Classifications

• • 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
  • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
  • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
• • 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
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/914—Hydrolases (3)
  • G01N2333/978—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
  • G01N2333/986—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in cyclic amides (3.5.2), e.g. beta-lactamase (penicillinase, 3.5.2.6), creatinine amidohydrolase (creatininase, EC 3.5.2.10), N-methylhydantoinase (3.5.2.6)

Definitions

• the present disclosure relates to methods and antibodies for detecting bacteria that are resistant to antibiotics and/or compounds which cause resistance of bacteria to antibiotics.
• Antibiotics are indispensable for treating microbial infections.
• the increasing occurrence of bacteria that are resistant to antibiotics has become a worldwide threat to effective medical treatment.
• Carbapenems are a group of beta lactam antibiotics. Resistant bacteria occur, but acquired carbapenem resistance is currently less common than other ⁇ -lactam resistance phenotypes. The use of these antibiotics should thus be reduced as much as possible to prevent bacteria from adapting to the selective pressure caused by exposure to carbapenem compounds and to ensure their future efficacy. Infections with bacteria that are resistant to carbapenems are usually hard to treat because drugs that then still can be used are rather ineffective and toxic. The need to control and to reverse the spread of such bacteria requires rapid diagnostic methods. Therefore, methods have been developed to detect carbapenem-resistant bacteria as this will allow the medical practitioner to timely escalate treatment and/or to take necessary infection control measures such as isolating affected patients. However, some of these tests may require cultivation, i.e. incubation over at least 24 hrs which hinders and delays treatment and infection control measures.
• antibiotics-resistant bacteria and more specifically carbapenem-resistant bacteria can be detected by anti-NDM, anti-KPC, anti-VIM, and/or anti-OXA-48 antibodies or binding fragments thereof which specifically recognize their cognate antigens NDM, KPC, VIM, and/or OXA-48 respectively.
• the data provided by this application further suggest that not only carbapenem-resistant bacteria can be detected, but also carbapenemases as such, for example if such carbapenemases have been secreted by carbapenem-resistant bacteria and thus are comprised in e.g. a bodily sample or if carbapenem-resistant bacteria have been subjected to e.g. denaturing conditions such as disinfection and/or decontamination.
• One aspect thus relates to a method of detecting at least one carbapenemase in a sample or carbapenem-resistant bacteria in a sample comprising at least the following steps:
• detection of said at least one carbapenemase and/or carbapenem-resistant bacteria is achieved by use of at least one antibody or binding fragment thereof, which specifically recognizes said at least one carbapenemase.
• the method can be performed such that at least two, three, or all four carbapenemases selected from the group consisting of NDM, KPC, VIM, and OXA-48 are detected and wherein each of said at least two, three, or all four carbapenemases selected from the group consisting of NDM, KPC, VIM, and OXA-48 are detected by use of at least one antibody or binding fragment thereof, which specifically recognizes said carbapenemase.
• Such antibodies or binding fragments thereof may be an Fab, an F(ab')2, an Fv, a single-chain antibody fragment, a diabody, a multispecific antibody, a bibody, a tribody, a variant or derivative of any one of these antibodies or binding fragments.
• Antibodies or binding fragments thereof may be monoclonal or polyclonal antibodies or binding fragments thereof. Monoclonal antibodies or binding fragments thereof are considered specifically in the context of the embodiments described hereinafter.
• Another aspect relates to anti-NDM-, anti-KPC-, anti-VIM-, and/or anti-OXA-48 antibodies or binding fragments thereof which can be used for performing the aforementioned method.
• An anti-NDM antibody or binding fragment thereof may comprise
• anti-NDM antibody or binding fragments thereof may comprise
• • at least a light chain variable sequence comprising a CDR1 of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or of a sequence, which is at least 70% identical thereof, a CDR2 of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or of a sequence, which is at least 70%> identical thereof, and a CDR3 of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or of a sequence, which is at least 70%> identical thereof.
• Selected anti-NDM antibodies or binding fragments thereof may comprise
• anti-NDM antibody or binding fragments thereof may comprise
• anti-KPC antibodies or binding fragments thereof may comprise
• Selected anti-KPC antibodies or binding fragments thereof may comprise
• anti-KPC antibodies or binding fragments thereof may comprise
• -VIM antibody or binding fragment thereof may comprise
• anti-VIM antibodies or binding fragments thereof may comprise
• • at least a light chain variable sequence comprising a CDRl of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, or of a sequence, which is at least 70% identical thereof, a CDR2 of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, or of a sequence, which is at least 70% identical thereof, and a CDR3 of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, or of a sequence, which is at least 70%> identical thereof.
• Selected anti-VIM antibodies or binding fragments thereof may comprise
• ⁇ at least a heavy chain variable sequence of SEQ ID NO: 20, or a sequence which is at least about 70% identical thereof, and at least a light chain variable sequence of SEQ ID NO: 24 or a sequence, which is at least about 70%> identical thereof.
• anti-VIM antibodies or binding fragments thereof may comprise
• An anti-OXA-48 antibody or binding fragment thereof may comprise
• anti-OXA-48 antibody or binding fragments thereof may comprise
• • at least a light chain variable sequence comprising a CDRl of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32, or of a sequence, which is at least 70% identical thereof, a CDR2 of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32, or of a sequence, which is at least 70% identical thereof, and a CDR3 of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32, or of a sequence, which is at least 70%> identical thereof.
• Selected anti- OXA-48 antibodies or binding fragments thereof may comprise
• anti-OXA-48 antibodies or binding fragments thereof may comprise
• Such antibodies or binding fragments thereof can be used in method for detecting
• carbapenemases and /or carbapenem-resistant bacteria by analyzing a sample from a human or animal being suspected to suffer from a bacterial colonization and/or infection.
• the sample can be subjected to denaturing conditions before the detection of NDM, KPC, VIM, and/or OXA-48 is performed.
• the methods may be implemented in specific assay formats such as lateral flow assays, dipstick assays and other ready-to-use assay formats.
• Another aspect relates to devices which are configured to allow performing methods as described above and hereinafter, i.e. devices which allow detecting at least one
• One embodiment thus relates to devices which allow utilizing specific assay formats such as lateral flow assays, dipstick assays and other ready-to-use assay formats.
• Such devices may comprise the antibodies as described herein and optionally further control antibodies, agents etc.
• kits of parts comprising the antibodies or binding fragment thereof as described herein and optionally one or more reagents such as further control antibodies, agents etc. and/or instructions, e.g. for performing the methods described herein.
• reagents such as further control antibodies, agents etc. and/or instructions, e.g. for performing the methods described herein.
• For detecting a carbapenemase selected from the group consisting of NDM, KPC, VIM, and OXA-48 one may consider using mixtures of anti-NDM, anti-KPC, anti-VIM, and/or anti- OXA-48 antibodies or binding fragments thereof, wherein a single monoclonal antibody or binding fragment thereof is used for each of NDM, KPC, VIM, and OXA-48 respectively.
