WO2022166354A1 - 一种抗oxa-23型碳青霉烯酶杂交瘤细胞株，单克隆抗体及应用 - Google Patents

一种抗oxa-23型碳青霉烯酶杂交瘤细胞株，单克隆抗体及应用 Download PDF

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WO2022166354A1
WO2022166354A1 PCT/CN2021/135404 CN2021135404W WO2022166354A1 WO 2022166354 A1 WO2022166354 A1 WO 2022166354A1 CN 2021135404 W CN2021135404 W CN 2021135404W WO 2022166354 A1 WO2022166354 A1 WO 2022166354A1
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antibody
seq
oxa
variable region
chain variable
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PCT/CN2021/135404
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French (fr)
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李可可
何永胜
苑庆华
陈晓玲
周跃辉
樊琳琳
王兴
臧丹戎
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天津一瑞生物科技股份有限公司
北京金山川科技发展有限公司
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Priority to EP21870557.2A priority Critical patent/EP4063498B1/en
Publication of WO2022166354A1 publication Critical patent/WO2022166354A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—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
    • C07K16/1218—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Acinetobacter
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]

Definitions

  • the invention belongs to the technical field of antibody preparation, in particular to an anti-OXA-23 type carbapenemase hybridoma cell line, a monoclonal antibody and applications thereof.
  • carbapenems have the most extensive antibacterial activity, can resist the hydrolysis of most ⁇ -lactamases and extended-spectrum ⁇ -lactamases, and can attenuate chromosome-mediated The role of the Amp C enzyme.
  • Acinetobacter has certain resistance to many antibiotics such as ⁇ -lactams, aminoglycosides, fluoroquinolones, etc., and carbapenems have always had good antibacterial activity against Acinetobacter.
  • the resistance rate of Acinetobacter baumannii to it is increasing year by year. In recent years, it has been reported that Acinetobacter carrying blaOXA-23 causes hospital sickness.
  • the invention provides an anti-OXA-23 type carbapenemase hybridoma cell line, a monoclonal antibody and applications thereof.
  • the technical solution adopted in the present invention is: an anti-OXA-23 type carbapenemase hybridoma cell line, named as HB12, with a deposit number of CGMCC No. 20285; or named as FA2, with a deposit number of CGMCC No. 20286.
  • An anti-OXA-23 type carbapenemase antibody named as antibody HB12, includes a light chain variable region and a heavy chain variable region, and the CDR region of the light chain variable region sequence includes such as SEQ ID NO:1, The three sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3, the CDR region of the heavy chain variable region sequence includes those shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 three sequences;
  • the CDR region of the light chain variable region sequence includes three sequences as shown in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, the CDR region of the heavy chain variable region sequence Include three sequences as shown in SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16;
  • amino acid sequence of the variable region of the light chain of antibody HB12 is shown in SEQ ID NO:7, and the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO:9;
  • the antibody FA2 light chain variable region amino acid sequence is shown in SEQ ID NO: 17, and the heavy chain variable region amino acid sequence is shown in SEQ ID NO: 19;
  • a nucleic acid molecule comprising a nucleotide sequence encoding an anti-OXA-23 type carbapenemase antibody.
  • nucleotide sequence of the nucleic acid molecule encoding the light chain variable region of the antibody HB12 is shown in SEQ ID NO: 8
  • nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region of the antibody HB12 is shown in SEQ ID NO: 10 shown;
  • nucleotide sequence of the variable region of the light chain of the nucleic acid molecule encoding antibody FA2 is shown in SEQ ID NO:18, and the nucleotide sequence of the variable region of the heavy chain of the nucleic acid molecule encoding the antibody FA2 is shown in SEQ ID NO:20;
  • the anti-OXA-23 carbapenemase antibody is used in an in vitro diagnostic kit or a microfluidic chip, and the in vitro diagnostic kit is a colloidal gold immunoassay kit, a chemiluminescence kit, a radioimmunoassay kit, an enzyme-linked immunosorbent assay kit or fluorescent immunoassay kit.
  • a double-antibody sandwich method immunocolloidal gold test strip is prepared, the antibody HB12 is a coated antibody, and the antibody FA2 is a gold-labeled antibody; or, the antibody HB12 is a gold-labeled antibody, and the antibody FA2 is a coated antibody.
  • the invention has the advantages and positive effects as follows: providing an anti-OXA-23 type carbapenemase antibody, which can be used to detect OXA-23 type carbapenemase, and the titer reaches 1:640000 or more; It is prepared into an in vitro diagnostic kit or a microfluidic chip, which can be used for early typing of drug-resistant strains, guiding medication, and assisting clinical infection control and treatment.
  • Fig. 1 is OXA-23 type carbapenemase protein electrophoresis image
  • Fig. 2 is an electrophoresis image of anti-OXA-23 type carbapenemase antibody protein.
  • the invention relates to an anti-OXA-23 type carbapenemase hybridoma cell line, the biological material is named HB12, belongs to the hybridoma cell line, and its preservation number is CGMCC No. 20285; The center, whose deposit date is August 27, 2020, was tested as alive.
  • the invention relates to an anti-OXA-23 type carbapenemase hybridoma cell line through mouse hybridoma monoclonal antibody screening and RT-PCR method to clone Ig variable region genes, and obtain stable secretion of anti-OXA-23 type carbapenems
  • the hybridoma cell line of the enzyme antibody and its variable region sequence were identified, and the binding specificity of the antibody was identified by ELISA.
