CN114755420A - Carbapenemase combined detection kit - Google Patents
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Abstract
The invention provides a carbapenemase joint detection kit, and belongs to the technical field of biology. The carbapenemase joint detection kit can detect five carbapenemases of KPC, OXA-48, NDM, IMP and VIM types at one time, and can be used for early typing of carbapenemase drug-resistant strains and guiding medication, thereby assisting clinical infection control and treatment. The kit is simple to operate and has good detection sensitivity and accuracy.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a carbapenemase joint detection kit.
Background
The carbapenem antibacterial drug has wide antibacterial spectrum, strong antibacterial effect and low adverse reaction incidence. With the use of a large amount of antibacterial agents, the resistance rate of gram-negative bacteria to carbapenem antibacterial agents is increasing, and the detection of carbapenem antibiotic-resistant Enterobacteriaceae (CRE) is increasing. The carbapenemase is used as a main drug resistance mechanism of bacteria, and a coding gene of the carbapenemase is positioned on a chromosome or a plasmid, so that the bacteria can resist beta-lactam antibacterial drugs such as meropenem, imipenem and the like through cloning propagation or gene level transfer. Carbapenemases can be classified into A, B, D types (Ambler classification) according to amino acid sequence homology and molecular structure characteristics, wherein A type is serine protease, and KPC (Klebsiella pneumoniae Carbapenemase) is mainly used; the B is metalloenzyme, mainly NDM (New Delhi metallo-beta lactamase), VIM (Verona-encoded metallo-beta-lactamase) and IMP (immunopotentiase); class D is the OXA enzyme (oxacillinase), mainly the OXA-48 enzyme. Among them, KPC, NDM, OXA-48 carbapenemases are the most major resistance mechanism of bacteria of Enterobacteriaceae to carbapenems.
At present, the commonly used methods for detecting carbapenemases mainly comprise a typing detection method and a gene detection method. The phenotype detection methods mainly comprise a Modified Hodgkin Test (MHT), a colorimetric-based Carba NP test, a mass spectrometry-based Meropenem dissolution test (MHA), a modified carbapenem inactivation method (mCIM) and the like, and the detection methods take a long time. Moreover, biochemical methods are time consuming and sensitive and specific, but cannot differentiate between subtypes. Laser desorption ionization time of flight mass spectrometry (MALDI-TOF) can be used for strain identification, subtype classification and drug-resistant gene detection, and has the main defect that instruments are high in cost and are not acceptable in many laboratories. The gene detection method is the 'gold standard' for carbapenemase detection, but has high labor and cost. For example, patent CN202110733877.1 discloses a primer and probe for detecting carbapenemase genes, which can be used for rapidly and sensitively detecting 6 carbapenemase genes in total of KPC, VIM, CTX, GES, NDM and IMP in a sample.
The rapid detection of the carbapenemase-producing multi-drug resistant and pan-drug resistant strains can be used for targeted and effective treatment. At present, there is a reagent application based on colloidal gold immunochromatography, for example, patent 201680030887.6 discloses a method for detecting one or more carbapenemases selected from OXA, particularly OXA-48, KPC, VIM and/or NDM carbapenemases from carbapenemase-producing enterobacteriaceae (CPE) that may be present in a biological sample. The patent CN202110166073.8 discloses an anti-OXA-23 carbapenemase hybridoma cell strain, a monoclonal antibody and application thereof, and the obtained anti-OXA-23 carbapenemase antibody can be used for detecting OXA-23 carbapenemase and can be prepared into an in vitro diagnostic kit.
Therefore, it is highly desirable to provide a method for detecting a fast carbapenemase based on a colloidal gold immunochromatography method for detecting KPC, OXA-48, NDM, IMP and VIM.
Disclosure of Invention
Aiming at the problems, the invention provides a carbapenemase joint detection kit. The carbapenemase joint detection kit can detect five carbapenemases of KPC, OXA-48, NDM, IMP and VIM types at one time, and can be used for early typing of carbapenemase drug-resistant strains, guiding medication and assisting clinical infection control and treatment.
In order to achieve the above object, the technical solution of the present invention is as follows:
in one aspect, the invention provides a carbapenemase combined detection kit, which comprises anti-KPC, OXA-48, NDM, IMP and VIM carbapenemase antigen binding protein.