• a carbapenemase selected from the group consisting of NDM, KPC, VIM, and OXA-48 may be achieved for each carbapenemase by use of at least one anti- NDM-, anti-KPC-, anti-VIM-, and/or anti-OXA-48 antibody or binding fragment thereof as described above and hereinafter, some embodiments such as the aforementioned lateral flow assays, dipstick assays, devices, and/or kits consider use of at least two antibodies or binding fragments for each of NDM, KPC, VIM, and OXA-48 thereof with one antibody as capture antibody and the other antibody as detection antibody.
• Such antibody combinations may also be used for any kind of assay format where the capture antibody is part of a stationary phase and where the detector antibody is part of a mobile phase.
• Any of the above mentioned anti- NDM, anti-KPC, anti-VIM, and/or anti-OXA-48 antibody or binding fragments thereof may be used as capture antibody and any of the remaining anti-NDM, anti-KPC, anti-VIM, and/or anti-OXA-48 antibody or binding fragment thereof may be used as a detector antibody.
• Monoclonal antibodies or binding fragments thereof are specifically considered in the context of such embodiments.
• a further embodiment therefore also relates to a combination of anti-NDM antibodies or binding fragments thereof and their use for performing the aforementioned methods, wherein an anti-NDM capture antibody or binding fragment thereof comprises at least a heavy chain variable sequence of SEQ ID NO: 2, or a sequence which is at least about 70% identical thereof, and at least a light chain variable sequence of SEQ ID NO: 6 or a sequence, which is at least about 70% identical thereof; and wherein an anti-NDM detector antibody or binding fragment thereof comprises at least a heavy chain variable sequence of SEQ ID NO: 3, or a sequence which is at least about 70% identical thereof, and at least a light chain variable sequence of SEQ ID NO: 7 or a sequence, which is at least about 70%> identical thereof.
• the anti-NDM capture antibody (e.g. in the specific example SEQ ID NO: 2 and 6) may also be used as the anti-NDM detector antibody and that the anti-NDM detector antibody (e.g. in the specific example SEQ ID NO: 3 and 7) may also be used as the anti-NDM capture antibody.
• a yet another embodiment relates to a combination of anti-NDM antibodies or binding fragments thereof and their use for performing the aforementioned methods, wherein an anti- NDM capture antibody or binding fragment thereof comprises at least a heavy chain variable sequence with a CDRl of SEQ ID NO: 2, or a sequence which is at least about 70% identical thereof, a CDR2 of SEQ ID NO: 2, or a sequence which is at least about 70% identical thereof, and a CDR3 of SEQ ID NO: 2, or a sequence which is at least about 70% identical thereof, and at least a light chain variable sequence with a CDRl of SEQ ID NO: 6 or a sequence, which is at least about 70% identical thereof, a CDR2 of SEQ ID NO: 6 or a sequence, which is at least about 70% identical thereof, and a CDR3 of SEQ ID NO: 6 or a sequence, which is at least about 70% identical thereof; and wherein an anti-NDM detector antibody or binding fragment thereof comprises at least a heavy chain variable sequence with a CDRl of SEQ ID NO
• the anti-NDM capture antibody (e.g. in the specific example CDRs 1 , 2, and 3 of SEQ ID NO: 2 and 6) may also be used as the anti- NDM detector antibody and that the anti-NDM detector antibody (e.g. in the specific example CDRs 1 , 2, and 3 of SEQ ID NO: 3 and 7) may also be used as the anti-NDM capture antibody.
• an anti- KPC capture antibody or binding fragment thereof comprises at least a heavy chain variable sequence of SEQ ID NO: 9, or a sequence which is at least about 70%> identical thereof, and at least a light chain variable sequence of SEQ ID NO: 13 or a sequence, which is at least about 70% identical thereof; and wherein an anti-KPC detector antibody or binding fragment thereof comprises at least a heavy chain variable sequence of SEQ ID NO: 1 1 , or a sequence which is at least about 70% identical thereof, and at least a light chain variable sequence of SEQ ID NO: 15 or a sequence, which is at least about 70%> identical thereof.
• the anti-KPC capture antibody (e.g. in the specific example SEQ ID NO: 9 and 13) may also be used as the anti-KPC detector antibody and that the anti-KPC detector antibody (e.g. in the specific example SEQ ID NO: 1 1 and 15) may also be used as the anti- KPC capture antibody.
• an anti- KPC capture antibody or binding fragment thereof comprises at least a heavy chain variable sequence with a CDRl of SEQ ID NO: 9, or a sequence which is at least about 70%> identical thereof, a CDR2 of SEQ ID NO: 9, or a sequence which is at least about 70% identical thereof, and a CDR3 of
• SEQ ID NO: 9 or a sequence which is at least about 70% identical thereof, and at least a light chain variable sequence with a CDRl of SEQ ID NO: 13 or a sequence, which is at least about 70% identical thereof, a CDR2 of SEQ ID NO: 13 or a sequence, which is at least about 70% identical thereof, and a CDR3 of SEQ ID NO: 13 or a sequence, which is at least about 70% identical thereof; and wherein an anti-KPC detector antibody or binding fragment thereof comprises at least a heavy chain variable sequence with a CDRl of SEQ ID NO: 1 1 , or a sequence which is at least about 70% identical thereof, a CDR2 of SEQ ID NO: 1 1 , or a sequence which is at least about 70% identical thereof, and a CDR3 of SEQ ID NO: 1 1 , or a sequence which is at least about 70% identical thereof, and at least a light chain variable sequence with a CDRl of SEQ ID NO: 15 or a sequence, which is at least about 70%> identical thereof,
• the anti-KPC capture antibody (e.g. in the specific example CDRs 1 , 2, and 3 of SEQ ID NO: 9 and 13) may also be used as the anti-KPC detector antibody and that the anti-VIM detector antibody (e.g. in the specific example CDRs 1 , 2, and 3 of SEQ ID NO: 1 1 and 15) may also be used as the anti-KPC capture antibody.
• a further embodiments relates to a combination of anti-VIM antibodies or binding fragments thereof and their use for performing the aforementioned methods, wherein an anti-VIM capture antibody or binding fragment thereof comprises at least a heavy chain variable sequence of SEQ ID NO: 17, or a sequence which is at least about 70% identical thereof, and at least a light chain variable sequence of SEQ ID NO: 21 or a sequence, which is at least about 70% identical thereof; and wherein an anti-VIM detector antibody or binding fragment thereof comprises at least a heavy chain variable sequence of SEQ ID NO: 19, or a sequence which is at least about 70% identical thereof, and at least a light chain variable sequence of SEQ ID NO: 23 or a sequence, which is at least about 70%> identical thereof.