  • the HB12 antibody produced by anti-OXA-23 type carbapenemase hybridoma cells includes a light chain variable region and a heavy chain variable region, and the CDR region of the light chain variable region sequence includes such as SEQ ID NO: 1, SEQ ID NO: 1, SEQ ID NO: 1, SEQ ID NO: 1, SEQ ID NO: 1
  • SEQ ID NO: 1 The three sequences shown in ID NO: 2 and SEQ ID NO: 3;
  • the CDR region of the heavy chain variable region sequence includes three sequences as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6;
  • the light chain variable region amino acid sequence is shown in SEQ ID NO: 7,
  • the heavy chain variable region amino acid sequence is shown in SEQ ID NO: 7,
  • nucleotide sequence of the light chain variable region encoding the anti-OXA-23 type carbapenemase antibody is shown in SEQ ID NO:8;
  • nucleotide sequence of the heavy chain variable region encoding the anti-OXA-23 type carbapenemase antibody is shown in SEQ ID NO: 10;
  • the FA2 antibody produced by the anti-OXA-23 type carbapenemase hybridoma cell line includes a light chain variable region and a heavy chain variable region, and the CDR region of the light chain variable region sequence includes, for example, SEQ ID NO: 11, Three sequences shown in SEQ ID NO: 12 and SEQ ID NO: 13;
  • the CDR region of the heavy chain variable region sequence includes three sequences as shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16;
  • the light chain variable region amino acid sequence is shown in SEQ ID NO: 17;
  • the heavy chain variable region amino acid sequence is shown in SEQ ID NO: 19;
  • nucleotide sequence of the light chain variable region encoding the anti-OXA-23 type carbapenemase antibody is shown in SEQ ID NO: 18;
  • nucleotide sequence of the heavy chain variable region encoding the anti-OXA-23 type carbapenemase antibody is shown in SEQ ID NO: 20;
  • Anti-OXA-23 carbapenemase antibody produced by HB12 hybridoma cell line and FA2 hybridoma cell line can be used to detect OXA-23 carbapenemase antigen, anti-OXA-23 carbapenemase monoclonal antibody
  • the cloned antibody has good performance in all aspects, so it is suitable as an immunodiagnostic reagent for the preparation of in vitro diagnostic kits.
  • the in vitro diagnostic kits can be colloidal gold immunoassay kits, chemiluminescence kits or fluorescent immunoassay kits; alternatively, anti-OXA-23 carbapenemase antibodies can be made into microfluidic chips for detecting OXA-23 carbapenemase antigens.
  • OXA-23 type carbapenemase diagnostic reagent by colloidal gold method, it is necessary to first extract the antigen producing OXA-23 type carbapenemase, and after obtaining the high-purity antigen, it can stimulate a good immune response in mice. , and then use hybridoma technology to screen antibodies with high affinity and specificity for the development of related in vitro diagnostic reagents.
  • the two antibodies involved in this scheme are especially suitable for making double-antibody sandwich method immunocolloidal gold test strips, wherein antibody 1AA4 is a coated antibody, and antibody AH9 is a gold-labeled antibody.
  • the prepared double-antibody sandwich method immunocolloidal gold test strip The paper strip is more sensitive, and at the same time, the antibody HB12 can also be used as a gold-labeled antibody, and the antibody FA2 can be used as a coating antibody.
  • the present invention will be further described below through specific embodiments. Among them, the experimental methods without specific description of the operation steps were carried out in accordance with the corresponding product instructions.
  • the instruments, reagents, and consumables used in the examples can be purchased from commercial companies unless otherwise specified.
  • Obtained sequence The OXA-23 type gene sequence was subjected to whole gene synthesis and ligated into Pet-28a(+) vector.
  • Expanded culture Expand the correct transformants, transfer them to LB liquid medium containing kanamycin resistance at a ratio of 1:100, and culture with shaking at 37°C.
  • Ultrasonic broken bacteria about 1 g of bacteria was added to 30 mL of PBS to resuspend the bacteria, and then ultrasonically broken, the power was 400W, ultrasonic for 3 s, and the interval was 5 seconds. After about 30 minutes, the bacteria liquid was not viscous and clarified, and then centrifuged to remove the bacterial fragments. Cleared through a 0.45 ⁇ m filter.
  • the 6-week-old female Balb/c mice were immunized with purified OXA-23 carbapenemase to prepare antibodies with a protein content of 0.1 mg/ml. They were divided into 2 groups according to the immunization dose, with 5 mice in each group. Rats; calculated according to the protein content, the immunization dose of the first group is 25ug/mouse, and the immunization dose of the second group is 50ug/mouse.
  • the first immunization For the first immunization, take an appropriate amount of OXA-23 carbapenemase and dilute it with distilled water to 300ul, add an equal amount 300 ul of Freund's complete adjuvant was emulsified uniformly, and the mice were injected subcutaneously at multiple points to immunize mice; two weeks later, the same dose was taken for the second immunization.
  • the second immunization was intraperitoneal injection to immunize mice, and the additional immunization was performed after two weeks, which was also intraperitoneal injection. Mice, blood was collected from the tail after 7 days, and the serum titer of the mice was determined by ELISA.
  • the specific steps are: OXA-23 0.2ug/ml, 100ul/well, coat ELISA plate overnight at 4°C, spin dry, and wash 3 times with PBST.
  • 5% skimmed milk powder 200ul/well, blocked at 37°C for 2h.
  • Blood was collected from the mouse tail at 3000 rpm, and the serum was collected after centrifugation. From 1:1000, it was diluted to 1:512000 with PBS for subsequent use.
  • Spin dry, wash 3 times with PBST add primary antibody diluted in PBS from 1:1000 times, 100ul/well, 37°C, 1h.
  • Spin dry, wash 5 times with PBST add 100ul TMB/well, 37°C, develop color for 10min, stop, and read the value.
  • mice were boosted with the same amount of inoculation as the previous immunization, without adjuvant, by intraperitoneal injection.
  • the feeder cells were prepared one day before fusion, and one Balb/c mouse, 6-8 weeks old, was taken and killed by cervical dislocation after the eyeball was bled, and then sterilized in 75% alcohol for 5 minutes, fixed on the plate, and sterile in the ultra-clean bench. Cut the abdominal skin. Inject 10 ml of HAT selection medium into the abdominal cavity of the mouse with a sterile syringe, rub the abdomen lightly with an alcohol cotton ball, and withdraw the medium.
  • HAT medium Add 40ml of HAT medium, spread it into 4 96-well cell culture plates, 100 ⁇ L/well, 37°C, 5% CO 2 cell culture incubator.
  • Myeloma cells (Sp2/0 cells) were recovered one week before fusion, cultured in PRMI-1640 medium containing 10% fetal bovine serum, and subcultured in a 37°C, 5% CO 2 incubator.
  • the cells in the logarithmic growth phase were collected into a centrifuge tube, counted, and the cells were diluted to 10 7 cells/ml for use.
  • the Balb/c mice that had been boosted for 3 days were taken, the eyeballs were removed for bloodletting to prepare positive serum, the cervical vertebrae were sacrificed, sterilized with 75% alcohol for 5 min. connective tissue.
  • the spleen on the microporous copper mesh add fresh RPMI-1640 culture medium, first use a syringe to suck the culture medium and inject it from the spleen section, blow off the spleen cells, after repeated several times, use the inner plug of the syringe to gently grind the remaining spleen. , until there is no obvious red tissue mass.
  • the spleen cell suspension in the plate was gently pipetted and then transferred to a 50 ml centrifuge tube, centrifuged at 1000 r/min for 5 min, and the spleen cells were collected and counted for later use.
