In this case, several tracer monoclonal antibodies specific for one of these antibiotics should advantageously be present on the deposit zone.
• the reaction zone can contain a test line on which is fixed an antibiotic A with an intact ⁇ -lactam ring, and a test line on which is fixed an antibiotic B with an intact ⁇ -lactam ring, B being different from A.
• monoclonal antibodies discriminating intact antibiotics A and B from antibiotics Hydrolyzed A and B have been advantageously deposited, so that the signal should appear on two lines if there are ⁇ -lactamases in the sample tested. It is also advantageous in this context to have antibodies that discriminate between the intact and hydrolyzed forms of carbapenems, these new-generation antibiotics which are known to be resistant to ESBL enzymes other than carbapenemase.
• Example 2 describes precisely how to obtain such antibodies, which can be used in the methods and kits of the invention.
• the antibodies discriminating between the intact and hydrolyzed forms of a carbapenem are deposited on the deposit zone, and an intact carbapenem antibiotic is immobilized on a Test line.
• the test of the invention makes it possible to identify the presence, in the sample, of carbapenemase enzymes, and therefore potentially of bacteria expressing these enzymes.
• the strip of the invention contains at least two test lines, on which are respectively immobilized the intact form of a non-carbapenem antibiotic (for example a cephalosporin) and the intact form of an antibiotic carbapenem, so as to identify in the sample the presence of ESBL enzymes other than carbapenemase (eg cephalosporinases, etc.) and carbapenemase.
• a non-carbapenem antibiotic for example a cephalosporin
• carbapenem for example a cephalosporin
• carbapenem for example a cephalosporin
• carbapenem eg cephalosporinases, etc.
• carbapenemase eg cephalosporinases, etc.
• the respective discriminating antibodies labeled (possibly with different markers), are for their part deposited on the deposit zone.
• the strip of the invention may also contain in its reaction zone, as explained above, other test lines, on which anti- ⁇ -lactamase enzyme antibodies
• the enzyme is not present in the sample, the antibiotic added to the sample will remain in intact form, the antibodies of the invention will bind to this intact antibiotic and will no longer be able to bind to the intact antibiotic of the LT. Conversely, if the enzyme is present in the sample, the added antibiotic will be hydrolysed, and the antibody of the invention will remain free to bind to the intact antibiotic immobilized on the LT. In these two cases, the free antibodies, ie, which have not been immobilized on the test line, will be captured at the level of the control line by the anti-antibody antibody of the invention.
• the strip test of the invention has been designed so that the signal at the level of the test line increases with the quantity of ⁇ -lactamases present in the sample.
• the strip test of the invention is very reliable, in that it links the appearance of a signal (and not its disappearance) to the quantity of enzyme. However, it is well known that it is easier and more reliable to detect the appearance of a signal than its decrease.
• a strip according to the invention can be prepared in the following way: - the antibody of the invention, preferably labeled, is deposited, and for example dried, on the deposit zone, -
• anti- ⁇ lactamase antibodies are also deposited on the deposition zone.
• the antibiotic with an intact ⁇ -lactam ring is immobilized on the LT, in the reaction zone (for example with a BSA-antibiotic system, which allows the immobilization of the antibiotic on the nitrocellulose while allowing it to interact with the labeled antibody),
• anti- ⁇ lactamase antibodies are also immobilized on another line of the reaction zone
• - antibodies recognizing the antibodies deposited on the strip deposit zone are immobilized, for example by adsorption, on the LC, opposite to the LT.
• the present invention therefore relates to a strip containing (cf.
• FIG. 5 1) a zone for depositing the sample, on which the antibody as defined above has been labeled, deposited and dried, as well as optionally labeled anti- ⁇ lactamase antibodies, 2) a reaction zone, comprising: - at least one test line on which the antibiotic with an intact ⁇ -lactam ring having been used to produce at least one of the antibodies deposited in the deposit zone 1) has been immobilized, and - optionally at least one test line on which antibodies anti - ⁇ lactamase have been immobilized, - a control line on which antibodies recognizing the antibody(ies) present in zone 1) have been immobilized, and 3) an absorption zone promoting antibody migration, said zone being located at the opposite end of the deposit zone 1).
• the labeled antibodies are deposited and stored on the surface of the strip preferably by drying. They can also be added to the sample just before depositing the latter on the strip.
• the reaction zone is preferably made of nitrocellulose or PVDF, cellulose, fiberglass.
• the immobilization of the antibiotic on the LT is preferably done by adsorption of the antibiotic coupled with BSA or another carrier molecule (for example casein, dextran, polylysine), which makes it possible to leaving accessible the ⁇ -lactam cycle which must capture the antibodies of the invention migrating towards the absorption zone.
• the antibodies recognizing the antibodies used on the strip of the invention are, for example, antibodies anti-constant part of murine immunoglobulin, protein A, protein G or any other system recognizing murine antibodies.
• the quantities of antibodies and antibiotics immobilized or labeled or as a substrate must in fact be rigorously controlled.
• Too large an amount of antibody would require a high concentration of substrate for occupation of all the binding sites and therefore a higher concentration of ⁇ -lactamase to have a significant drop in the occupation of these sites.