Specifically, the anti-NDM carbapenemase antigen binding protein comprises the following complementarity determining regions:
(1) heavy chain complementarity determining region 1HCDR1 comprising the amino acid sequence set forth in any one of SEQ ID NO. 1 or SEQ ID NO. 7, or a variant sequence thereof;
(2) a heavy chain complementarity determining region 2HCDR2 comprising an amino acid sequence set forth in any one of SEQ ID NO. 2 or SEQ ID NO. 8, or a variant sequence thereof;
(3) a heavy chain complementarity determining region 3HCDR3 comprising an amino acid sequence set forth in any one of SEQ ID NO. 3 or SEQ ID NO. 9, or a variant sequence thereof;
(4) a light chain complementarity determining region 1LCDR1 comprising the amino acid sequence set forth in SEQ ID NO. 4 or a variant sequence thereof;
(5) a light chain complementarity determining region 2LCDR2 comprising an amino acid sequence set forth in any one of SEQ ID NO. 5 or SEQ ID NO. 10, or a variant sequence thereof;
(6) a light chain complementarity determining region 3LCDR3 comprising an amino acid sequence set forth in any one of SEQ ID NO 6 or SEQ ID NO 11, or a variant sequence thereof;
preferably, the variant sequence is a CDR sequence having substitution, deletion or addition of one or several amino acids compared to the CDR from which it is derived; the substitutions are conservative substitutions.
Further specifically, the anti-NDM carbapenemase antigen binding protein comprises:
(1) a heavy chain variable region VH comprising an amino acid sequence shown in any one of SEQ ID NO 12 or SEQ ID NO 14; and/or a light chain variable region VL comprising an amino acid sequence set forth in any one of SEQ ID NO 13 or SEQ ID NO 15;
or (2), a VH having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to any one VH of (1); and/or a VL having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a VL in any of (1);
or (3) a VH having one or more amino acid substitutions, deletions or additions, or any combination thereof, compared to any VH in (1); and/or a VL having one or several amino acid substitutions, deletions or additions, or any combination thereof, as compared to any VL in (1); the substitutions are conservative substitutions.
Further specifically, the anti-NDM carbapenemase antigen binding protein comprises:
(1) a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO 12; and/or a light chain variable region VL comprising the amino acid sequence set forth in SEQ ID NO 13; and/or
(2) A heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO. 14; and/or a light chain variable region VL comprising the amino acid sequence set forth in SEQ ID NO. 15.
More specifically, the anti-NDM carbapenemase antigen-binding protein includes a full-length antibody, Fab ', F (ab') 2, Fv, scFv, di-scFv, bispecific antibody, multispecific antibody, heavy chain antibody and/or single domain antibody, or a monoclonal antibody and/or polyclonal antibody produced from the above antibody.
Specifically, the kit further comprises a gold-labeled pad, a nitrocellulose membrane, absorbent paper, a bottom plate and a plastic card.
More specifically, the gold-labeled pad is coated with five antigen binding proteins of gold-labeled carbapenemases KPC, OXA-48, NDM, IMP and VIM, five antigen binding proteins of carbapenemases KPC, OXA-48, NDM, IMP and VIM are coated on a nitrocellulose membrane T line (detection line), and a C line (quality control line) is coated with a goat anti-mouse IgG antibody.
In another aspect, the invention provides a method for detecting five carbapenemases of KPC, OXA-48, NDM, IMP and VIM types by using the kit, wherein the method is used for non-disease diagnosis or treatment.
In another aspect, the invention provides the application of the kit in the detection of carbapenemase.
Specifically, the carbapenemases include five carbapenemases of KPC, OXA-48, NDM, IMP and VIM types.