  • the immunized mouse splenocytes and Sp2/0 cells were mixed at a ratio of 10:1 according to the number of cells, added to a 50 ml centrifuge tube, centrifuged at 1000 r/min for 5 min, the supernatant was discarded, and the two cells were thoroughly mixed by rubbing gently in the palm of the hand.
  • the blue-cap bottle is filled with 37°C hot water, and the preheated 1ml DMSO/PEG is added dropwise to the fusion tube within 1min. Slowly and then fast, while adding, gently rotate the centrifuge tube.
  • the positive cells in the 96-well plate were subcloned by the limiting dilution method: first, prepare feeder cells according to the above method, take the hybridoma cells to be cloned for cell counting, and dilute the cells with HT medium to 5-8 cells/ ml, add 100 ⁇ L/well to a 96-well cell plate that has been plated with feeder cells, clone a 96-well cell plate for each hybridoma cell, and culture in a 37°C, 5% CO 2 cell incubator.
  • the number of clones in the cell wells was counted, marked, and replaced with a new medium after 7 days, and the cells were detected when the cells covered 1/3 to 1/2 of the entire bottom of the well.
  • the cell wells of the 96-well plate After 2-3 times of cloning, when all the cell wells of the 96-well plate are positive, it can be expanded, cultured, established, and frozen. Hybridoma cells confirmed by positive detection were expanded for culture and cryopreserved. The specific process is as follows: the vigorously growing hybridoma cells in good condition are gently blown off the cell flask with anti-blood-free DMEM, centrifuged at 1000 r/min for 5 min, and the supernatant is discarded.
  • a cryopreservation solution (containing 40% RPMI-1640 medium, 50% fetal bovine serum, 10% DMSO) was added, and the cells were blown off and then dispensed into cell cryopreservation tubes. Put the cryovials in a freezing box and place them in a -70°C freezer. One day later, transfer the cryovials to liquid nitrogen and make a record.
  • Purify ascites with Protein-G column the steps are as follows: take 2ml (n) of ascites, centrifuge at 10000g, take the clarified part, add 2ml (1:1) washing buffer, mix well, drain the column with 20% ethanol and then use 8mL The washing solution is balanced, the sample is passed through the column, the flow rate is 8S/drop, and the sample is loaded repeatedly for 3 times, and then the precipitate is washed with 15mL washing buffer, and the flow rate is 8S/drop. After washing, wash with 10mL of elution buffer.
  • the subclass identification of monoclonal antibody was carried out by the capture ELISA method, as follows: after diluting the subclass identification reagent of monoclonal antibody at 1:1000, add it to the enzyme label well, 100 ⁇ L/well, and incubate at 37°C for 1h; Wash three times with PBST and pat dry; Dilute the antibody 1:1000 times and add samples, 100 ⁇ L/well, incubate at 37°C for 1 h; Wash three times with PBST and pat dry; HRP enzyme-labeled goat anti-mouse IgG secondary antibody is diluted 1:6000 before adding sample, 100 ⁇ L/well, incubate at room temperature for 30 min; develop color for 10-20 min.
  • the OD450 reading value was significantly higher than that of the subtype reagents added in other wells as the subtype of the monoclonal antibody.
  • the antibody subtype of antibody HB12 is IgG2a
  • the antibody subtype of FA2 is IgG2b.
  • the antibody titer after purification was determined by indirect ELISA. The steps were as follows: OXA-23 carbapenemase was diluted to 0.2ug/mL, 100ul/well, and an uncoated control was set up. Dry, washed 3 times with PBST; 5% nonfat milk powder, 200ul/well, blocked at 37°C for 2 hours; dried, washed 3 times with PBST, and added with 1:1000 times the antibody (concentration of 1 mg/ml) for doubling dilution , a total of 12 gradients, and set up an uncoated control 100ul/well, 37°C, 1h.
  • SDS-PAGE method was used to identify the molecular weight and purity of the antibody; for gel preparation, the separation gel was 12%, and the stacking gel was 5%; sample preparation, 20ul sample + 20ul buffer, mixed, and boiled for 3min; each well was loaded with 20ul, and set up at the same time Protein pre-stained Marker control; 80 volts for 30 minutes, 120 volts for 2 hours; after electrophoresis, put into Coomassie brilliant blue solution for staining; destain, deionized water boiled for 5 minutes, a total of 3 times; the purified monoclonal antibody was SDS-PAGE identification, the band is clear, no heteroband, as shown in Figure 2, there are clear bands at 50KDa and 25KDa.
  • Ig variable region gene was cloned by RT-PCR method. Total RNA was extracted, single-stranded cDNA was synthesized, the total RNA of HB12 and FA2 hybridoma cell lines were extracted by Trizol method (purchased from Invitrogen), and total RNA was reversed into cDNA with M-MLV reverse transcriptase (purchased from Invitrogen). library.
  • the PCR reaction system (50 ⁇ l) was prepared as follows: cDNA: 2 ⁇ l; upstream primer (10 ⁇ M): 2 ⁇ l; downstream primer (10 ⁇ M): 2 ⁇ l; NTP mixture: 2 ⁇ l; pfu DNA polymerase (5U/ ⁇ l): 1 ⁇ l; 10X pfu Buffer II: 5 ⁇ l; ddH2O: make up to 50 ⁇ l.
  • Reaction conditions pre-denaturation at 95 °C for 5 min; repeat the following cycle 35 times: 95 °C for 30 s, 58 °C for 30 s, 72 °C for 1 min; finally, extension at 72 °C for 10 min.
  • VL and VH fragments were separated and recovered by agarose gel electrophoresis.
  • the recovered VL and VH fragments were respectively connected with pMD19-T (simple) vector (Takara company), and the connection system was: VL PCR product/VH PCR product 70ng each; pMD19-T (simple) vector 1 ⁇ l; Solution I connection The reaction solution was 5 ⁇ l; ddH2O was supplemented to 10 ⁇ l.
  • the ligation product was transformed into E.coli DH5 ⁇ competent bacteria, and after overnight incubation at 37°C, a single colony was picked, shaken at 37°C for 2 hours, and then identified by bacterial liquid PCR.
  • the cDNA of the corresponding antibody was used as a positive control.
  • the reaction system (25 ⁇ l) was prepared as follows: bacterial solution: 1 ⁇ l; upstream primer (10 ⁇ M): 1 ⁇ l; downstream primer (10 ⁇ M): 1 ⁇ l; dNTP Mixture (2.5Mm each) 2 ⁇ l; Taq DNA polymerase (5U/ ⁇ l): 0.5 ⁇ l ; 10 ⁇ Taq Buffer (Mg2+plus): 2.5 ⁇ l; replenish water to 25 ⁇ l.