• too high a concentration of labeled or immobilized antibiotics would induce very high competition with respect to the binding of the substrate by the antibody, which would induce the appearance of a signal even in the absence of ⁇ -lactamase or then to the use of an excess of substrate in order to maintain the total occupation of the binding sites of the antibody by this same substrate. This excess of substrate would lead to a strong drop in sensitivity or to a longer incubation time. Examples illustrating these optimization steps are given below.
• the quantity of antibody to be deposited on the deposit zone must be such that it makes it possible to saturate all the binding sites for the intact antibiotic used in the method or on the strip of the invention. Moreover, he understands that its minimum quantity is that which makes it possible to obtain a maximum signal on the test line containing the antibiotic.
• the optimum quantity of antibody to be deposited on the deposit zone is such that 10 ⁇ L of the solution having an absorbance relative to colloidal gold – DO – is deposited. between 0.1 and 10.
• the optimal amount of antibiotic immobilized on the LT line (1 ⁇ L/cm) is ideally between 1 ⁇ g and 1mg/mL.
• a concentration of about 0.1 mg/mL gives excellent results.
• K d between 1 pM and approximately 10 nM
• kits of the invention containing the strip of the invention Before being deposited on the deposit zone, an antibiotic with an intact ⁇ -lactam ring must be added to the sample to be tested, prior to bringing it into contact with the strip of the invention.
• the present invention also relates to a kit containing, in addition to the strip of the invention, the antibiotic with an intact ⁇ -lactam ring which was used to obtain the antibody of the invention, and which is immobilized on the deposit zone 1) of the strip.
• This antibiotic is preferably contained in a separate container, isolated from the strip.
• the kit of the invention will contain a strip on which said anti-cefotaxime antibody intact antibiotic is deposited and said intact antibiotic is immobilized, together with a vial or tube containing intact cefotaxime antibiotic.
• the kit of the invention will contain a strip on which said anti-carbapenem antibody intact antibiotic is deposited and said intact antibiotic is immobilized, together with a vial or tube containing intact carbapenem antibiotic.
• the kit of the invention may also contain a container comprising a functional ⁇ -lactamase.
• the user will thus be able, for example, to compare the difference in signal obtained in the sample to be tested (ie, when the endogenous ⁇ -lactamase is optionally present), or when an exogenous enzyme is added. This step is used to check that the test is working properly.
• this kit may also contain a control sample not containing ⁇ -lactamase, the means for detecting the labeled antibody of the invention (for example, antibodies recognizing the constant part of an immunoglobulin of mice) and/or instructions explaining to users the details of the experiments to be carried out to detect bacteria producing functional ⁇ -lactamases quickly and efficiently.
• the present invention relates more particularly to a kit containing: - at least one strip according to the invention, and - a separate container containing the antibiotic with an intact ⁇ -lactam ring used to obtain the antibody immobilized on the deposit zone 1) of the strip, - optionally, a separate container containing ⁇ -lactamase enzyme and/or a sample containing no ⁇ -lactamase enzyme.
• the present invention relates to a method allowing the detection of ⁇ -lactamases, in a sample likely to contain them. Said sample has been described above.
• said method uses the strip or the kit of the invention containing this strip, as described above. This method involves the following steps: a) Bringing an antibiotic with an intact ⁇ -lactam ring into contact with the sample to be tested, said antibiotic being specifically recognized by the antibody of the invention labeled and deposited on the strip, and being immobilized on the LT line of the strip.
• the sample (containing the intact antibiotic and the bacteria) is deposited on the strip deposit area.
• the antibiotic not having been hydrolyzed (because there is no ⁇ -lactamase activity in the sample), a complex is formed between the latter and the antibody of the invention. Having migrated to the test line, the antibody thus complexed will not be able to bind to the antibiotic which is immobilized there. On the other hand, the antibody will be immobilized at the level of the control line (LC) by the anti-antibody antibody of the invention. So only the LC will be visible. The test will be negative and it can be concluded that there are no bacteria producing ⁇ -lactamase in the sample to be tested.
• LC control line
• Case 2 The sample to be tested contains bacteria producing a ⁇ -lactamase enzyme. After an incubation time, the sample no longer contains intact antibiotic since the ⁇ -lactamase enzyme has hydrolyzed it. When it is deposited on the drop zone of the strip, no complex is formed between the hydrolyzed antibiotic and the antibody of the invention.
• the antibody of the invention whose binding sites are free, migrates towards the test line where it can bind to the intact antibiotic immobilized at the level of the test line.
• the antibody of the invention in excess is for its part immobilized at the level of the control line (LC) by the anti-antibody antibody. In this case, the two lines (the test line and the control line) are visible.
• the test is declared positive, concluding with the presence of a bacterium producing a ⁇ -lactamase (ESBL, or other ⁇ -lactamase) in the sample.
• a bacterium producing a ⁇ -lactamase for example LT1: intact antibiotic; LT2: anti-CTX-M (figure 10)
• two LTs for example LT1: intact antibiotic; LT2: anti-CTX-M
• four cases then arise, depending on what the bacterium present produces as ⁇ -lactamase (figure 11) : Case 1:
• the sample to be tested contains bacteria that do not produce ⁇ -lactamase. After an incubation time, the sample (containing the intact antibiotic and the bacteria) is deposited on the strip deposit area.
• the antibiotic Since the antibiotic has not been hydrolyzed (because there is no ⁇ -lactamase activity in the sample), a complex forms between it and the antibody of the invention.