Compared with the prior art, the invention has the advantages that:
(1) the detection test paper in the kit (colloidal gold immunochromatography) of the invention fixes anti-KPC, OXA-48, NDM, IMP and VIM type carbapenemase monoclonal antibody (coating antibody) in a detection area of a nitrocellulose membrane, fixes goat anti-mouse IgG polyclonal antibody in a control area, and pre-coats anti-KPC, OXA-48, NDM, IMP and VIM type carbapenemase monoclonal antibody (marking antibody) -colloidal gold on an immune gold pad. For the test, 100. mu.L of the buffer mixed with the specimen was added dropwise and placed horizontally. The anti-KPC, OXA-48, NDM, IMP and VIM carbapenemase monoclonal antibody (labeled antibody) -colloidal gold on the immuno-gold pad is dissolved, if KPC, OXA-48, NDM, IMP and VIM carbapenemase are contained in the sample, anti-KPC, OXA-48, NDM, IMP and VIM carbapenemase monoclonal antibody (labeled antibody) -colloidal gold is combined with KPC, OXA-48, NDM, IMP and VIM carbapenemase in the sample to form an antigen-antibody-gold complex, the complex moves backwards along the test strip, firstly reaches a detection zone coated with anti-KPC, OXA-48, NDM, IMP and VIM carbapenemase monoclonal antibody (coated antibody), the antigen-antibody-gold complex is combined with VIXA, OXA-48, NDM, IMP and VIM carbapenemase monoclonal antibody (coated antibody), and left on the test line, forming a red line, which indicates a positive result. The shade of the line color is proportional to the carbapenemase concentration in the sample. If no colored line in the area indicates that the sample does not contain the carbon qingrenes of KPC, OXA-48, NDM, IMP and VIM or that the carbon qingrenes of KPC, OXA-48, NDM, IMP and VIM are lower than the lowest detection limit of the test paper. The complex continues to move to the control zone coated with goat anti-mouse IgG polyclonal antibody, and the free antibody-gold complex will bind with goat anti-mouse IgG polyclonal antibody and enrich in the control zone, forming a red line. The appearance of the control line proves that the test strip has normal detection function. The control zone of the test strip should show a control line from light red to purple red in the validation test, regardless of whether the sample contains KPC, OXA-48, NDM, IMP and VIM carbapenemases. The sample continues to move through the control zone into the absorption zone. The absorption zone functions to adsorb the remaining complex, move it within the strip, and eliminate the background color. And (4) counting time from the addition of the diluted sample mixed solution into the test strip, and reading the result within 10-15 minutes.
(2) The invention provides a carbapenemase NDM antibody, which has better affinity with the carbapenemase NDM, and can be prepared into a detection kit for early typing of carbapenemase drug-resistant strains and guiding drug use, thereby assisting clinical infection control and treatment.
(3) The kit provided by the invention is simple to operate and has better detection sensitivity and accuracy.
Detailed Description
The present invention will be described in further detail with reference to specific examples, which are not intended to limit the present invention, but to illustrate the present invention. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.
Example 1 preparation of NDM monoclonal antibody
1. Immunization of mice
An NDM protein (amino acid sequence is shown in the national center for Biotechnology information NCBI BAJ 10873) is adopted to immunize female Balb/c mice of about 6 weeks old for antibody preparation. The concentration of NDM protein is 1mg/mL, and the immunization dose is 50 mug/mouse. For the first immunization, NDM protein and Freund's complete adjuvant are mixed according to the volume ratio of 1: 1, mixing, emulsifying, and injecting into abdominal cavity. 14d and 28d, NDM protein was mixed with freund's incomplete adjuvant in a volume ratio of 1: 1, mixing, emulsifying, injecting into abdominal cavity, and performing second and third immunization. At 42d, NDM protein was dissolved in PBS for a fourth immunization. Titre tests were performed at 21d and 35d, respectively, to test for immune efficacy.
2. Cell fusion and culture
Immunization 45d the spleen cells and myeloma cells of the extracted mice were mixed in a count ratio of 1: 5. After mixing, RPMI1640 medium was added to 50mL, and the mixture was centrifuged at 1500rpm for 5min to remove the supernatant. The cell pellet was fused at 37 deg.C, 1mL of pre-warmed fusion agent was added, and after 2min of standing, 2mL of pre-warmed RPMI1640 medium was added slowly. After mixing, centrifugation is carried out at 1500rpm for 5min, and the supernatant is removed. The fused cell sap was plated on feeder cells-containing cell plates with 5% CO2Culturing at 37 deg.C in incubator.
3. Screening of hybridoma positive clone cells
And after 7 days of culture, replacing the cell culture solution with a fresh HAT culture medium, continuing to culture, performing secondary screening on positive cells by adopting a limiting dilution method after 14 days, diluting the cells for 3-4 times, and selecting the cells with the highest positive value for expanded culture to obtain the hybridoma positive clone cell strain.