  • the reaction conditions were the same as before.
  • the PCR-positive clones of the bacteria were selected for expansion and culture, and the positive cloned plasmids were extracted with a plasmid extraction kit (Takara Company), and sent to the detection sequence. At least 5 clone samples were submitted for each chain of each antibody, and at least three samples were sequenced with the same result.
  • the heavy chain and light chain variable region sequences of antibodies FA2 and HB12 were successfully cloned, and they were aligned with the characteristics of typical antibody variable region sequences.
  • the OXA-23 carbapenemase test card was prepared by the colloidal gold method, and the double-antibody sandwich method immunocolloidal gold test strip was prepared.
  • the preparation method was as follows:
  • Step 1 Add 0.1M K2CO3 solution to the colloidal gold solution while stirring, adjust the pH, add anti-OXA-23 carbapenemase monoclonal antibody FA2, add 10% bovine serum albumin solution, 2% PEG20000 After stirring, centrifuge at low speed to take the supernatant, and then centrifuge at high speed to take the precipitate, and use colloidal gold resuspension to make up the volume to form gold-labeled antibody;
  • step 2 the gold-labeled antibody is sprayed on the glass cellulose membrane, and dried to make a gold-labeled pad;
  • Step 3 Add 1% thimerosal sodium solution to anti-OXA-23 type carbapenemase monoclonal antibody HB12, mix well to form a detection line coating solution, and then add PBS and 1% thimerosal to goat anti-mouse IgG Sodium solution, after mixing, the coating liquid of quality control line is formed, and the coating liquid of quality control line and the coating liquid of detection line are drawn on the nitrocellulose membrane, and the coating membrane is obtained after drying;
  • Step 4 Stick the coating film on the bottom plate, put the gold label pad and absorbent paper on the coating film, and cut after lamination to obtain a colloidal gold method OXA-23 type carbapenemase detection card.

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Abstract

提供一种抗OXA-23型碳青霉烯酶杂交瘤细胞株，通过小鼠杂交瘤单克隆抗体筛选及RT-PCR法克隆Ig可变区基因，获得稳定分泌抗OXA-23型碳青霉烯酶抗体的杂交瘤细胞株及其可变区序列，并用ELISA方式对抗体结合特异性进行了鉴定；该抗体能够用于检测OXA-23型碳青霉烯酶，效价达到了1:640000以上，可制备成体外诊断试剂盒或微流体芯片，能够用于耐药菌株的早期分型，指导用药，辅助临床感染控制和治疗。

Description

一种抗OXA-23型碳青霉烯酶杂交瘤细胞株，单克隆抗体及应用 技术领域
本发明属于抗体制备技术领域，尤其是涉及一种抗OXA-23型碳青霉烯酶杂交瘤细胞株，单克隆抗体及应用。
背景技术
在β-内酰胺类抗生素中，碳青霉烯类具有最广泛的抗菌活性，能够抵抗绝大部分β-内酰胺酶、超广谱β-内酰胺酶的水解作用，并且能够减弱染色体介导的Amp C酶的作用。不动杆菌对许多抗生素如β-内酰胺类、氨基糖苷类、氟喹诺酮类等均有一定的耐药性，而碳青霉烯类抗生素对不动杆菌一直具有较好的抗菌活性。但随着临床上碳青霉烯类抗生素的广泛应用，鲍曼不动杆菌对其耐药率在逐年上升。近年不断有携带blaOXA-23的不动杆菌导致院感的报道。有文献报道国内耐碳青霉烯鲍曼不动杆菌80％为产OXA-23型碳青霉烯酶，使临床上耐碳青霉烯类药物的不动杆菌的治疗十分困难。据2011年卫生部耐药性监测网CHINET数据显示，我国鲍曼不动杆菌对碳青霉烯类药物亚胺培南和美罗培南的耐药率分别为56.8％和58.7％，表明耐碳青霉烯类鲍曼不动杆菌给临床抗感染治疗带来严峻的挑战。