• Labeled anti-CTX-M antibodies do not bind CTX-M enzyme. Having migrated to test line 1 (LT1), the antibody of the invention thus complexed will not be able to bind to the antibiotic which is immobilized there. Since the labeled anti-CTX-M antibodies are not complexed with the enzyme, they cannot be fixed by the second anti-CTXM antibody on test line 2 (LT2). On the other hand, the antibodies will be immobilized at the level of the control line (LC) by the antibody anti- antibody. Thus, only the LC will be visible.
• LC control line
• the test will be negative and it can be concluded that there are no bacteria producing ⁇ -lactamase in the sample to be tested.
• Case 2 The sample to be tested contains bacteria producing a ⁇ -lactamase but not a CTX-M enzyme. After an incubation time, the sample no longer contains intact antibiotic since the ⁇ -lactamase enzyme has hydrolyzed it. When the sample is deposited on the strip deposit zone, no complex is formed between the hydrolyzed antibiotic and the antibody of the invention. Labeled anti-CTX-M antibodies do not bind CTX-M enzyme. The antibody of the invention, the binding sites of which are free, migrates towards the test line where it can bind to the intact antibiotic immobilized at the level of test line 1 (LT1).
• LT1 level of test line 1
• test line 2 test line 2
• the anti-CTX-M antibodies and the antibody of the invention in excess are for their part immobilized at the level of the control line (LC) by the anti-antibody antibody.
• the test 1 and control lines are visible.
• the test is declared positive, concluding with the presence of a bacterium producing a ⁇ -lactamase (ESBL, or other ⁇ -lactamase), but which is not a CTX-M enzyme, capable of hydrolyzing the antibiotic in the 'sample.
• ESBL ⁇ -lactamase
• CTX-M enzyme capable of hydrolyzing the antibiotic in the 'sample.
• Case 3 The sample to be tested contains bacteria producing a CTX-M type ⁇ -lactamase enzyme.
• the sample no longer contains intact antibiotic since the ⁇ -lactamase enzyme has hydrolyzed it.
• the sample is deposited on the strip deposit zone, no complex is formed between the hydrolyzed antibiotic and the antibody of the invention.
• Anti-CTX-M antibodies bind the CTX-M enzyme present.
• the antibody of the invention migrates towards the test line where it can bind to the intact antibiotic immobilized at the level of test line 1 (LT1).
• the anti-CTX-M antibodies present on test line 2 (LT2) bind the CTX-M enzyme complexed with the labeled anti-CTXM antibodies.
• the anti-CTX-M antibodies and the antibody of the invention in excess are for their part immobilized at the level of the control line (LC) by the anti-antibody antibody.
• the lines test 1, test 2 and control are visible.
• the test is declared positive, concluding with the presence of a bacterium producing a ⁇ -lactamase (ESBL, or other ⁇ -lactamase), of the CTX-M type, capable of hydrolysing the antibiotic in the sample.
• ESBL ⁇ -lactamase
• the antibiotic in intact form or “substrate” is added to the sample to be tested. The amount of intact antibiotic can be adjusted to optimize test sensitivity.
• the quantity of substrate added to the sample it is necessary for the quantity of substrate added to the sample to allow the occupation of all the binding sites of the antibodies used, without being in excess.
• a signal appears when the quantity of the substrate is no longer sufficient to occupy all of the binding sites of the antibodies of the invention.
• An excess of substrate would not make it possible to detect low enzyme concentrations or would require long incubation times that are incompatible with a test that is intended to be quick and simple.
• the person skilled in the art by following the indications mentioned in the present invention (and in particular in the examples) will be able to determine the quantity of intact antibiotic to be added to the sample in the initial stage of the method, so that it allows the occupation of all the binding sites of the antibodies used, without being in excess.
• an anti-cefotaxime antibody according to the invention it is possible to add, for example, between 10 ng/mL and 50 ng/mL of cefotaxime antibiotic in the original sample. More generally, for other antibodies with a strong affinity for the intact antibiotic that they specifically recognize (KD between 1 pM and approximately 10 nM), it will be possible to add between 1 ng/mL and 1 ⁇ g/mL, between 10ng/mL and 1 ⁇ g/mL, between 50ng/mL and 1 ⁇ g/mL, between 100ng/mL and 1 ⁇ g/mL or between 100ng/mL and 0.5 ⁇ g/mL of intact antibiotic in the initial sample (containing bacteria producing or not a ⁇ -lactamase).
• Step b) incubation can be done at room temperature.
• the duration of this step can be adapted so that the sensitivity of the test is optimal.
• this incubation step can last between 10 minutes and one hour. Good results have been obtained with a duration of 30 minutes, under the conditions tested.
• KD KD between 1 pM and approximately 10 nM
• an incubation period between 10 minutes and one hour is optimal.
• the antibodies should be allowed time to migrate to the test and control lines. This migration step can last between 5 minutes and 30 minutes.
• cefotaxime is used (cf.
• Step d) of reading the result can therefore be done, in the case where cefotaxime is used, approximately 40 minutes after bringing the sample and the antibiotic into contact.
• KD KD between 1 pM and approximately 10 nM
• a reading of the result can take approximately 10 minutes to 1 hour (from preferably between 20 minutes and 40 minutes) after bringing the sample into contact with the strip. All the steps of this method can be carried out at room temperature. To be exploitable, the method of the invention must be able to give reliable results in a minimum time, ideally in less than one hour.