• the anti-VIM capture antibody (e.g. in the specific example SEQ ID NO: 17 and 21) may also be used as the anti-VIM detector antibody and that the anti-VIM detector antibody (e.g. in the specific example SEQ ID NO: 19 and 23) may also be used as the anti- VIM capture antibody.
• an anti- VIM capture antibody or binding fragment thereof comprises at least a heavy chain variable sequence with a CDRl of SEQ ID NO: 17, or a sequence which is at least about 70%> identical thereof, a CDR2 of SEQ ID NO: 17, or a sequence which is at least about 70% identical thereof, and a CDR3 of SEQ ID NO: 17, or a sequence which is at least about 70% identical thereof, and at least a light chain variable sequence with a CDRl of SEQ ID NO: 21 or a sequence, which is at least about 70% identical thereof, a CDR2 of SEQ ID NO: 21 or a sequence, which is at least about 70% identical thereof, and a CDR3 of SEQ ID NO: 21 or a sequence, which is at least about 70% identical thereof; and wherein an anti-VIM detector antibody or binding fragment thereof comprises at least a heavy chain variable sequence with a CDRl of SEQ ID NO:
• the anti-VIM capture antibody (e.g. in the specific example SEQ ID NO: 17 and 21) may also be used as the anti-VIM detector antibody and that the anti-VIM detector antibody (e.g. in the specific example SEQ ID NO: 19 and 23) may also be used as the anti-VIM capture antibody.
• a further embodiment relates to a combination of anti-OXA-48 antibodies or binding fragments thereof and their use for performing the aforementioned methods, wherein an anti- OXA-48 capture antibody or binding fragment thereof comprises at least a heavy chain variable sequence of SEQ ID NO: 27, or a sequence, which is at least about 70%> identical thereof, and at least a light chain variable sequence of SEQ ID NO: 31 or a sequence, which is at least about 70% identical thereof; and wherein an anti-OXA-48 detector antibody or binding fragment thereof comprises at least a heavy chain variable sequence of SEQ ID NO: 25, or a sequence, which is at least about 70% identical thereof, and at least a light chain variable sequence of SEQ ID NO: 29 or a sequence, which is at least about 70%> identical thereof.
• the anti-OXA-48 capture antibody (e.g. in the specific example CDRs 1 , 2, and 3 of SEQ ID NO: 27 and 31) may also be used as the anti-OXA-48 detector antibody and that the anti-OXA-48 detector antibody (e.g. in the specific example CDRs 1 , 2, and 3 of SEQ ID NO: 25 and 29) may also be used as the anti-OXA-48 capture antibody.
• an anti-OXA-48 capture antibody or binding fragment thereof comprises at least a heavy chain variable sequence with a CDRl of SEQ ID NO: 27, or a sequence which is at least about 70%> identical thereof, a CDR2 of SEQ ID NO: 27, or a sequence which is at least about 70% identical thereof, and a CDR3 of SEQ ID NO: 27, or a sequence which is at least about 70% identical thereof, and at least a light chain variable sequence with a CDRl of SEQ ID NO: 31 or a sequence, which is at least about 70% identical thereof, a CDR2 of SEQ ID NO: 31 or a sequence, which is at least about 70% identical thereof, and a CDR3 of SEQ ID NO: 31 or a sequence, which is at least about 70% identical thereof; and wherein an anti-OXA-48 detector antibody or binding fragment thereof comprises at least a heavy chain variable sequence with a
• the anti-OXA-48 capture antibody (e.g. in the specific example CDRs 1 , 2, and 3 of SEQ ID NO: 27 and 31) may also be used as the anti-OXA-48 detector antibody and that the anti-OXA-48 detector antibody (e.g. in the specific example CDRs 1 , 2, and 3 of SEQ ID NO: 25 and 29) may also be used as the anti-OXA-48 capture antibody.
• said detector antibody or binding fragment thereof can be coupled to a detectable label.
• anti-NDM-, anti-KPC-, anti-VIM-, and/or anti-OXA-48 antibodies or binding fragments thereof may be used such that
• a) detection of NDM has a specificity of at least about 85%, at least about 90%, at least about 95%, at least about 96%>, at least about 97%, at least about 99% or at least about 99% and a sensitivity of at least about 80%, at least about 90%, at least about 95%), at least about 96%, at least about 97%, at least about 99% or at least about 99%,
• KPC has a specificity of at least about 85%, at least about 90%, at least about 95%), at least about 96%, at least about 97%, at least about 99% or at least about 99% and a sensitivity of at least about 80%, at least about 90%, at least about 95%), at least about 96%, at least about 97%, at least about 99% or at least about
• VIM has a specificity of at least about 85%, at least about 90%, at least about 95%), at least about 96%, at least about 97%, at least about 99% or at least about 99% and a sensitivity of at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 99% or at least about 99%,
• OXA-48 has a specificity of at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 99% or at least about 99% and a sensitivity of at least about 80%, at least about 90%, at least about 95%), at least about 96%, at least about 97%, at least about 99% or at least about 99%.
• the methods disclosed herein may be used for analyzing bodily samples include swabs and bodily fluids such as blood samples, urine samples, sputum, exudates, transudation, stool, liquid stool, etc. Such bodily samples may be tested without prior enrichment steps such as cultivation.
• the methods disclosed herein may be explicitly useful for detecting colonization or occult or asymptomatic infections in a subject without a clinically diagnosed condition or infection, for example in a screening procedure.
• the method disclosed herein may be used to screen healthy subjects who might have been exposed to carbapenemase-positive bacteria, who might have had contact to colonized/infected subjects etc., or who might be subjected to screening or infection control measures for any other reason.
• the methods disclosed herein further allow detecting the presence of a carbapenemase as such.
• carbapenemase as such.
• carbapenem-resistant bacteria was secreted by carbapenem-resistant bacteria and ultimately be found in a sample, which would not comprise the carbapenem-resistant bacteria, the methods described may still reveal the presence of carbapenemases.
• carbapenem-resistant bacteria from an individual, which suffers from an undetected infection with carbapenem-resistant bacteria would have found their way into liquids that would subsequently form the basis of a swap sample. Even though the carbapenem-resistant bacteria may not be alive anymore in such a sample due to e.g.
• the carbapenemase(s) may still be detected.
• Such information will be valuable to e.g. a medical practitioner as it would signal the danger of potential contamination of the hospital setting and/or of past exposure of staff and other patients with carbapenem-resistant bacteria.
• Figure 1 schematically shows an ArrayTube® and its spots
• Figure 2 shows the results for identification of potential anti-OXA-48 antibodies using the ArrayTube® platform
• a “disease” in the sense of the disclosure is any condition of a subject, also referred to as patient, human or animal, that is altered in comparison to the condition of a subject which is regarded healthy.
• bacterial infection is to be understood according to its general meaning in the field of medicine (or veterinary medicine) and denotes a state in which one or more species of pathogenic bacteria are present in an organism and may have already or may subsequently lead to a clinical picture, which may be regarded as disease or unhealthy.