实验室检测碳青霉烯酶的方法众多。主要包括改良Hodge试验、Carba NP试验、改良碳青霉烯灭活试验(modified carbapenem inactivation method，mCIM)、酶抑制剂增强试验、免疫金标试验以及分子生物学方法等。免疫诊断试剂是体外诊断试剂中发展最快的的细分领域，利用抗原与抗体之间的特异性结合进行定性或定量检测。
发明内容
本发明提供一种抗OXA-23型碳青霉烯酶杂交瘤细胞株，单克隆抗体及应用。
本发明采用的技术方案是：一种抗OXA-23型碳青霉烯酶杂交瘤细胞株，命名为HB12，保藏编号为CGMCC No.20285；或者命名为FA2，保藏编号为CGMCC No.20286。
一种抗OXA-23型碳青霉烯酶抗体，命名为抗体HB12，包括轻链可变区和重链可变区，轻链可变区序列的CDR区中包括如SEQ ID NO:1、SEQ ID NO:2 和SEQ ID NO:3所示的三个序列，重链可变区序列的CDR区中包括如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的三个序列；
SEQ ID NO:1
KASQDVSIAVA(CDR-L1)
SEQ ID NO:2
WASTRHT(CDR-L2)
SEQ ID NO:3
QQHYSTPYL(CDR-L3)
SEQ ID NO:4
GYTFTSYWMY(CDR-H1)
SEQ ID NO:5
EIYPTNGRTNYNEKFKT(CDR-H2)
SEQ ID NO:6
YYYGSYAMDY(CDR-H3)
或者，
命名为抗体FA2，轻链可变区序列的CDR区中包括如SEQ ID NO:11、SEQ ID NO:12和SEQ ID NO:13所示的三个序列，重链可变区序列的CDR区中包括如SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示的三个序列；
SEQ ID NO:11
KGSQDVRTAVA(CDR-L1)
SEQ ID NO:12
WASTRHT(CDR-L2)
SEQ ID NO:13
QQHYSIPYT(CDR-L3)
SEQ ID NO:14
GFSLTSYGVH(CDR-H1)
SEQ ID NO:15
VIWRGGNTDYNAAFMSRLS(CDR-H2)
SEQ ID NO:16
SLTTSTFDY(CDR-H3)
优选地，抗体HB12轻链可变区氨基酸序列为SEQ ID NO:7所示，重链可变区氨基酸序列为SEQ ID NO:9所示；
SEQ ID NO:7轻链可变区氨基酸序列
Figure PCTCN2021135404-appb-000001
SEQ ID NO:9重链可变区氨基酸序列
Figure PCTCN2021135404-appb-000002
或者，
抗体FA2轻链可变区氨基酸序列为SEQ ID NO:17所示，重链可变区氨基酸序列为SEQ ID NO:19所示；
SEQ ID NO:17轻链可变区氨基酸序列
Figure PCTCN2021135404-appb-000003
SEQ ID NO:19重链可变区氨基酸序列
Figure PCTCN2021135404-appb-000004
优选地，由保藏编号为CGMCC No.20285和CGMCC No.20286的抗OXA-23型碳青霉烯酶杂交瘤细胞株产生。
一种核酸分子，包含编码抗OXA-23型碳青霉烯酶抗体的核苷酸序列。
优选地，核酸分子编码抗体HB12的轻链可变区的核苷酸序列如SEQ ID NO:8所示，核酸分子编码抗体HB12的重链可变区的核苷酸序列如SEQ ID NO:10所示；
SEQ ID NO:8轻链可变区碱基序列
Figure PCTCN2021135404-appb-000005
SEQ ID NO:10重链可变区碱基序列
Figure PCTCN2021135404-appb-000006
或者，
核酸分子编码抗体FA2的轻链可变区的核苷酸序列如SEQ ID NO:18所示，核酸分子编码抗体FA2的重链可变区的核苷酸序列如SEQ ID NO:20所示；
SEQ ID NO:18轻链可变区碱基序列
Figure PCTCN2021135404-appb-000007
SEQ ID NO:20重链可变区碱基序列
Figure PCTCN2021135404-appb-000008
Figure PCTCN2021135404-appb-000009
抗OXA-23型碳青霉烯酶抗体在检测OXA-23型碳青霉烯酶抗原中的应用。
优选地，将抗OXA-23型碳青霉烯酶抗体用于体外诊断试剂盒或微流体芯片，体外诊断试剂盒为胶体金免疫试剂盒、化学发光试剂盒、放射免疫试剂盒、酶联免疫试剂盒或荧光免疫试剂盒。
优选地，制备双抗体夹心法免疫胶体金试纸条，抗体HB12为包被抗体，抗体FA2为金标抗体；或者，抗体HB12为金标抗体，抗体FA2为包被抗体。
本发明具有的优点和积极效果是：提供一种抗OXA-23型碳青霉烯酶抗体，能够用于检测OXA-23型碳青霉烯酶，效价达到了1:640000以上；将其制备成体外诊断试剂盒或微流体芯片，能够用于耐药菌株的早期分型，指导用药，辅助临床感染控制和治疗。
附图说明
图1是OXA-23型碳青霉烯酶蛋白电泳图；
图2是抗OXA-23型碳青霉烯酶抗体蛋白电泳图。
具体实施方式
下面对本发明的实施例做出说明。
本发明涉及抗OXA-23型碳青霉烯酶杂交瘤细胞株，生物材料命名为HB12，属杂交瘤细胞，其保藏编号是CGMCC No.20285；保藏地为中国微生物菌种保藏管理委员会普通微生物中心，其保藏日期为2020年8月27日，检测为存活。另有一株抗OXA-23型碳青霉烯酶杂交瘤细胞株，生物材料命名为FA2，属杂交瘤细胞，其保藏编号是CGMCC No.20286；保藏地为中国微生物菌种保藏管理委员会普通微生物中心，其保藏日期为2020年8月27日，检测为存活。
本发明涉及抗OXA-23型碳青霉烯酶杂交瘤细胞株通过小鼠杂交瘤单克隆抗体筛选及RT-PCR法克隆Ig可变区基因，获得稳定分泌抗OXA-23型碳青霉烯酶抗体的杂交瘤细胞株及其可变区序列，并用ELISA方式对抗体结合特异性 进行了鉴定。
抗OXA-23型碳青霉烯酶杂交瘤细胞生产的HB12抗体，包括轻链可变区和重链可变区，轻链可变区序列的CDR区中包括如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的三个序列；
SEQ ID NO:1
KASQDVSIAVA(CDR-L1)
SEQ ID NO:2
WASTRHT(CDR-L2)
SEQ ID NO:3
QQHYSTPYL(CDR-L3)
重链可变区序列的CDR区中包括如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的三个序列；
SEQ ID NO:4