• the test of the invention has a specificity of 100% and a sensitivity of 100%, which is excellent. It may be advantageous to prepare the sample to be tested before using the strip of the invention, in particular if the sample is solid. If the sample is solid (for example soil), it is possible to dilute it by adding a buffer before proceeding to the step of bringing it into contact with the antibiotic.
• This buffer can contain, for example, NaCl, a molecule known to reduce non-specific interactions (PVP, PVA, BSA) and detergent (Tween 20). Its pH is preferentially 8. The concentration of NaCl is preferentially close to 150 mM.
• a cell lysis is carried out, in order to release the ⁇ -lactamase enzyme possibly contained in the bacteria of the sample and to make its activity visible more quickly.
• Conventional lysis buffers can be used (cf. example below). If the sample is liquid (for example a biological fluid), it is possible to dilute it by adding a buffer before proceeding to the step of placing it in contact with the antibiotic.
• This buffer can contain, for example, NaCl, a protein known to reduce non-specific interactions (PVP, PVA, BSA) and detergent (Tween 20). Its pH is preferentially 8. The concentration of NaCl is preferentially close to 150 mM. A person skilled in the art knows how to obtain such usable samples.
• Figures Figure 1 describes the principle of immunoenzymatic test 1 used in the application to select the antibody of interest that can be used in the system of the invention. This test involves non-hydrolyzed Cefotaxime-biotin (NH), the antibody derived from a hybridoma or from plasma of mice immunized with cefotaxime and Streptavidin-Acetylcholinesterase (G4). Acetylcholinesterase reacts with a chromogen to produce a colored product.
• NH Cefotaxime-biotin
• G4 Streptavidin-Acetylcholinesterase
• Figure 2 describes the principle of three other immunoenzymatic tests of interest used to select the antibody of interest that can be used in the system of the invention.
• Test 2 uses: Hydrolyzed cefotaxime-biotin (H) + Antibodies from hybridoma or plasma from mice immunized with cefatoxine + Streptavidin-G4
• Test 3 uses: Non-hydrolyzed cefotaxime-biotin (NH) + Non-hydrolyzed cefotaxime (NH) + Hybridoma or plasma antibodies from mice immunized with cefaoxime + Streptavidin-G4
• Test 4 uses: Non-hydrolyzed cefotaxime-biotin ( NH) + Cefotaxime hydrolyzed (H) + Hybridoma or plasma antibodies from mice immunized with cefatoxime + Streptavidin-G4
• Figures 3A and 3B represent the competition curves obtained with cefotaxime hydrolyzed or not, and the various monoclo
• FIG. 4 represents the competition curve obtained with cefotaxime, hydrolyzed or not, with one of the unselected monoclonal antibodies.
• FIG. 5 represents the various elements constituting the strips conventionally used for analyte detection purposes.
• Figure 6 describes the two cases expected when the sample contains (positive test) or does not contain (negative test) ESBL bacteria or ⁇ -lactamase enzymes.
• FIG. 7 depicts the plastic cassette that can be used to protect the tape of the invention.
• Figure 8 depicts the structures of the carbapenem compounds used in Example 2 (B).
• Figure 9 presents the principle of tests 1 to 4 which are described in example 2 (C).
• FIG. 10 shows a strip according to the invention, containing two test lines and a control line.
• the first test line corresponds to an area where Cefotaxime-BSA has been immobilized
• the second test line corresponds to an area where anti-CTX-Ms antibodies have been immobilized.
• the control line corresponds to an area where secondary antibodies, recognizing the other labeled antibodies used in the invention, have been immobilized.
• anti-cefotaxime antibodies of the invention and anti-CTX-Ms antibodies, all labeled with colloidal gold, have been deposited.
• Figure 11 describes the three cases expected when the sample does not contain bacteria with an ESBL or ⁇ -lactamase enzymes (negative test), or contains ESBL bacteria or ⁇ -lactamase enzymes other than CTX-M ( test positive on one line), or contains ESBL bacteria or CTX-M type ⁇ -lactamase enzymes (test positive on both lines).
• Example 1 Detection of bacteria resistant to cefotaxime A. Design and production of the immunogen Cefotaxime is a small molecule incapable of inducing an immune response, essential for obtaining antibodies.
• BSA bovine serum albumin
• SATA N-succinimidyl S-acetylthioacetate
• Cefotaxime-BSA was used to immunize mice. To carry out the immunizations, injections by subcutaneous route of 50 ⁇ g of cefotaxime-BSA/mouse were carried out every three weeks for three months (4 immunizations in total). After 2 months of rest for the mice, new injections of cefotaxime-BSA were carried out intravenously in the latter: 50 ⁇ g of product/mouse, once a day for three days. After two days of rest, mouse spleen cells were fused with NS1 mouse myeloma cells, and anti-cefotaxime antibodies specific in myeloma culture supernatants were detected using an enzyme immunoassay. B.
• Non-hydrolyzed cefotaxime-biotin was obtained by coupling chloroacetamido-cefotaxime and biotin coupled to a polyethylene glycol arm (PEG) and a thiol function (Biotin-PEGx-Thiol ), using the procedure previously described for the immunogen.