4. Preparation of monoclonal antibodies
Taking female Balb/c mice of about 10 weeks old, injecting incomplete Freund's adjuvant into abdominal cavity according to 1 mL/mouse dosage, and after 3 days, according to 5 × 105Single cell/single agentAnd measuring hybridoma cells cultured in an intraperitoneal injection way to logarithmic phase for about 7-14 days, observing the abdomen of the mouse, extracting ascites after the abdomen of the mouse is raised, and extracting the ascites every 3 days until no ascites is generated or the state of the mouse is poor. Purifying mouse ascites to obtain monoclonal antibody.
The invention prepares 2 monoclonal antibodies, and the sequence information of the antibodies is shown in the following table 1.
TABLE 1
| Monoclonal antibodies | NDM antibody A | NDM antibody B |
| Heavy chain CDR1 | SEQ ID NO:1 | SEQ ID NO:7 |
| Heavy chain CDR2 | SEQ ID NO:2 | SEQ ID NO:8 |
| Heavy chain CDR3 | SEQ ID NO:3 | SEQ ID NO:9 |
| Light chain CDR1 | SEQ ID NO:4 | SEQ ID NO:4 |
| Light chain CDR2 | SEQ ID NO:5 | SEQ ID NO:10 |
| Light chain CDR3 | SEQ ID NO:6 | SEQ ID NO:11 |
| Heavy chain variable region | SEQ ID NO:12 | SEQ ID NO:14 |
| Light chain variable region | SEQ ID NO:13 | SEQ ID NO:15 |
Example 2 NDM antibody affinity assays
The binding capacity of the NDM antibody prepared in the embodiment 1 of the invention and the NDM protein is detected by an ELISA method. The specific experimental process is as follows: NDM protein was diluted to a concentration of 1. mu.g/mL and added to a 96-well plate at 100. mu.L/well overnight at 4 ℃. The 96-well plate was washed three times with PBST, added with 2% BSA in PBS, and left at 37 ℃ for 1 h. NDM antibody is diluted in a concentration gradient manner, and then added into a 96-well plate respectively, and incubated for 1h at 37 ℃. PBST was washed three times, goat anti-mouse secondary antibody was added, and incubated at 37 ℃ for 30-60 min. After PBST is washed for three times, TMB developing solution is added for 10min, the developing is stopped, the OD value of each hole is measured, and the dissociation constant Kd is calculated.
The NDM antibody prepared by the invention has good binding capacity with NDM protein through detection, wherein the median Kd of the antibody A is 5.2pM, and the median Kd of the antibody B is 3.9 pM.
Example 3 carbapenemase Combined detection kit
The invention uses colloidal gold method to prepare carbapenemase detection card and double antibody sandwich method immune colloidal gold reagent kit. In the kit, the antibody A can be used as a coated antibody, and the antibody B can be used as a gold-labeled antibody; meanwhile, the antibody B can be used as a coating antibody, and the antibody A can be used as a gold-labeled antibody.
The following kit adopts an antibody A as a coating antibody and an antibody B as a gold-labeled antibody; among them, NDM antibody A and antibody B are shown in Table 1 above; KPC antibody A was purchased from Guangzhou Bokang Biotechnology Inc. under the product number of C661, and KPC antibody B was purchased from Guangzhou Bokang Biotechnology Inc. under the product number of C663; VIM antibody A was purchased from Guangzhou Bokang Biotechnology Inc., the product number is C691, VIM antibody B was purchased from Guangzhou Bokang Biotechnology Inc., the product number is C692; IMP antibody A is purchased from Guangzhou Bokang biotechnologies Inc., with the product number of C681, and IMP antibody B is purchased from Guangzhou Bokang biotechnologies Inc., with the product number of C682; OXA-48 antibody A was purchased from Guangzhou Bokang Biotechnology Inc. under the accession number C652, and OXA-48 antibody B was purchased from Guangzhou Bokang Biotechnology Inc. under the accession number C653.
1. Preparing a detection reagent strip:
(1) preparing a detection line coating solution: the antibody A was prepared to 2mg/mL with 0.01M phosphate buffer pH7.0-7.6 and 3% sucrose solution, respectively, and used.
(2) Preparing a control line coating solution: the goat anti-mouse IgG antibody was prepared to 2mg/mL using 0.01M phosphate buffer pH7.0-7.6 and 3% sucrose solution for use.