GYTFTSYWMY(CDR-H1)
SEQ ID NO:5
EIYPTNGRTNYNEKFKT(CDR-H2)
SEQ ID NO:6
YYYGSYAMDY(CDR-H3)
轻链可变区氨基酸序列为SEQ ID NO:7所示，
SEQ ID NO:7轻链可变区氨基酸序列
Figure PCTCN2021135404-appb-000010
重链可变区氨基酸序列为SEQ ID NO:7所示，
SEQ ID NO:9重链可变区氨基酸序列
Figure PCTCN2021135404-appb-000011
编码抗OXA-23型碳青霉烯酶抗体的轻链可变区的核苷酸序列如SEQ ID  NO:8所示；
SEQ ID NO:8
Figure PCTCN2021135404-appb-000012
编码抗OXA-23型碳青霉烯酶抗体的重链可变区的核苷酸序列如SEQ ID NO:10所示；
SEQ ID NO:10
Figure PCTCN2021135404-appb-000013
抗OXA-23型碳青霉烯酶杂交瘤细胞株生产的FA2抗体，包括轻链可变区和重链可变区，轻链可变区序列的CDR区中包括如SEQ ID NO:11、SEQ ID NO:12和SEQ ID NO:13所示的三个序列；
SEQ ID NO:11
KGSQDVRTAVA(CDR-L1)
SEQ ID NO:12
WASTRHT(CDR-L2)
SEQ ID NO:13
QQHYSIPYT(CDR-L3)
重链可变区序列的CDR区中包括如SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示的三个序列；
GYSEQ ID NO:14
GFSLTSYGVH(CDR-H1)
SEQ ID NO:15
VIWRGGNTDYNAAFMSRLS(CDR-H2)
SEQ ID NO:16
SLTTSTFDY(CDR-H3)
轻链可变区氨基酸序列为SEQ ID NO:17所示；
SEQ ID NO:17轻链可变区氨基酸序列
Figure PCTCN2021135404-appb-000014
重链可变区氨基酸序列为SEQ ID NO:19所示；
SEQ ID NO:19重链可变区氨基酸序列
Figure PCTCN2021135404-appb-000015
编码抗OXA-23型碳青霉烯酶抗体的轻链可变区的核苷酸序列如SEQ ID NO:18所示；
SEQ ID NO:18
Figure PCTCN2021135404-appb-000016
编码抗OXA-23型碳青霉烯酶抗体的重链可变区的核苷酸序列如SEQ ID  NO:20所示；
SEQ ID NO:20
Figure PCTCN2021135404-appb-000017
HB12杂交瘤细胞株和FA2杂交瘤细胞株所生产的抗OXA-23型碳青霉烯酶抗体可用于检测OXA-23型碳青霉烯酶抗原，抗OXA-23型碳青霉烯酶单克隆抗体在各方面均有较佳表现，从而适合作为免疫诊断试剂用于制备体外诊断试剂盒，体外诊断试剂盒可为胶体金免疫试剂盒、化学发光试剂盒、放射免疫试剂盒、酶联免疫试剂盒或荧光免疫试剂盒；又或者可将抗OXA-23型碳青霉烯酶抗体制成微流体芯片，用于检测OXA-23型碳青霉烯酶抗原。
开发胶体金法的OXA-23型碳青霉烯酶诊断试剂，首先需要提取产OXA-23型碳青霉烯酶的抗原，获得高纯度的抗原后，在小鼠体内激起良好的免疫反应，进而采用杂交瘤技术，筛选高亲和力和特异性的抗体，用于相关体外诊断试剂的开发。本方案所涉及的两种抗体尤其适合搭配制成双抗体夹心法免疫胶体金试纸条，其中抗体1AA4为包被抗体，抗体AH9为金标抗体，制得的双抗体夹心法免疫胶体金试纸条灵敏性更高，同时，也可将抗体HB12做为金标抗体，抗体FA2为包被抗体。下面通过具体实施例对本发明做出进一步说明。其中，未具体说明操作步骤的实验方法，均按照相应商品说明书进行，实施例中所用到的仪器、试剂、耗材如无特殊说明，均可从商业公司购买得到。
实施例1：抗OXA-23型碳青霉烯酶抗体的制备
1.1抗原表达
获得序列：将OXA-23型基因序列进行全基因合成并连接到Pet-28a(+)载体上。
转化到宿主菌：将含有目的基因的载体Pet-28a(+)转化到克隆宿主大肠杆菌(BL21系列的Rosetta TM(DE3)菌)。
扩大培养：将验证正确的转化子进行扩大培养，并按1:100比例转接到含有卡那霉素抗性的LB液体培养基中，37℃摇床震荡培养。
诱导：当菌液OD值在0.4-0.6之间时，加入IPTG终浓度为1mM，37℃培养3小时，离心收集菌体。
超声破碎菌体：约1g菌体加入30mL PBS重悬菌体后超声破碎，功率为400W，超声3s，间隔5s，约30分钟后，菌液不黏稠且澄清后，离心去掉菌体碎片，上清过0.45μm滤膜。
纯化：将上清液上样于提前填好填料并用平衡缓冲液平衡过的镍柱中，上样结束后进行咪唑浓度梯度洗脱，洗涤液咪唑浓度为150mM、250mM,洗脱液咪唑浓度500mM时可获得纯度较高目的蛋白，分子量和预计33.kD相符，SDS-PAGE见图1，用BCA法定量后分装进行后续实验。
1.2小鼠免疫
用纯化的OXA-23型碳青霉烯酶免疫6周龄左右的雌性Balb/c小鼠，进行抗体制备，蛋白含量为0.1mg/ml，按照免疫剂量分为2组，每组5只小鼠；按照蛋白含量计算，第一组免疫剂量为25ug/只，第二组免疫剂量为50ug/只，首免，取适量OXA-23型碳青霉烯酶经蒸馏水稀释至300ul，加入等量弗氏完全佐剂300ul乳化均匀，皮下多点注射免疫小鼠；两周后，取相同剂量进行二免，二免为腹腔注射免疫小鼠，两周后再追加免疫一次，亦为腹腔注射免疫小鼠，7天后鼠尾采血，ELISA测定小鼠血清效价。
具体步骤为：OXA-23 0.2ug/ml，100ul/孔，4℃过夜包被ELISA板，甩干，PBST洗涤3次。5％脱脂乳粉，200ul/孔，37℃封闭2h。小鼠鼠尾采血，3000转/min，离心后收集血清，从1:1000开始用PBS进行倍比稀释至1:512000，备用。甩干，PBST洗涤3次，1:1000倍起加入PBS稀释的一抗，100ul/孔，37℃，1h。甩干，PBST洗涤3次,加PBS 1:6000倍稀释的羊抗鼠二抗，100ul/孔，37℃，45min。甩干，PBST洗涤5次,加100ul TMB/孔，37℃，显色10min，终止，读值。
1.3细胞融合