• a subject may also be infected, or colonized, with pathogenic bacteria, but does not show any change in the clinical picture or not show symptoms of a disease.
• Such an infection is denoted as colonization or occult or asymptomatic infection.
• the method disclosed herein may be particularly useful in detecting both, bacterial colonization and infection in a subject.
• a pathogenic or facultative pathogenic/opportunistic bacterium is present in a subject.
• detecting the presence of bacteria in a bodily sample such as a swab or a bodily fluid of a subject may be used to detect a bacterial colonization or infection of the subject from which the bodily sample was obtained.
• Diagnosing an infection denotes the determination of a specific disease of a subject by analyzing the symptoms, clinical condition and optionally also other factors, such as laboratory and microscopic indications.
• the method disclosed herein may be explicitly useful for diagnosing a pathogen infection, such as a bacterial infection, by detecting proteins with a specific enzymatic activity, namely carbapenemases, being indicative of an infection of the subject from a bodily sample of said subject.
• a “subject” or “patient” may be a human or non-human, e.g. animal organism.
• the methods described herein may be applicable to both human and veterinary bacterial infections.
• a subject is typically a living organism, the methods described herein may be used in post-mortem analyses as well.
• Typical subjects are "patients”, e.g. living humans or animals that are receiving medical care or treatment for a disease or condition. These subjects also include persons with no defined illness, being investigated for signs of pathology as well as healthy subjects subjected to screening as described above.
• sample includes any type of sample such as bodily samples, which can be analyzed by the methods described herein.
• the terms "bodily sample” is to be understood according to its meaning in the field of medicine and denotes any possible fluid which may be obtained from a subject, such as a human or animal. "Obtaining” or “to obtain” are to be understood according to their general meaning in the field of medicine and medical diagnostics and denote the retrieval or taking of a sample of from a subject, such as taking of a blood sample from a subject or a urinary or sputum or any other kind of e.g. bodily fluid probe of a subject depending on the bodily fluid which is to be obtained.
• Typical bodily samples include bodily fluids.
• Bodily fluids may be blood samples, urine samples, tears, lacrimal fluid, saliva, nasal fluid, sputum, ear fluid, genital fluid, breast fluid, milk, colostrum, placental fluid, amniotic fluid, perspirate, synovial fluid, ascites fluid, cerebrospinal fluid, bile, gastric fluid, aqueous humor, vitreous humor, gastrointestinal fluid, exudate, transudate, pus, pleural fluid, bronchoalveolar lavage, bronchial secretions, pericardial fluid, semen, upper airway fluid, peritoneal fluid, liquid stool, fluid harvested from a site of an infection or immune response or pooled fluids harvested from multiple collection sites.
• the blood sample may be a whole blood sample, plasma or serum sample or a blood sample unable to coagulate or laced with a substance preventing coagulation.
• a sample may be more readily analyzed following a fractionation or purification procedure, for example separation of whole blood into serum or plasma components.
• the sample of a bodily fluid may be a sputum sample.
• Other bodily samples may also include swabs from wounds, from intact skin and mucous membranes including oropharyngeal, nasal, rectal and genital/vaginal swabs.
• bodily samples may also include stool, fecal samples, tissue from biopsies, swabs or materials from synthetic implants such as catheters, pacemakers, endoprostheses, surgical screws etc. as well as tissue from necropsies, organ swabs and samples from post mortem analyses. Even though this is not considered necessary for the methods described herein, bodily samples may also be first cultivated in a medium suitable for growing bacteria. The bacterial cells of such cultures, cell extracts of such cultures or the culture supernatants may then be used as samples. However, as mentioned bodily samples may also be used without prior cultivation, e.g. either directly or after dilution or certain preparatory steps such as
• the methods described herein are not conducted on the human or animal body.
• the term "providing a sample” thus does not include the step of actively drawing a sample from the human or animal body. Rather, such samples have been obtained before by a medical practitioner and are then provided to conduct the methods described herein.
• bacteria or "bacterium” denote a microorganism and are to be understood according to their general meaning in the field of microbiology and biology. Both terms further include all microorganisms which are susceptible or have an acquired or intrinsic resistance to a treatment with a particular antibiotic. This includes typical pathogens but also bacteria that colonize the human body without causing symptomatic disease.
• susceptible to a treatment with an antibiotic denotes that the bacterium either does not grow/divide any longer after contact with the antibiotic at standardized concentrations or dies after contact with an antibiotic at standardized concentrations.
• carbapenem denotes a class of ⁇ -lactam antibiotics in which the sulfur atom in position 1 of the ⁇ -lactam structure has been replaced with a carbon atom, and an unsaturation has been introduced, thus having a molecular structure which renders them resistant to most ⁇ -lactamases, which have a broad spectrum of antibacterial activity.
• carbapenems are one of the antibiotics of last resort for many bacterial infections, such as Escherichia coli (E. coli) and Klebsiella pneumoniae.
• Carbapenems include for instance the approved antibiotics Imipenem, Meropenem,
• Bacteria, which are resistant to antibiotic treatment with carbapenems, are designated as "carbapenem-resistant bacteria". Such resistance can be mediated by so-called
• carbapenemases i.e. ⁇ -lactamases, which can hydro lyze the ⁇ -lactam ring of carbapenems and thus render these antibiotics ineffective.
• Such carbapenemases have been detected in various bacteria including Klebsiella pneumoniae and categorized in different classes, namely so-called Class A carbapenemase, Class B metallo-P-lactamases and Class D serine-carbapenemases (for review see Queenan and Bush (2007) Clin.l Microbiol. Rev. 20(3): 440-458).
• NDM has the GenBank number FN396876.1 and the protein number CAZ39946.1
• KPC has the GenBank number EU447304.1 and the protein number ACA34343.1
• VIM has the GenBank number Yl 8050.2 and the protein number CAB46686.1
• OXA-48 has the GenBank number AY236073.2 and the protein number AAP70012.1
• NDM refers to a carbapenemase of SEQ ID NO: 33 or a protein having
• KPC refers to a carbapenemase of SEQ ID NO: 34 or a protein having substantially the same enzymatic activity as a carbapenemase of SEQ ID NO: 34 and having a sequence identity of at least 70%, optionally at least 80%, optionally at least 90%>, optionally at least 95%, optionally at least 96%, optionally at least 97%, optionally at least 98%, or optionally at least 99% with SEQ ID NO: 34.
• VIM refers to a carbapenemase of SEQ ID NO: 35 or a protein having
• OXA-48 refers to a carbapenemase of SEQ ID NO: 36 or a protein having substantially the same enzymatic activity as a carbapenemase of SEQ ID NO: 36 and having a sequence identity of at least 70%, optionally at least 80%, optionally at least 90%, optionally at least 95%, optionally at least 96%, optionally at least 97%, optionally at least 98%, or optionally at least 99% with SEQ ID NO: 36.