• PEG polyethylene glycol arm
• Biotin-PEGx-Thiol a thiol function
• Chloroacetamido-cefotaxime (31.6 mg, 0.06 mmol, 1 eq.) and Biotin-PEGx-Thiol (94 mg, 0.119 mmol, 2 eq.) were dissolved in 0.5 ml of DMF and 2 ⁇ l of triethylamine, and were then added to the mixture under Argon. The reaction was stirred for 3 days. After the reaction was complete, the mixture was evaporated under reduced vacuum. Then, the product was purified by reverse phase chromatography on a water/acetonitrile gradient from 0 to 40% (isolated peak at 26% acetonitrile).
• the molecular weight of this tracer was checked by mass spectrometry, where a 15-minute purification cycle on a C18 column is carried out, then the sample is ionized on a quadrupole.
• the hydrolyzed cefotaxime-biotin was obtained by enzymatic reaction with beads coupled to KPC-2 (Klebsiella pneumoniae carbapenemase), which is a recombinant ⁇ -lactamase. To do this, 5 mg of beads (tosylactivated Dynabeads M-280) were washed with 0.1 M borate buffer pH 9.5. 100 ⁇ g of the recombinant protein KPC-2 were added to the beads in a volume of 150 ⁇ l.
• the Beads-KPC-2 were removed using a magnet.
• the supernatant was recovered and purified by reverse phase chromatography on a water/acetonitrile gradient from 0 to 40% (isolated peak at 23% acetonitrile).
• the molecular weight of this tracer was checked by mass spectrometry, where a 15-minute purification cycle on a C18 column is carried out, then the sample is ionized on a quadrupole.
• Non-hydrolyzed cefotaxime (Sigma-Aldrich) was purified by reverse phase chromatography on a water/acetonitrile gradient from 0 to 20% (isolated peak at 8.5% acetonitrile). The molecular weight of this product was checked by mass spectrometry, where a 15-minute purification cycle on a C18 column is carried out, then the sample is ionized on a quadrupole. Hydrolyzed cefotaxime was also obtained by enzymatic reaction with beads coupled to KPC-2. The same bead-KPC-2 coupling protocol, cited above, was carried out.
• mice were immunized with cefotaxime-BSA. To do this, injections by subcutaneous route of 50 ⁇ g of cefotaxime-BSA/mouse were carried out every three weeks for three months (4 immunizations in total). After 3 months of rest for the mice and in order to select the mice presenting the best immune response, their antibodies were analyzed with a first test. In this test, murine antibodies collected during the immunization protocol were captured by a first murine anti-antibody antibody (AffiniPure Goat Anti-Mouse IgG + IgM (H+L); Jackson Immunoresearch LABORATORIES) immobilized on the wall of wells of a microtiter plate.
• a first murine anti-antibody antibody AffiniPure Goat Anti-Mouse IgG + IgM (H+L); Jackson Immunoresearch LABORATORIES
• Ellman's medium comprises a mixture of 7.5 10 -4 M acetylthiocholine iodide (enzyme substrate) and 2.5 10 -4 M 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) (reagent for the colorimetric measurement of thiol) in a 0.1 M phosphate buffer, pH 7.4.
• Enzyme activity was expressed in Ellman units (EU).
• EU Ellman units
• One EU is defined as the amount of enzyme producing an increase in absorbance of one unit for 1 min in 1 ml of medium, for an optical path length of 1 cm: it corresponds to approximately 8 ng of enzyme.
• splenocytes spleen cells
• NSI murine myeloma cells NSI murine myeloma cells
• Test 1 In this test, the antibodies present in the culture supernatants are captured by a first anti-murine antibody antibody immobilized on the wall of the wells of a microtiter plate. Unhydrolyzed cefotaxime-biotin is added to each well. After incubation at 4° C. overnight and after washing, streptavidin-G4 is added to reveal the presence of non-hydrolyzed cefotaxime coupled to biotin and therefore of non-hydrolyzed anti-cefotaxime antibodies.
• Test 2 In this test, the antibodies present in the culture supernatants are captured by a first anti-murine antibody antibody immobilized on the wall of the wells of a microtiter plate. Hydrolyzed cefotaxime-biotin is added to each well. After incubation at 4° C. overnight and after washing, streptavidin-G4 is added to reveal the presence of hydrolyzed cefotaxime coupled to biotin and therefore the presence of anti-hydrolyzed cefotaxime antibodies.
• Test 3 In this test, non-hydrolysed cefotaxime-biotin is placed in competition with non-hydrolysed cefotaxime with respect to recognition by the specific antibodies present in the culture supernatants. After incubation at 4° C. overnight and after washing, streptavidin-G4 is added to reveal the presence of intact cefotaxime coupled to biotin.
• Test 4 In this test, non-hydrolyzed cefotaxime-biotin is placed in competition with hydrolyzed cefotaxime at the same concentration as the non-hydrolyzed cefotaxime used in test 3, with respect to recognition by the specific antibodies present in culture supernatants. After incubation at 4° C. overnight and after washing, streptavidin-G4 is added to reveal the presence of intact cefotaxime coupled to biotin.
• the wells were selected for which a signal was obtained for test 1 and no signal for test 2, a drop in the most significant signal of the signal for test 3 and no drop in signal for test 4. At the end of the selection process, 18 hybridomas were stored to produce monoclonal antibodies.
• tests 3 and 4 were performed with different concentrations of unhydrolyzed and hydrolyzed cefotaxime as competitor.