(3) Scribing: the detection line coating solution and the comparison line coating solution prepared above are respectively loaded into a film spraying machine, and the detection line and the comparison line are scribed on a nitrocellulose membrane (the specification of the membrane is 25mm multiplied by 310mm) according to the amount of 0.1 muL/mm.
(4) Coating: the above nitrocellulose membrane was incubated at 37 ℃ for 2 h.
(5) And (3) sealing: and (3) putting the incubated cellulose nitrate membrane into 0.5% BSA blocking solution for soaking for 30min, washing twice with distilled water, and drying in a vacuum drier at 37 ℃ for 16h with the vacuum degree of 0.1.
(6) And (3) packaging: and (5) putting the dried nitrocellulose membrane into an aluminum foil bag in a drying chamber (the humidity is less than 20%), and sealing. And a desiccant is added for moisture prevention.
2. Preparation of conjugate pad
Selection of gold-labeled antibody concentration and pH: by adopting a cross matching method, the most suitable labeling concentration of the carbapenemase antibody is 2mg/mL, and the pH value is 7.0-7.6.
(1) Preparing colloidal gold: taking AuCl4Diluting to 0.01% water solution, boiling, adding 1% trisodium citrate water solution 0.7mL under stirring, changing golden yellow chloroauric acid water solution into mauve within 2min, boiling for 15min, cooling, and recovering to original volume with distilled water.
(2) Coupling: the prepared colloidal gold solution is firstly used with 0.2M K2CO3Adjusting pH to the optimum range of 7.0-7.6, adding antibody B according to the optimum labeling concentration ratio, and stirring at room temperature for 30 min.
(3) And (3) sealing: 10% PEG20000 was added to make the final concentration 1%, and stirred for 20 min.
(4) Coating: soaking 10mm × 310mm nitrocellulose membrane in the above sealed solution for 5-10 min.
(5) And (3) drying: and (3) putting the nitrocellulose membrane into a vacuum drier for drying for 20 hours, wherein the vacuum degree is 0.1.
(6) And (3) packaging: in a drying chamber (humidity < 20%), the dried nitrocellulose membrane is packed into an aluminum foil bag which is filled with a drying agent in advance, and the machine is sealed.
3. Sample buffer
The sample buffer used was Phosphate Buffer (PB): weighing 2.84g of disodium hydrogen phosphate, dissolving in high-purity water, and fixing the volume of the volumetric flask to 100mL to obtain 0.2M disodium hydrogen phosphate solution; weighing 2.40g of sodium dihydrogen phosphate, dissolving in high-purity water, and metering the volume of the volumetric flask to 100mL to obtain 0.2M sodium dihydrogen phosphate solution; filtering with 0.22 μm filter head, and storing at 2-8 deg.C. 19mL of 0.2M sodium dihydrogen phosphate solution and 81mL of 0.2M disodium hydrogen phosphate solution were measured, 1900mL of high-purity water was added, and the mixture was mixed to obtain 0.01M Phosphate Buffer (PB) having a pH of 7.4, and the mixture was stored under a closed condition at 2-8 ℃ for further use.
4. Detection of
The kit and the plate containing the cloned strain to be tested were returned to room temperature (15-30 ℃) before the experiment was started. And opening the packaging bag and taking out the detection card.
And (3) a checking step:
(1) a sterile EP tube was prepared to add 10 drops of the sample treatment solution dropwise.
(2) The clonal strains were dipped using a disposable bacterial loop and the loop was inserted into a sterile EP tube containing the sample treatment fluid.
(3) The solution was stirred well to homogenize.
(4) 100 μ L of the diluted sample was applied to the loading hole of the test card.
(5) The results were read after waiting 15 minutes.
5. And respectively taking a negative sample and a positive sample, and detecting by using the kit and the detection method. The results of the measurements are shown in Table 2 below.
TABLE 2
Note: + indicates a positive detection; -indicates a negative test.