• BLAST polynucleotide searches are performed with the BLASTn program.
• the "Max Target Sequences” box may be set to 100, the “Short queries” box may be ticked, the “Expect threshold” box may be set to 10 and the “Word Size” box may be set to 28.
• the scoring parameters the "Match/mismatch Scores” may be set to 1,-2 and the "Gap Costs” box may be set to linear.
• the "Low complexity regions” box may not be ticked, the "Species-specific repeats” box may not be ticked, the "Mask for lookup table only” box may be ticked, and the "Mask lower case letters” box may not be ticked.
• the "Max Target Sequences” box may be set to 100
• the "Short queries” box may be ticked
• the "Expect threshold” box may be set to 10
• the "Word Size” box may be set to 3.
• the scoring parameters the "Matrix” box may be set to "BLOSUM62”
• the "Gap Costs” Box may be set to "Existence: 11 Extension: 1”
• the “Compositional adjustments” box may be set to "Conditional compositional score matrix adjustment”.
• the "Low complexity regions” box may not be ticked
• the "Mask for lookup table only” box may not be ticked
• the "Mask lower case letters” box may not be ticked
• antibody is to be understood according to its common general meaning describing a polypeptide of Y-shaped form being made of two so-called heavy chains and two so-called light chains, with each heavy chain comprising a so-called variable region and three constant regions and each light chain comprising a so-called variable region and one constant region.
• anti-NDM antibody relates to antibodies, which specifically bind to NDM. This means that the antibody binds NDM with higher affinity than other antigens including bacterial beta-lactamases, optionally with a higher affinity of a factor of at least about 2, of at least about 5, of at least about 10, of at least about 50, of at least about 10 2 , of at least about 10 3 , of at least about 10 3 , of at least about 10 4 , of at least about 10 5 , or of at least about 10 6 .
• an "anti-NDM antibody” specifically binds to NDM, but will not detectably bind to other bacterial carbapenemases or other bacterial ⁇ -lactamases, if the same assays is chosen for detection of antibody-antigen interaction.
• anti-KPC antibody relates to antibodies, which specifically bind to NDM. This means that the antibody binds KPC with higher affinity than other antigens including bacterial beta-lactamases, optionally with a higher affinity of a factor of at least about 2, of at least about 5, of at least about 10, of at least about 50, of at least about 10 2 , of at least about 10 3 , of at least about 10 3 , of at least about 10 4 , of at least about 10 5 , or of at least about 10 6 .
• an "anti-KPC antibody” specifically binds to KPC, but will not detectably bind to other bacterial carbapenemases or other bacterial ⁇ -lactamases, if the same assays is chosen for detection of antibody-antigen interaction.
• anti-VIM antibody relates to antibodies, which specifically bind to VIM.
• the antibody binds VIM with higher affinity than other antigens including bacterial beta-lactamases, optionally with a higher affinity of a factor of at least about 2, of at least about 5, of at least about 10, of at least about 50, of at least about 10 2 , of at least about 10 3 , of at least about 10 3 , of at least about 10 4 , of at least about 10 5 , or of at least about 10 6 .
• an "anti-VIM antibody” specifically binds to VIM, but will not detectably bind to other bacterial carbapenemases or other bacterial ⁇ -lactamases, if the same assay is chosen for detection of antibody-antigen interaction.
• anti-OXA-48 antibody relates to antibodies, which specifically bind to OXA-48. This means that the antibody binds OXA-48 with higher affinity than other antigens including bacterial beta-lactamases, optionally with a higher affinity of a factor of at least about 2, of at least about 5, of at least about 10, of at least about 50, of at least about 10 2 , of at least about 10 3 , of at least about 10 3 , of at least about 10 4 , of at least about 10 5 , or of at least about 10 6 .
• an "anti-OXA-48 antibody” specifically binds to OXA-48, but will not detectably bind to other bacterial carbapenemases or other bacterial ⁇ -lactamases, if the same assay is chosen for detection of antibody-antigen interaction.
• anti-NDM-, anti-KPC-, anti-VIM-, and anti-OXA-48 or binding fragments thereof as described herein specifically bind to NDM, KPC, VIM, and OXA-48 respectively, they can simultaneously be used in the methods described herein allowing for parallel detection of the different carbapenemases. Detection may be achieved e.g. by using mixtures of monoclonal antibodies or binding fragments thereof and/or polyclonal antibodies or binding fragments thereof for each of NDM, KPC, VIM, and OXA-48 respectively or by using single
• anti-NDM-, anti-KPC-, anti-VIM-, and anti-OXA-48 antibodies may also interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibodies, and in particular, in the constant region of the antibody or fragment.
• Such binding which is not mediated by the variable regions of anti-NDM,- anti-KPC-, anti-VIM-, and anti- OXA-48 antibodies, is not understood as being indicative of non-specific binding to antigens other than NDM, KPC, VIM, and OXA-48.
• binding fragment of an antibody refers to a fragment of a full-length antibody which results e.g. from deletion of N-terminal or C-terminal amino acids of the full-length antibody and which maintains the capacity to bind the cognate antigen with about the same specificity and/or K D as the full-length antibody.
• anti-NDM-, anti-KPC-, anti-VIM-, and anti-OXA-48 antibodies are described herein as being defined by their CDRs, i.e. CDR1, CDR2, and CDR3 of the heavy and/or light chain variable region, this refers to the underlined sequences in SEQ ID Nos: 1 to 32 of Table 1.
• the first sequence underlined in the heavy or light chain variable region of SEQ ID NO: 1 (reading the sequence from the N-terminus to the C-terminus) is CDR1
• the second sequence underlined in the heavy or light chain variable region of SEQ ID NO: 1 is CDR 2
• the third sequence underlined in the heavy or light chain variable region of SEQ ID NO: 1 is CDR3.
• SEQ ID NO: 2-32 The same applies correspondingly to SEQ ID NO:
• Anti-NDM-, anti-KPC-, anti-VIM-, and anti-OXA-48 antibodies or binding fragments thereof may typically bind their cognate antigens NDM, KPC, VIM and OXA-48 with an affinity having a K D in the micromolar to low picomolar range.
• the K D may be in the range of about 0.1 * 10 -12 to about 1 * 10 -6 , optionally in the range of about 0.1 * 10 -12 to about 0.1 * 10 -7 , optionally in the range of about 0.1 * 10 -12 to about 10* 10 -9 , optionally in the range of about 0.1 * 10 -12 to about 1 * 10 -9 .