融合前三天进行小鼠加强免疫，接种量同前次免疫，不加佐剂，腹腔注射。融合前一天准备饲养层细胞，取6-8周龄小鼠Balb/c 1只，取眼球放血后颈椎脱位致死，放于75％酒精中消毒5min，固定于盘上，在超净台中无菌剪开腹部皮肤。用无菌注射器吸取HAT选择培养液10ml注入小鼠腹腔，用酒精棉球轻揉腹部，抽回培养基。加入40ml HAT培养液中，铺入到4块96孔细胞培养板中，100μL/孔，37℃，5％CO 2细胞培养箱中培养。融合前一周复苏骨髓瘤细胞(Sp2/0细胞)，用含10％胎牛血清的PRMI-1640培养基培养，37℃，5％CO 2培养箱中传代培养。将处于对数生长期的细胞收集至离心管中，细胞计数，把细胞稀释为10 7个/ml备用。取加强免疫3天的Balb/c小鼠，摘眼球放血制备阳性血清，脱颈椎处死，75％酒精消毒5min，在超净工作台无菌取出脾脏，在无菌平皿中冼涤数次，剥离结缔组织。将脾脏放在微孔铜网上，加入新鲜的RPMI-1640培养液，先用注射器吸取培养液由脾脏一段注入，吹下脾细胞，反复数次之后，用注射器的内塞轻轻将剩余脾脏研磨，直到无明显的红色组织块。将平皿中脾细胞悬液轻轻吹打后转移到50ml离心管中，1000r/min离心5min，收集脾细胞，计数后备用。将免疫鼠脾细胞与Sp2/0细胞按细胞数量10:1混合，加入50ml的离心管内，1000r/min离心5min，弃上清，在手心轻轻摩擦使两种细胞充分混匀，将离心管至于100mL蓝盖瓶内，蓝盖瓶内装有37℃热水，将预热好的1ml DMSO/PEG在1min内逐滴加入融合管内，先慢后快，边加边轻轻旋转离心管。然后立即加入无抗无血RPMI-1640培养液终止反应，第一分钟加1ml，第二分钟加2ml，第三分钟加3ml，第四分钟加4ml。37℃水浴5min，后800r/min离心5min，弃上清，将沉淀以HAT悬起，混匀到40ml含37℃预热的20％小牛血清的HAT选择培养液中，铺入已加有饲养细胞的96孔细胞板中，100μL/孔，将培养板放入37℃，5％CO 2培养箱培养。7d后将用新鲜的HAT培养基对细胞板半换液，10天后用HT培养基全换液。将96孔板中检测阳性的细胞采用有限稀释法进行亚克隆：首先按照上述方法制备饲养层细胞，取待克隆杂交瘤细胞进行细胞计数，用HT培养基将细胞稀释至5-8个细胞/ml，加入到已铺饲养细胞的96孔细胞板中100μL/孔，每株杂交瘤细胞克隆一块96孔细胞板，37℃、5％CO 2细胞培养箱中培养。约5天后数出细胞孔里的克隆数，标记，7天时并换新的培养基，待细胞铺满整个孔底的1/3～1/2时检测。经过2-3次克隆化，待96孔板 所有细胞孔均为阳性时，即可进行扩大培养，定株，冻存。将检测阳性确定定株的杂交瘤细胞扩大培养并冻存。具体过程如下：将生长旺盛、状态良好的杂交瘤细胞用无抗无血DMEM轻轻从细胞瓶上吹下，1000r/min离心5min，弃去上清。加入冻存液(含40％RPMI-1640培养液、50％胎牛血清、10％DMSO)，将细胞吹散后分装到细胞冻存管中。将冻存管放入冻存盒置于-70℃冰箱中，一天后将冻存管转移入液氮中，做好记录。
1.4腹水制备
取10-12周龄雌性Balb/c小鼠，腹腔注射无菌液体石蜡，0.5mL/只，7天后腹腔注射培养至对数期的杂交瘤细胞，5×10 6个细胞/只。每天注意观察，约7-10天，待小鼠腹部出现明显隆起后，用75％酒精棉球消毒下腹部皮肤，用16号针头刺入腹腔，收集腹水。待腹水再生积聚后，再次收集。将收集的腹水3000r/min离心10min，取中间澄清部分，用滤纸过滤后，分装，-70℃保存。
1.5抗体纯化
用Protein-G柱子进行腹水纯化，步骤如下：取腹水2ml(n)，10000g离心，取澄清部分，加入2ml(1:1)洗涤缓冲液，混匀，柱子用20％乙醇流尽后用8mL洗涤液平衡，样本过柱子，流速为8S/滴沉，反复上样3次，然后用15mL洗涤缓冲液进行洗涤淀，流速为8S/滴沉，洗涤完毕后用10mL的洗脱缓冲液进行洗脱，洗脱完毕会用1M Tris PH＝9调PH至7.4，然后用浓缩注进行浓缩，于50kd透析袋，PBS，4℃透析过夜。获得抗体HB12和抗体FA2。
实施例2：抗OXA-23型碳青霉烯酶抗体的鉴定
2.1抗体亚类鉴定
按照SIGMA试剂盒说明书，以捕获ELISA的方法进行单抗的亚类鉴定，具体如下：将单抗亚类鉴定试剂1:1000稀释后，加入酶标孔中，100μL/孔，37℃孵育1h；PBST洗三次，拍干；将抗体1:1000倍稀释后加样，100μL/孔，37℃孵育1h；PBST洗三次，拍干；HRP酶标羊抗鼠IgG二抗以1:6000稀释后加样，100μL/孔，室温孵育30min；显色10～20min。以OD450读值明显高于其他孔所加亚类试剂为单抗所属亚类类型。抗体HB12的抗体亚型为IgG2a、FA2的抗体亚型为IgG2b。
2.2抗体效价测定
采用间接ELISA法进行纯化后抗体效价测定，步骤如下：OXA-23型碳青霉烯酶分别稀释至0.2ug/mL，100ul/孔，同时设立不包被对照，4℃过夜包被，甩干，PBST洗涤3次；5％脱脂乳粉，200ul/孔，37℃封闭2h；甩干，PBST洗涤3次，加入从1:1000倍开始进行倍比稀释的抗体(浓度为1mg/ml)，共计12个梯度，同时设立不包被对照100ul/孔，37℃，1h。甩干，PBST洗涤3次，加PBS1:6000倍稀释的羊抗鼠二抗，100ul/孔，37℃，45min。甩干，PBST洗涤5次，加100ulTMB/孔，37℃，显色10min，终止，读值。纯化后抗体稀释至1mg/ml，检测其效价达到1:640000以上。
2.3抗体纯度及分子量鉴定
采用SDS-PAGE法进行抗体分子量及纯度鉴定；制胶，分离胶为12％，浓缩胶为5％；制样，20ul样品+20ul buffer，混匀，煮沸3min；每孔上样20ul，同时设立蛋白预染Marker对照；80伏30min，120伏2h；电泳完毕后，放入考马斯亮蓝溶液进行染色；脱色，去离子水煮沸脱色，每次5min，共计3次；纯化后的单克隆抗体经SDS-PAGE鉴定，条带清晰，无杂带，如图2所示，在50KDa和25KDa处各有清晰的条带。
实施例3：抗OXA-23型碳青霉烯酶抗体的基因验证
通过RT-PCR法克隆Ig可变区基因。提取总RNA，合成单链cDNA，用Trizol法(试剂盒购自Invitrogen)分别提取HB12和FA2杂交瘤细胞株的总RNA，用M-MLV逆转录酶(购自Invitrogen)将总RNA逆转为cDNA文库。
重链骨架区上游引物
P1：5’SAGGTGMAGCTKCASSARTCWGG3’
重链可变区下游引物
P2：5’TGGGGSTGTYGTTTTGGCTGMRGAGACRGTGA3’
轻链前导肽上游引物
P3：5’ATGGATTTTCAAGTGCAGATTTTCAG3’
轻链可变区下游引物
P4：5’GGATACAGTTGGTGCAGCATCAGCCCGTTT3’
配制PCR反应体系(50μl)为：cDNA:2μl；上游引物(10μM):2μl；下游引物(10μM):2μldNTP mixture:2μl；pfu DNA聚合酶(5U/μl):1μl；10X pfu BufferⅡ:5μl；ddH2O:补足至50μl。