As can be seen from Table 2, the detection result of the negative sample is negative, and the result of the positive sample is positive, which proves that the kit prepared by the application can be used for rapidly detecting KPC, OXA-48, NDM, IMP and VIM carbapenemases and KPC, OXA-48, NDM, IMP and VIM carbapenemases positive samples, and has low detection limit and convenient operation.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
SEQUENCE LISTING
<110> Guangdong Confucius biotechnology Limited
<120> carbapenemase combined detection kit
<130> 20220527
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Glu Val Ser Tyr Gly Asp Met Glu
1 5
<210> 8
<211> 13
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 8
Val Arg Ala Gly Ser Gly Asp Thr His Lys Tyr Asn Glu
1 5 10
<210> 9
<211> 6
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 9
Gly Gly Glu Ile Ser Lys
1 5
<210> 10
<211> 7
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 10
Glu Ala Arg Asn Thr Thr Gly
1 5
<210> 11
<211> 11
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 11
Gln Gln Asp Asn Ser Trp Pro Tyr Ile Gly Thr
1 5 10
<210> 12
<211> 119
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 12
Gln Val Gln Leu Thr Gln Ser Gly Ala Glu Phe Leu Ala Lys Pro Gly
1 5 10 15
Ala Lys Asp Ser Cys Lys Ala Val Gly Tyr Thr Phe His Glu Val Ser
20 25 30
Tyr Gly Ile Met Glu Thr Val Lys Gln Arg Thr Gly Gln Gly Leu Glu
35 40 45
Trp Ile Gly Val Tyr Ala Gly Ser Gly Asp Thr His Ser Tyr Asn Glu
50 55 60
Tyr Ala Thr Ile Leu Thr Ala Asp Lys His Ser Ser Thr Ala Tyr Arg
65 70 75 80
Gln Leu Asn Ser Leu Thr Thr Ala Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Tyr Tyr Glu Arg Gly Gly Glu Val Ser Asn Ala Lys Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 13
<211> 109
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 13
Asp Ile Gln Leu Thr Gln Ser Pro Leu Thr Leu Ala Val Thr Pro Gly
1 5 10 15
Asp Ser Val Ser Leu Ser Cys Gln Val Trp Leu Thr Cys Arg Asp Gly
20 25 30
Lys Ser Pro Tyr Asn Val Leu His Gln Arg Ser Glu Arg Ser Phe Arg
35 40 45
Cys Leu Ala Lys Glu Ala Arg Asn Thr Ile Gly Ile Cys Ser Pro Arg
50 55 60
Phe Ala Gly Ser Glu Thr Asp Phe Thr Leu Gln Ile Lys Glu Val Glu
65 70 75 80
Thr Val Asp Phe Gly Arg Cys Gln Arg Asp Asn Ser Trp Pro Tyr Ile
85 90 95
Gly Thr Phe Gly Asn Tyr Thr Lys Ala Glu Lys Ile Lys
100 105
<210> 14
<211> 117
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 14
Gln Val Gln Leu Thr Gln Ser Gly Ala Glu Phe Leu Ala Lys Pro Gly
1 5 10 15
Ala Lys Asp Ser Cys Lys Ala Val Gly Tyr Thr Phe His Glu Val Ser
20 25 30
Tyr Gly Asp Met Glu Thr Val Lys Gln Arg Thr Gly Gln Gly Leu Glu
35 40 45
Trp Ile Gly Val Arg Ala Gly Ser Gly Asp Thr His Lys Tyr Asn Glu
50 55 60
Tyr Ala Thr Ile Leu Thr Ala Asp Lys His Ser Ser Thr Ala Tyr Arg
65 70 75 80
Gln Leu Asn Ser Leu Thr Thr Ala Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Tyr Tyr Glu Arg Gly Gly Glu Ile Ser Lys Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 15
<211> 109
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 15
Asp Ile Gln Leu Thr Gln Ser Pro Leu Thr Leu Ala Val Thr Pro Gly
1 5 10 15
Asp Ser Val Ser Leu Ser Cys Gln Val Trp Leu Thr Cys Arg Asp Gly
20 25 30
Lys Ser Pro Tyr Asn Val Leu His Gln Arg Ser Glu Arg Ser Phe Arg
35 40 45
Cys Leu Ala Lys Glu Ala Arg Asn Thr Thr Gly Ser Cys Ser Pro Arg
50 55 60
Phe Ala Gly Ser Glu Thr Asp Phe Thr Leu Gln Ile Lys Glu Val Glu
65 70 75 80
Thr Val Asp Phe Gly Arg Cys Gln Gln Asp Asn Ser Trp Pro Tyr Ile
85 90 95
Gly Thr Phe Gly Asn Tyr Thr Lys Ala Glu Lys Ile Lys
100 105
Claims (10)
1. A carbapenemase combined detection kit is characterized in that: the kit comprises anti-KPC, OXA-48, NDM, IMP and VIM type carbapenemase antigen binding protein;
the anti-NDM carbapenemase antigen binding protein comprises the following complementarity determining regions:
(1) heavy chain complementarity determining region 1HCDR1 comprising the amino acid sequence set forth in any one of SEQ ID NO. 1 or SEQ ID NO. 7, or a variant sequence thereof;