• the anti-NDM-, anti-KPC-, anti-VIM-, and anti-OXA-48 antibodies or binding fragments thereof will typically detect their cognate antigens at a concentration of the antigen of at least about 0.01 ng/ml, optionally at least about 0.02 ng/ml, optionally at least about 0.03 ng/ml, optionally at least about 0.04 ng/ml, optionally at least about 0.05 ng/ml, optionally at least about 0.06 ng/ml, optionally at least about 0.07 ng/ml, optionally at least about 0.08 ng/ml, optionally at least about 0.09 ng/ml, optionally at least about 0.1 ng/ml, optionally at least about 0.2 ng/ml, optionally at least about 0.3 ng/ml, optionally at least about 0.4 ng/ml, optionally at least about 0.5 ng/ml, optionally at least about 0.6 ng/ml, optionally at least about 0.7 ng/
• a Fab fragment consists of the VL, VH, CL and CHI domains.
• An F(ab') 2 fragment comprises two Fab fragments linked by a disulfide bridge at the hinge region.
• An Fv is the VL and VH domains of a single arm of an antibody.
• a scFv may comprise a V H region at the amino -terminal end and a V L region at the carboxy-terminal end.
• scFv may comprise a V L region at the amino -terminal end and a VH region at the carboxy-terminal end.
• VL and VH the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883).
• scFv single chain Fv
• a scFv may optionally further comprise a polypeptide linker between the heavy chain variable region and the light chain variable region.
• polypeptide linkers generally comprise between 1 and 50 amino acids, alternatively between 3 and 12 amino acids, alternatively 2 amino acids.
• An example of a linker peptide for linking heavy and light chains in a scFv comprises the 5 amino acid sequence Gly-Gly-Gly-Gly-Ser.
• Other examples comprise one or more tandem repeats of this sequence (for example, a polypeptide comprising two to four repeats of Gly-Gly-Gly-Gly-Ser) to create linkers.
• Multispecific antibodies include diabodies.
• Diabodies are bivalent antibodies in which V H and V L domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., EP 404,097; WO 93/11161; Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444- 6448 and Poljak et a/ ⁇ (1994) Structure 2: 1121-1123). Diabodies can be bispecific or monospecific.
• bispecific Fab-scFv (“bibody”) and trispecific Fab-(scFv)(2) (“tribody”) are described in Schoonjans et al. (2000) J. Immunol. 165: 7050-7057 and Willems et al. (2003) J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 786: 161-176.
• a scFv molecule is fused to one or both of the VL-CL (L) and VH-CHi (Fd) chains, e.g., to produce a tribody two scFvs are fused to C-term of Fab while in a bibody one scFv is fused to C-term of Fab.
• a variant of an antibody, a binding fragment thereof, a Fab, etc. comprises one or more amino acid sequence substitutions, deletions or insertions that have the same or substantially the same affinity and specificity of epitope binding as one or more of the exemplary antibodies, fragments and sequences disclosed herein.
• a variant of an antibody may result from one or more changes to an anti-NDM antibody having a heavy chain variable region of SEQ ID NO: 1 and a light chain variable region of SEQ ID NO: 5. Substitutions can be conservative or non-conservative substitutions.
• Variants can thus encompass any of the e.g. specifically mentioned amino acid sequences of the light or heavy chains with one or more conservative substitutions ⁇ e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative substitutions).
• Consideration for selecting conservative substitutions include the context in which any particular amino acid substitution is made, the hydrophobicity or polarity of the side-chain, the general size of the side chain, and the pK value of side-chains with acidic or basic character under physiological conditions. For example, lysine, arginine, and histidine are often suitably substituted for each other.
• amino acids frequently suitably substituted for each other include, but are not limited to, the group consisting of glutamic and aspartic acids; the group consisting of phenylalanine, tyrosine, and tryptophan; and the group consisting of serine, threonine, and, optionally, tyrosine.
• glutamic and aspartic acids include glutamic and aspartic acids; the group consisting of phenylalanine, tyrosine, and tryptophan; and the group consisting of serine, threonine, and, optionally, tyrosine.
• Derivatives include antibodies, binding fragments thereof, variants, Fab, etc. which have been chemically modified. Examples include covalent attachment of one or more polymers, such as water soluble polymers, N-linked, or O-linked carbohydrates, sugars, phosphates, and/or other such molecules such as detectable labels such as fluorophores.
• polymers such as water soluble polymers, N-linked, or O-linked carbohydrates, sugars, phosphates, and/or other such molecules such as detectable labels such as fluorophores.
• sensitivity when used in the context of evaluating results of the methods described herein relates to the proportion of subjects known to have a bacterial infection with carbapenemase-resistant bacteria, who will test positive for it. Mathematically, this can be expressed as:
• Sensitivity (number of true positives)/((number of true positives)+(number of false negatives))
• the methods as described herein allow for a specificity of at least about 85%, at least about 90%), at least about 95%, at least about 96%>, at least about 97%, at least about 99% or at least about 99%.
• the methods as described herein further allow for a sensitivity of at least about 80%), at least about 90%>, at least about 95%, at least about 96%, at least about 97%, at least about 99% or at least about 99%.
• the values mentioned for specificity and sensitivity may be combined in all variations.
• anti-NDM, anti-KPC, anti-VIM, and anti-OXA-48 antibodies described herein can be used to detect carbapenemases and/or carbapenem-resistant bacteria under denaturing conditions. Denaturing conditions due to thermal or chemical stress lead to a destruction of the three-dimensional structure of NDM, KPC, VIM and OXA-48.
• Denaturing condition thus can include subjecting the sample to be tested to e.g. temperatures > 90° C for ⁇ 5 min, e.g. to - 99° C for 5, 6, 7, 8, 9, or 10 min. Denaturing conditions also include subjecting the sample to be tested to microbicidal agents such as, e.g. Bacillol®, Sterilium®, etc., other disinfectants or to several such treatments combined.
• microbicidal agents such as, e.g. Bacillol®, Sterilium®, etc., other disinfectants or to several such treatments combined.
• NDM, KPC, VIM and/or OXA-48, the anti-NDM, anti-KPC, anti-VIM, and/or anti-OXA-48 antibodies can be labeled with a one or more detectable marker or label for performing the detection reaction.
• the detection can also take place by e.g. secondary antibodies which bind to the anti-NDM, anti-KPC, anti-VIM, and/or anti- OXA-48 antibodies and which may be detectably labeled.
• one or more detectable label refers to any compound or moiety that comprises one or more appropriate chemical substances or enzymes, which directly or indirectly generate a detectable compound or signal in a chemical, physical or enzymatic reaction. Such a label may thus be necessary for or will facilitate detection of the reporter compound of interest, i.e. NDM, KPC, VIM and/or OXA-48. As used herein, the term is to be understood to include both detectable labels as such (also referred to as “markers”) as well as any compounds coupled to one or more such detectable markers.
• moieties interfering with the generation of a detectable signal by a label may also belong to the detectable labels.
• Detectable markers or labels include any compound, which directly or indirectly generates a detectable compound or signal in a chemical, physical or enzymatic reaction. Labeling can be achieved by methods well known in the art (see, for example, Sambrook, J. et al., supra; and Lottspeich, F., and Zorbas H., supra).