反应条件：95℃预变性5min；重复如下循环35次：95℃30s，58℃30s，72℃1min；最后，72℃延伸10min。
琼脂糖凝胶电泳分离并回收VL、VH片段。将回收后的VL、VH片段分别与pMD19-T(simple)载体(Takara公司)进行连接，连接体系为：VL PCR产物/VH PCR产物各70ng；pMD19-T(simple)载体1μl；Solution I连接反应液5μl；ddH2O补足至10μl。
4℃连接过夜。
连接产物转化入E.coli DH5α感受态细菌中，37℃过夜培养后，挑取单个菌落，37℃震摇2小时后进行菌液PCR鉴定，以对应抗体的cDNA为阳性对照。配制反应体系(25μl)为：菌液：1μl；上游引物(10μM)：1μl；下游引物(10μM)：1μl；dNTP Mixture(各2.5Mm)2μl；Taq DNA聚合酶(5U/μl)：0.5μl；10×Taq Buffer(Mg2+plus)：2.5μl；补水至25μl。反应条件同前。
选取菌PCR阳性的克隆扩大培养，用质粒提取试剂盒(Takara公司)提取阳性克隆质粒，送检测序。每个抗体的每条链至少送检5个克隆样品，至少三个样品测序结果相同为止。成功克隆得到抗体FA2、HB12的重链、轻链可变区序列，经比对符合典型抗体可变区序列特征。
实施例4：OXA-23型碳青霉烯酶检测试剂盒的制备
使用胶体金法制备OXA-23型碳青霉烯酶测卡，制备双抗体夹心法免疫胶体金试纸条，制备方法如下：
步骤一向胶体金溶液中边搅拌边加入0.1M K2CO3溶液，调节pH值后加入抗OXA-23型碳青霉烯酶单克隆抗体FA2，搅拌后加入10％的牛血清白蛋白溶液，2％PEG20000，搅拌后低速离心取上清，再高速离心后取沉淀，用胶体金重悬液定容形成金标抗体；
步骤二将金标抗体喷于玻璃纤维素膜，烘干制成金标垫；
步骤三向抗OXA-23型碳青霉烯酶单克隆抗体HB12中加入1％的硫柳汞钠 溶液，混匀后形成检测线包被液，再向羊抗鼠IgG中加入PBS和1％的硫柳汞钠溶液，混匀后形成质控线包被液，将质控线包被液和检测线包被液划在硝酸纤维素膜上，烘干后获得包被膜；
步骤四将包被膜贴在底板上，将金标垫和吸水纸搭上包被膜，层压后切割获得胶体金法OXA-23型碳青霉烯酶检测卡。
以上对本发明的实施例进行了详细说明，但所述内容仅为本发明的较佳实施例，不能被认为用于限定本发明的实施范围。凡依本发明申请范围所作的均等变化与改进等，均应仍归属于本发明的专利涵盖范围之内。
Figure PCTCN2021135404-appb-000018
Figure PCTCN2021135404-appb-000019
Figure PCTCN2021135404-appb-000020
Figure PCTCN2021135404-appb-000021
Figure PCTCN2021135404-appb-000022
Figure PCTCN2021135404-appb-000023
Figure PCTCN2021135404-appb-000024
Figure PCTCN2021135404-appb-000025
Figure PCTCN2021135404-appb-000026
Figure PCTCN2021135404-appb-000027

Claims (9)

  1. 一种抗OXA-23型碳青霉烯酶杂交瘤细胞株，其特征在于：命名为HB12，保藏编号为CGMCC No.20285；或命名为FA2，保藏编号为CGMCC No.20286。
  2. 一种抗OXA-23型碳青霉烯酶抗体，其特征在于：抗体HB12，包括轻链可变区和重链可变区，轻链可变区序列的CDR区中包括如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的三个序列，重链可变区序列的CDR区中包括如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的三个序列；
    或者，
    抗体FA2，轻链可变区序列的CDR区中包括如SEQ ID NO:11、SEQ ID NO:12和SEQ ID NO:13所示的三个序列，重链可变区序列的CDR区中包括如SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示的三个序列。
  3. 根据权利要求2所述的抗OXA-23型碳青霉烯酶抗体，其特征在于：抗体HB12轻链可变区氨基酸序列为SEQ ID NO:7所示，重链可变区氨基酸序列为SEQ ID NO:9所示；
    或者，
    抗体FA2轻链可变区氨基酸序列为SEQ ID NO:17所示，重链可变区氨基酸序列为SEQ ID NO:19所示。
  4. 根据权利要求2或3所述的抗OXA-23型碳青霉烯酶抗体，其特征在于：抗体HB12由保藏编号为CGMCC No.20285的抗OXA-23型碳青霉烯酶杂交瘤细胞株产生；
    或者，
    抗体FA2由保藏编号为CGMCC No.20286的抗OXA-23型碳青霉烯酶杂交瘤细胞株产生。
  5. 一种核酸分子，其特征在于：包含编码权利要求2或3所述的抗OXA-23型碳青霉烯酶抗体的核苷酸序列。
  6. 根据权利要求5所述的核酸分子，其特征在于：所述核酸分子编码抗体HB12的轻链可变区的核苷酸序列如SEQ ID NO:8所示，所述核酸分子编码抗体HB12的重链可变区的核苷酸序列如SEQ ID NO:10所示；
    或者，
    所述核酸分子编码抗体FA2的轻链可变区的核苷酸序列如SEQ ID NO:18所示， 所述核酸分子编码抗体FA2的重链可变区的核苷酸序列如SEQ ID NO:20所示。
  7. 权利要求2-4中任一所述的抗OXA-23型碳青霉烯酶抗体在检测OXA-23型碳青霉烯酶抗原中的应用。
  8. 根据权利要求7所述的抗OXA-23型碳青霉烯酶抗体在检测OXA-23型碳青霉烯酶抗原中的应用，其特征在于：将抗OXA-23型碳青霉烯酶抗体用于体外诊断试剂盒或微流体芯片，所述体外诊断试剂盒为胶体金免疫试剂盒、化学发光试剂盒、放射免疫试剂盒、酶联免疫试剂盒或荧光免疫试剂盒。
  9. 根据权利要求7所述的抗OXA-23型碳青霉烯酶抗体在检测OXA-23型碳青霉烯酶抗原中的应用，其特征在于：制备双抗体夹心法免疫胶体金试纸条，抗体HB12为包被抗体，抗体FA2为金标抗体；
    或者，抗体HB12为金标抗体，抗体FA2为包被抗体。
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