(2) a heavy chain complementarity determining region 2HCDR2 comprising an amino acid sequence set forth in any one of SEQ ID NO. 2 or SEQ ID NO. 8, or a variant sequence thereof;
(3) a heavy chain complementarity determining region 3HCDR3 comprising an amino acid sequence set forth in any one of SEQ ID NO. 3 or SEQ ID NO. 9, or a variant sequence thereof;
(4) a light chain complementarity determining region 1LCDR1 comprising the amino acid sequence set forth in SEQ ID NO. 4 or a variant sequence thereof;
(5) a light chain complementarity determining region 2LCDR2 comprising an amino acid sequence set forth in any one of SEQ ID NO 5 or SEQ ID NO 10, or a variant sequence thereof;
(6) a light chain complementarity determining region 3LCDR3 comprising the amino acid sequence set forth in any one of SEQ ID NO 6 or SEQ ID NO 11, or a variant sequence thereof;
the variant sequence is a CDR sequence having substitution, deletion or addition of one or several amino acids compared to the CDR from which it is derived; the substitutions are conservative substitutions.
2. The kit of claim 1, wherein: the anti-NDM carbapenemase antigen binding protein comprises:
(1) a heavy chain variable region VH comprising an amino acid sequence set forth in any one of SEQ ID NO 12 or SEQ ID NO 14; and/or a light chain variable region VL comprising an amino acid sequence set forth in any one of SEQ ID NO 13 or SEQ ID NO 15;
or (2), a VH having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of the VH in (1); and/or a VL having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a VL in any of (1);
or (3) a VH having one or more amino acid substitutions, deletions or additions, or any combination thereof, compared to any VH in (1); and/or a VL having one or several amino acid substitutions, deletions or additions, or any combination thereof, as compared to any VL in (1); the substitutions are conservative substitutions.
3. The kit of claim 2, wherein: the anti-NDM carbapenemase antigen binding protein comprises:
(1) a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO 12; and/or a light chain variable region VL comprising the amino acid sequence set forth in SEQ ID NO 13; and/or
(2) A heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO. 14; and/or a light chain variable region VL comprising the amino acid sequence set forth in SEQ ID NO. 15.
4. The kit of claim 3, wherein: the anti-NDM carbapenemase antigen-binding protein comprises a full-length antibody, Fab ', F (ab') 2, Fv, scFv, di-scFv, bispecific antibody, multispecific antibody, heavy chain antibody and/or single domain antibody, or a monoclonal antibody and/or polyclonal antibody prepared from the antibodies.
5. The kit of claim 1, wherein: the kit also comprises a gold-labeled pad, a nitrocellulose membrane, absorbent paper, a bottom plate and a plastic card.
6. The kit of claim 5, wherein: the gold-labeled pad is coated with five antigen binding proteins of gold-labeled carbapenemase KPC, OXA-48, NDM, IMP and VIM types.
7. The kit of claim 6, wherein: the nitrocellulose membrane detection line is coated with five antigen binding proteins of carbapenemase KPC, OXA-48, NDM, IMP and VIM types, and the quality control line is coated with goat anti-mouse IgG antibody.
8. A carbapenemase detection method, which is characterized in that: use of a kit according to any one of claims 1 to 7 for the detection of five carbapenemases of KPC, OXA-48, NDM, IMP and VIM type, for non-disease diagnostic or therapeutic purposes.
9. Use of the kit of any one of claims 1 to 7 for the detection of carbapenemases.
10. Use according to claim 9, characterized in that: the carbapenemases include KPC, OXA-48, NDM, IMP and VIM type five carbapenemases.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| EP4261278A4 (en) * | 2022-03-02 | 2024-04-17 | Tianjin Era Biology Tech Co Ltd | Mouse anti-ndm carbapenemase hybridoma cell strain, monoclonal antibody and use |
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