• the labels can be selected inter alia from fluorescent labels, enzyme labels, colored labels, chromogenic labels, luminescent labels, radioactive labels, haptens, biotin, metal complexes, metals, and colloidal gold or silver. All these types of labels are well established in the art.
• An example of a physical reaction that is mediated by such labels is the emission of fluorescence or phosphorescence upon irradiation or excitation.
• Alkaline phosphatase, horseradish peroxidase, ⁇ -galactosidase, and ⁇ -lactamase are examples of enzyme labels, which catalyze the formation of chromogenic reaction products.
• the detectable labels are fluorescent labels. Numerous fluorescent labels are well established in the art and commercially available from different suppliers(see, for example, The Handbook - A Guide to Fluorescent Probes and Labeling Technologies, 10th ed. (2006), Molecular Probes,
• Direct detectable markers or labels that may be conjugated to the anti-NDM-, anti-KPC-, anti- VIM-, and/or anti-OXA-48 antibodies thus include fluorescent or luminescent tags, metals, dyes, radionuclides, and the like, attached to the antibody.
• Indirect markers or labels may thus include various enzymes well known in the art, such as the aforementioned alkaline phosphatase, horseradish peroxidase and the like.
• Antibodies attached to a second molecule, such as a detectable label are referred to herein also as contextantibody conjugates".
• detection takes place by labeling the antigens with one or more detectable markers as described.
• a sample suspected to comprise the antigen may be fluorescently labeled or with other labels such as biotin.
• the interaction between the antigens and the anti-NDM, anti-KPC, anti-VIM, and/or anti-OXA-48 antibodies can then be detected.
• Label free detection is also considered.
• the interaction between the antigens and the anti- NDM, anti-KPC, anti-VIM, and/or anti-OXA-48 antibodies may be detected e.g. mass spectrometry analysis.
• Such approaches include competitive and non-competitive assays. These assays can utilize e.g. labeled anti-NDM, anti-KPC, anti-VIM, and/or anti-OXA-48 antibodies in various sandwich, competitive, or non-competitive assay formats, to generate a signal that is related to the presence or amount of NDM, KPC, VIM and/or OXA-48.
• labeled anti-NDM, anti-KPC, anti-VIM, and/or anti-OXA-48 antibodies in various sandwich, competitive, or non-competitive assay formats, to generate a signal that is related to the presence or amount of NDM, KPC, VIM and/or OXA-48.
• immobilized anti-NDM, anti-KPC, anti-VIM, and/or anti-OXA-48 antibodies for NDM, KPC, VIM and/or OXA-48 is specifically contemplated by the embodiments described herein.
• anti-NDM-, anti-KPC-, anti-VIM-, and/or anti-OXA-48 antibodies could be immobilized onto a variety of solid supports, such as magnetic or chromatographic matrix particles, the surface of an assay place (such as microtiter wells), pieces of a solid substrate or membrane (such as plastic, nylon, metals, glass, paper), on an array and the like, by a variety of means known in the art.
• solid supports such as magnetic or chromatographic matrix particles, the surface of an assay place (such as microtiter wells), pieces of a solid substrate or membrane (such as plastic, nylon, metals, glass, paper), on an array and the like, by a variety of means known in the art.
• Preparation of solid phases and detectable label antibody conjugates can comprise the use of chemical cross-linkers.
• Cross-linking reagents contain at least two reactive groups, and are divided generally into homo functional cross-linkers (containing identical reactive groups) and heterofunctional cross-linkers (containing non-identical reactive groups).
• Homobifunctional cross-linkers that couple through amines, sulfhydryls or react non- specifically are available from many commercial sources.
• Maleimides, alkyl and aryl halides, alpha-haloacyls and pyridyl disulfides are thiol reactive groups.
• Maleimides, alkyl and aryl halides, and alpha-haloacyls react with sulfhydryls to form thiol ether bonds, while pyridyl disulfides react with sulfhydryls to produce mixed disulfides.
• the pyridyl disulfide product is cleavable. Imdidoesters are also useful.
• Heterobifunctional cross-linkers possess two or more different reactive groups that allow for sequential conjugations minimizing undesirable polymerization or self-conjugation.
• Heterobifunctional reagents are also used when modification of amines is problematic.
• Other moieties such as sulfhydryls, carboxyls, phenols and carbohydrates may be more appropriate targets.
• array formats like lateral flow assay, dipstick-based assay, bead-based assays, array-based assay, PIMA®-based assays, Triage®-based assays and the like are considered.
• the capture antibody will usually be attached to a solid support forming a stationary phase.
• the sample to be tested will then be moved by a mobile (liquid) phase comprising the detector antibody with a detectable label across the stationary phase.
• the antigen within the sample will bind to the detector antibody, which when encountering the capture antibody, will form a sandwich format that can then be detected.
• Such a set up may be used for lateral flow and dipstick assays.
• Such set ups can be configured to detect the presence of NDM, KPC, VIM or OXA-48 in a sample or to detect various combinations or all of NDM, KPC, VIM and OXA-48 in a sample.
• the set up to detect various combinations or all of NDM, KPC, VIM and OXA-48 in a sample can be useful to detect the simultaneous presence of different carbapenem-resistant bacteria.
• a set up for detecting the presence of NDM, KPC, VIM or OXA-48 in a sample can include the use of just an anti-NDM, anti-KPC, anti-VIM, or anti-OXA-48 antibodies in e.g. the form of capture and detection antibodies.
• a set up for detecting the presence of NDM, KPC, VIM and OXA-48 in a sample can include the simultaneous use of anti-NDM, anti-KPC, anti-VIM, and anti-OXA-48 antibodies in e.g. the form of capture and detection antibodies with the anti-NDM, anti-KPC, anti-VIM, and anti-OXA-48 antibodies being immobilized at different sites on the support or stationary phase.
• Assays can be thoroughlyrapid", which, as used herein, refers to an assay that allows to obtain a result or report within about less than about 6 hours, less than about 4 hours, less than about 3 hours, less than about 2 hours, less than about 1 hour, less than about 50 min, less than about 40 min, less than about 30 min, less than about 20 min, less than about 10 min, less than about 9 min, less than about 8 min, less than about 7 min, less than about 6 min, less than about 5 min, less than about 4 min, less than about 3 min, less than about 2 min, or less than about 1 min.
• Detection in less than about 5 min, such as less than about 4 min, less than about 3 min, less than about 2 min, or less than about 1 min may be achieved with e.g. lateral flow assays.
• lateral flow assays Embodiments will now be described with respect to specific examples, which are however not to be construed as limiting the invention in any way.