• the solutions were deposited in a 96-well microplate, on the wall of which an anti-mouse antibody antibody (identical to that used in the antibody selection experiments) was immobilized beforehand, at a rate of 25 ⁇ l of marker (Cefotaxime-biotin intact ) and 25 ⁇ l of competitor (non-hydrolyzed or hydrolyzed Cefotaxime). Then 50 ⁇ l of the antibody solution was added.
• the microplates were incubated overnight at 4° C., then, after washing, 100 ⁇ l of Streptavidin-G4 were added for 1 hour at room temperature and with stirring. After washing the wells, 200 ⁇ l of chromogen (Ellman's medium) were deposited.
• the absorbance was read after 1 hour of incubation with stirring, at 414 nm on a spectrophotometer.
• the Bo signal corresponds to the absorbance obtained in the absence of a competitor (maximum absorbance).
• Signal B is the absorbance obtained in the medium where competitor and marker are interacting with the antibody. In order for the figure to be readable, only the results obtained with a few antibodies have been represented (FIGS. 3A and 3B).
• FIG. 4 demonstrates, for this non-retained antibody, a greater drop in signal for hydrolyzed cefotaxime than for non-hydrolyzed cefotaxime, which means that the latter form is less well recognized by the non-retained antibody.
• Antibodies 1; 2; 3; 4 and 5 were then used on test strips by competition.
• the strips are made up of 4 distinct parts:
• PC conjugate paper
• labeled antibody also called tracer
• This paper can be accessory in the case where the tracer antibody is used in liquid format. In this case 10 ⁇ l of tracer antibody are added to 100 ⁇ l of sample before depositing on the strip.
• the amount of tracer The chosen antibody is coupled to colloidal gold (colored marker), its absorbance could be measured at 530 nm. Therefore, in the results, the term absorbance (OD) relating to colloidal gold is used. Several ODs were thus tested. The objective was to determine the smallest quantity of tracer enabling a significant signal to be observed 10 minutes after depositing the sample on the strip.
• the amount of intact BSA-cefotaxime on the LT once the antibody OD has been fixed, several concentrations of intact BSA-cefotaxime on the LT were tested (1 ⁇ L/cm). The objective was to fix the concentration, non-limiting for the signal observed at the level of the LT, the weakest.
• the amount of non-hydrolyzed cefotaxime to add to the samples was once the two previous parameters have been determined, the objective was to set the minimum concentration of intact cefotaxime above which no signal is observed on the LT. At the same time, hydrolyzed cefotaxime was added to verify that the antibody is indeed specific for the non-hydrolyzed form. For this, a range of non-hydrolyzed cefotaxime and a range of hydrolyzed cefotaxime were prepared with strip buffer: 1000 ng/ml; 100ng/ml; 10ng/ml; 1ng/ml.
• a color intensity scale was used to evaluate the results obtained on the test strips. This scale has been set from 1 to 10, where each value is characteristic of increasing signal strength.
• 96-well microplates were used.
• 10 ⁇ l of tracer in liquid form were added to 100 ⁇ l of buffer containing or not cefotaxime.
• a strip composed of a sample paper, a nitrocellulose membrane and an absorbent paper was placed in the well. A 10 min incubation was performed, then the signal intensity was assessed with the color intensity scale.
• the OD in tracer was first optimized. On the LT, a default concentration of 1mg/ml of intact BSA-Cefotaxime has been deposited. The test results are shown in Table 1.
• the OD:1 was selected because it is located in the 8.5/9 intensity scale. Different concentrations of non-hydrolyzed BSA-Cefatoxime were then deposited on the LT (1 ⁇ L/cm). The DO:1 concentration previously selected for the tracer was used.
• Table 2 Optimization of BSA-Cefotaxime concentration on LT. The intensity of the signal increases only very slightly beyond a concentration of 0.1mg/ml in BSA-Cefotaxime. This is why this concentration was selected. In order to adjust the parameters more precisely, different amounts of tracer were tested with this amount of BSA-cefotaxime. Table 1: Second tracer OD optimization.
• the concentration of unhydrolyzed cefotaxime to be used in the samples was then determined.
• a control is carried out (condition at ong/ml), to see the maximum signal that can be obtained on the LT (Table 4).
• the signal is equivalent to the control (the tracers bind to the LT).
• the hydrolyzed cefotaxime is not recognized by the tracer antibody, the binding sites of which are free to interact with the cefotaxime of LT.
• antibodies have been tested.
• the conditions of use of the antibodies are as follows: Antibody 1, DO0.5, 0.3mg/ml of BSA-Cefotaxime; Antibody 2, DO1, 0.3mg/ml of BSA-Cefotaxime; Antibody 3, DO0.5, 0.1mg/ml of BSA-Cefotaxime; Antibody 4, DO0.5, 0.3mg/ml of BSA-Cefotaxime; Antibody 5, DO0.5, 0.3mg/ml of BSA-Cefotaxime (Table 5).
• Table 5 Optimization of the concentration of intact cefotaxime to be added to the samples for different antibodies. 0: No signal was observed on the LT.
• antibody 3 showed the best performance in the strip format because it makes it possible to detect enzymatic activity for the lowest enzyme concentration.
• this result shows that other antibodies could have been used in this method.
• Antibody 3 was used for further developments and optimizations.
• the concentrations used are: 10 ng/ml; 3ng/ml; lng/ml; 0.3ng/ml; 0.1ng/ml in enzymes, and are incubated with intact cefotaxime at room temperature. Two controls are also carried out containing either only intact cefotaxime (no signal should be observed on the LT because all of the tracer binding sites are occupied), or only strip buffer (maximum signal that can be obtained on the LT because all of the tracer binding sites are free).
• the tracer is first added in liquid form (10 ⁇ l at OD:0.5 are added to 100 ⁇ l of the enzymatic solution) then, secondly, in dried format on conjugated paper (10 ⁇ l at OD:0.9 ).
• the conjugate paper was then inserted on the strip between the sample paper and the nitrocellulose membrane. Therefore, two types of strips were used, with or without conjugate paper (PC). It was indicated, for each experiment, which type of strip was used.
• the LT on the nitrocellulose membrane is composed of 0.1mg/ml of unhydrolyzed BSA-Cefotaxime. The different conditions were prepared and then incubated at room temperature. Once the incubation time had elapsed, 100 ⁇ l of solution were taken and deposited, either in a microplate well, or in a deposit well of a plastic cassette.
• Table 6 Kinetics of hydrolysis at ambient temperature of cefotaxime by a CTXM-2 enzyme, liquid tracer (N: negative test, no signal at LT; P: positive test, signal visible at LT).
• Table 7 Kinetics of hydrolysis at 37° C. of intact cefotaxime by CTXM-2, liquid tracer.
• the tracer was dried on the PC. It has been observed that after resolubilization, part of the tracer antibody is adsorbed by the PC. To compensate for this absorption, the quantity of tracer used must be increased. The final OD in tracer on the PC was 0.9 for this first test. The tests were carried out in 96-well microplates, with 100 ⁇ l of sample. The concentration of non-hydrolyzed cefotaxime was 10 ng/ml. New hydrolysis kinetics were achieved with this protocol (Table 8).
• Table 8 Kinetics of hydrolysis at AT of cefotaxime not hydrolyzed by CTXM-2.
• Table 9 Kinetics of hydrolysis of intact cefotaxime by a CTXM-2 enzyme (OD:1).
• Table 10 Kinetics of hydrolysis of cefotaxime not hydrolyzed by CTXM-2 (DO1.5).
• the increase in tracer OD made it possible to lower the concentration of enzymes necessary to have a visible signal on the LT.
• DO 1.5 shows better results with detection at lng/ml from 15 minutes.
• the concentration of non-hydrolyzed cefotaxime was 10 ng/ml in strip buffer. Two controls were also carried out containing either only non-hydrolysed cefotaxime or only strip buffer. The plotter has been dried on the PC.
• the LT was composed of 0.1mg/ml of unhydrolyzed BSA-Cefotaxime. The migration took place in a plastic cassette.
• the incubation time of 30 minutes was selected. By applying this incubation period, all of the resistant colonies are indeed positive and the non-resistant colonies are negative.
• the strip test is therefore well suited for use on bacterial colonies.
• bacteria were divided into two groups: ⁇ -lactamase producers that do not hydrolyze cefotaxime and ⁇ -lactamase producers that hydrolyze cefotaxime (Table 12).
• test strip of the invention made it possible to obtain a sensitivity of 100% and a specificity of 100% for the detection of a cephalosporinase activity in 40 minutes (incubation+migration). These performances are perfect for use in clinical and veterinary diagnostics and also in the context of environmental assessment.
• Example 2 Detection of bacteria resistant to a carbapenem
• Alkyne carbapenem (S. Saijalolov, Chem. Eur. J., 2021) is a small molecule incapable of inducing an immune response essential for obtaining antibodies. It was therefore necessary to couple this antibiotic to a larger molecule immunogenic, bovine serum albumin (BSA).
• BSA bovine serum albumin
• step 1 consisted of producing SMC-BSA, then step 2 coupled amine-carbapenem (lannazzo L et al, 2016) with SATA (N-succinimidyl S-acetylthioacetate), and step 3 coupled SMC-BSA with SATA-carbapenem.
• step 3 coupled SMC-BSA with SATA-carbapenem.
• Immunogen A was used to immunize mice. To carry out the immunizations, injections by subcutaneous route of 50 ⁇ g of immunogen A/mouse were carried out every three weeks for three months (4 immunizations in total).
• mice After 2 months of rest for the mice, new injections of immunogen A were carried out intravenously in the latter: 50 ⁇ g of product/mouse, once a day for three days. After two days of rest, mouse spleen cells were fused with NSI mouse myeloma cells, and specific anti-carbapenem antibodies in myeloma culture supernatants were detected using a enzyme immunoassay.
• Non-hydrolyzed carbapenem-biotin was obtained by coupling amine-carbapenem and biotinamidohexanoic acid N-hydroxy-succinimide ester (NHS-LC-Biotin). The chemical reaction occurs between the NH2 group of the amine carbapenem and the N-hydroxy-succinimide group of biotin. The resulting product is called tracer A.
• HA tracer Hydrolyzed carbapenem-biotin (HA tracer) was obtained by enzymatic reaction with beads coupled to KPC-2 (Klebsiella pneumoniae carbapenemase), which is a recombinant ⁇ -lactamase.