• Example 1 Identification of anti-NDM-, anti-KPC-, anti-VIM-, and/or anti-OXA-48 antibodies
• potential anti-NDM-, anti-KPC-, anti-VIM-, and/or anti-OXA-48 antibodies were spotted onto the array at concentrations of 0.5 mg/ml, 0.2 mg/ml, 0.1 mg/ml, 0.05 mg/ml and 0.01 mg/ml for each antibody.
• antibodies were spotted at concentrations of 0.2 mg/ml, 0.1 mg/ml, 0.05 mg/ml and 0.01 mg/ml and BSA with BSA being present in an amount to give a final concentration of antibody and BSA of 0.5 mg/ml.
• the antigens were then added at concentration of 100 pg/ml in buffer and 0.25% Triton and incubating for 30 min at 300 rpm. After incubation a washing step with 150 ⁇ l buffer for 5 min at 400 rpm was performed.
• FIG. 1 A general lay out of the array is shown in Figure 1.
• Table 2 displays spotting for potential anti-OXA-48 antibodies.
• Table 3 depicts the binding of the various antibody combinations to their antigen.
• Figure 2 shows the read out of the array for potential anti- OXA-48 antibodies. The same approach was taken for anti-NDM-, anti-KPC-, and anti- VIM antibodies.
• the 186 strains were cultivated, cells were tested by the combination of anti-OXA-48 antibody 1461(SEQ ID Nos: 27 and 31) as capture antibody and anti-OXA-48 antibody 1281 (SEQ ID Nos: 25 and 29) as detector antibody using the Array Tube® platform as described in Example 1.
• the assay for OXA-48 has thus a sensitivity of about 97% and a specificity of about 98%.
• the sensitivity may be even higher if genotyping would reveal that the two OXA- 181 positive strains had originally been wrongly classified.
• VIM-positive strains were tested. These had previously been categorized as VIM- positive, which could be confirmed by genotyping as described above. Further 186 strains were tested, which had previously been categorized as VIM-negative, which was confirmed by genotyping as described above.
• the 222 strains were cultivated, and cells were tested by the combination of anti-VIM antibody 1071 (SEQ ID Nos: 17 and 21) as capture antibody and anti-KPC antibody 1721 (SEQ ID Nos: 19 and 23) as detector antibody using the Array Tube® platform as described in Example 1.
• the assay for VIM has thus a sensitivity of about 97% and a specificity of about 100%.
• the 94 strains were cultivated, and cells were tested by the combination of anti-KPC antibody 1351 (SEQ ID Nos: 9 and 13) as capture antibody and anti-KPC antibody 1591 (SEQ ID Nos: 11 and 15) as detector antibody using the Array Tube® platform as described in Example 1.
• the assay for KPC has thus a sensitivity of about 100% and a specificity of about 100%.
• 34 NDM-positive strains were tested. These had previously been categorized as NDM- positive, which could be confirmed by genotyping as described above. Further 60 strains were tested, which had previously been categorized as NDM-negative, which was confirmed by genotyping as described above.
• the 94 strains were cultivated, cells were tested by the combination of anti-NDM antibody 1041 (SEQ ID Nos: 2 and 6) as capture antibody and anti-NDM antibody 1121 (SEQ ID Nos: 3 and 7) as detector antibody using the ArrayTube® platform as described in Example 1.
• the assay for NDM has thus a sensitivity of about 100% and a specificity of about 100%.
• Supernatants of cells expressing NDM, KPC, VIM, or OXA-48 were subjected to denaturing conditions including heating for 10 min at 99°C, treatment with disinfectants Bacillol® at 1 :6 dilution or Sterilium® at 1 :6 dilution or higher dilutions. Testing for NDM, KPC, VIM and OXA-48 was done as described in Example 2. The results are shown in Table 4. For Bacillol®, signals could be detected for dilutions down to 1 : 100.000.
• Bacillol (l :6) +++ +++ +++ +++ n.t. Sterilium (l :6) n.t. n.t. n.t. +++ not
• Example 5 Further approaches a) Bodily samples may be taken and cultivated overnight on 2xTY (trypton-yeast extract), bile-chrysoidin-glycerol + 4-methylumbelliferyl-beta-D-glucuronid (CGC- MUG), McConkey or Columbia blood agar plates or, respectively, in other growth media used in diagnostic procedures.
• 2xTY trypton-yeast extract
• CGC- MUG 4-methylumbelliferyl-beta-D-glucuronid
• McConkey Columbia blood agar plates or, respectively, in other growth media used in diagnostic procedures.
• An array or a lateral flow test, on which anti-NDM, anti-KPC, anti-VIM, and/or anti- OXA-48 antibodies as described e.g. in Example 2 have been immobilized at defined locations as capture antibodies, can then be provided.
• the anti-NDM, anti-KPC, anti-VIM, and/or anti-OXA-48 antibodies can thus simultaneously be used for detection of all four antigens in parallel.
• an array such as the ArrayTube® platform
• fluorescence detection 10 ⁇ l 60 nm Red-Beads coupled to streptavidin dilute 1 : 100 in buffer and 0.25% Triton were added at 300 rpm for 30 min. Then, a washing step for 5 min in buffer and 0.25% Triton X at 400 rpm was carried out. The buffer was removed and exchanged by 100 ⁇ l buffer and 0.25%> Triton and images were taken with a FluoReader.
• bodily samples may be taken and cultivated overnight in 2xTY, CGC-MUG, McConkey or Columbia blood agar plates or, respectively, in other growth media used in diagnostic procedures. Cell free culture supematants are produced by centrifuging liquid growth media or other liquids containing bacterial cells for 5 min at 6000rpm.
• An array or a lateral flow test, on which anti-NDM, anti-KPC, anti-VIM, and/or anti- OXA-48 antibodies as described e.g. in Example 2 have been immobilized at defined locations as capture antibodies, can then be provided.
• the anti- NDM-, anti-KPC-, anti-VIM-, and/or anti-OXA-48 antibodies can thus simultaneously be used for detection of all four antigens in parallel.
• bodily samples may be taken and cultivated overnight culture in 2xTY- or GN-medium (http://www.neogen.com). Cell free culture
• supematants are produced from liquid media by centrifuging cells for 5 min at 6000rpm.
• the supematants can then be denaturated for example with Sterillium® and/or
• An array or a lateral flow test, on which anti-NDM, anti-KPC, anti-VIM, and/or anti- OXA-48 antibodies as described e.g. in Example 2 have been immobilized at defined locations as capture antibodies, can then be provided.
• the anti-NDM, anti-KPC, anti-VIM, and/or anti-OXA-48 antibodies can thus simultaneously be used for detection of all four antigens in parallel.
• An array or a lateral flow test, on which anti-NDM, anti-KPC, anti-VIM, and/or anti- OXA-48 antibodies as described e.g. in Example 2 have been immobilized at defined locations as capture antibodies, can then be provided.
• the anti-NDM, anti-KPC, anti-VIM, and/or anti-OXA-48 antibodies can thus simultaneously be used for detection of all four antigens in parallel.

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