• KPC-2 Klebsiella pneumoniae carbapenemase
• 50 ⁇ l of the solution of Beads-KPC-2 at 20 mg/ml were added to 1 ml of a solution of tracer A NH at 2 mg/ml. After a reaction of 16 hours at 25°C, the Beads-KPC-2 were removed using a magnet. The supernatant containing the AH tracer was recovered and purified by reverse phase chromatography. The molecular weight of this tracer was verified by mass spectrometry.
• a second non-hydrolyzed carbapenem-biotin was produced by another chemical process. It was produced by coupling an alkyne-carbapenem and biotin coupled to a polyethylene glycol arm (PEG) and an azide function (Biotin-dPEG®7-azide). The resulting product is called tracer B NH.
• PEG polyethylene glycol arm
• Biotin-dPEG®7-azide Biotin-dPEG®7-azide
• BH tracer Hydrolyzed carbapenem-biotin (BH tracer) was obtained by enzymatic reaction with beads coupled to KPC-2 (Klebsiella pneumoniae carbapenemase), which is a recombinant ⁇ -lactamase. To do this, 50 ⁇ l of the solution of Beads-KPC-2 at 20 mg/ml were added to 1 ml of a solution of tracer A NH at 2 mg/ml. After a reaction of 16 hours at 25°C, the Beads-KPC-2 were removed using a magnet. The supernatant containing the BH tracer was recovered and purified by reverse phase chromatography. The molecular weight of this tracer was verified by mass spectrometry. Production of non-hydrolyzed and hydrolyzed carbapenems
• the non-hydrolyzed carbapenem used for antibody selection is meropenem (Sigma-Aldrich).
• the non-hydrolysed meropenem was purified by reverse phase chromatography on a water/acetonitrile gradient from 0 to 20% (isolated peak at 7.8% acetonitrile).
• mice were immunized with immunogen A. To do this, injections by the subcutaneous route of 50 ⁇ g of immunogen A/mouse were carried out every three weeks for three months (4 immunizations in total). After 3 months of rest for the mice and in order to select the mice presenting the best immune response, their antibodies were analyzed with a first test. In this test, the murine antibodies collected during the immunization protocol were captured by a first murine anti-antibody antibody (Aff ⁇ niPure Goat Anti-Mouse IgG + IgM (H+L); Jackson Immunoresearch LABORATORIES) immobilized on the wall of the well of a microtiter plate, by incubating for 4 hours at room temperature with gentle agitation.
• a first murine anti-antibody antibody Aff ⁇ niPure Goat Anti-Mouse IgG + IgM (H+L); Jackson Immunoresearch LABORATORIES
• Ellman's medium comprises a mixture of 7.5 10-4 M acetylthiocholine iodide (enzyme substrate) and 2.5 10-4 M 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) (reagent for the colorimetric measurement of thiol) in a 0.1 M phosphate buffer, pH 7.4.
• Enzyme activity was expressed in Ellman units (EU).
• EU Ellman units
• One EU is defined as the amount of enzyme producing an increase in absorbance of one unit for 1 min in 1 ml of medium, for an optical path length of 1 cm: it corresponds to approximately 8 ng of enzyme.
• mice having the best immune response received new intravenous injections of immunogen A: 50 ⁇ g of product/mouse, once a day for three days. After two days of rest, they were sacrificed and their splenocytes (spleen cells) were hybridized with NSI murine myeloma cells in order to obtain hybridomas.
• Test 1 In this test, the antibodies present in the culture supernatants are captured by a first anti-murine antibody antibody immobilized on the wall of the wells of a microtiter plate. Incubation is carried out for 4 hours at room temperature with stirring. After washing, tracer A NH-biotin is added to each well. After incubation at 4° C. overnight and after washing, streptavidin-G4 is added to reveal the presence of the A NH tracer and therefore of non-hydrolyzed anti-carbapenem antibodies.
• Test 2 In this test, the antibodies present in the culture supernatants are captured by a first anti-murine antibody antibody immobilized on the wall of wells of a microtiter plate. Incubation is carried out for 4 hours at room temperature with stirring. After washing, AH tracer is added to each well. After incubation at 4° C. overnight and after washing, streptavidin-G4 is added to reveal the presence of the AH tracer and therefore the presence of hydrolyzed anti-carbapenem antibodies.
• Test 3 In this test, tracer A NH is placed in competition with non-hydrolyzed meropenem with respect to recognition by the specific antibodies present in the culture supernatants. To do this, the antibodies present in the culture supernatants are captured by a first anti-murine antibody antibody immobilized on the wall of the wells of a microtitration plate. Incubation is carried out for 4 hours at room temperature with stirring. After washing, tracer A NH and non-hydrolyzed meropenem are added to each well. After incubation at 4° C. overnight and after washing, streptavidin-G4 is added to reveal the presence of the A NH tracer.
• Test 4 In this test, tracer A NH is placed in competition with hydrolyzed meropenem at the same concentration as the non-hydrolyzed meropenem used in test 3, with respect to recognition by the specific antibodies present in the supernatants of culture. To do this, the antibodies present in the culture supernatants are captured by a first anti-murine antibody antibody immobilized on the wall of the wells of a microtitration plate. Incubation is carried out for 4 hours at room temperature with stirring. After washing, tracer A NH and hydrolyzed meropenem are added to each well. After incubation at 4° C. overnight and after washing, streptavidin-G4 is added to reveal the presence of the A NH tracer.

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