CN114456265B - anti-HFABP monoclonal antibody and application thereof - Google Patents

anti-HFABP monoclonal antibody and application thereof Download PDF

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CN114456265B
CN114456265B CN202210032615.7A CN202210032615A CN114456265B CN 114456265 B CN114456265 B CN 114456265B CN 202210032615 A CN202210032615 A CN 202210032615A CN 114456265 B CN114456265 B CN 114456265B
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monoclonal antibody
hfabp
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刘如石
郑姣
邱义兰
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Hunan Xuxiang Biotechnology Co ltd
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Abstract

The invention discloses an anti-HFABP monoclonal antibody and application thereof. The expressed HFABP is used as antigen to immunize BALB/c mice, 17 monoclonal antibodies of anti-human HFABP protein are obtained through screening, the monoclonal antibodies of each strain are labeled and paired by magnetic beads and alkaline phosphatase, the paired antibodies capable of achieving the optimal detection effect are obtained, the paired antibodies are further subjected to heavy chain and light chain sequence determination, and the CDR sequences of the heavy chain and the light chain of the antibodies are obtained through conventional method analysis. The prepared antibody can be applied to a chemiluminescent enzyme immunoassay (CLEIA) method, can accurately and quantitatively detect the content of HFABP in a patient sample, can be used for early detection and monitoring of myocardial ischemia injury, and has the advantages of strong specificity, high sensitivity, good accuracy, rapidness, simplicity and the like.

Description

anti-HFABP monoclonal antibody and application thereof
Technical Field
The invention belongs to the field of immunology, and particularly relates to an anti-HFABP monoclonal antibody, and a preparation method and application thereof.
Background
Human Heart Fatty Acid Binding Protein (HFABP) is a low molecular weight protein of 15 kDa, consisting of 132 amino acids. Is mainly located in the heart and occupies 5-15% of cytosolic protein pool, and these proteins are involved in the transportation of long chain fatty acids in cells, which are the main sources of energy for heart to mitochondria. H-FABP is mainly found in cardiomyocytes and normally ranges between 0.0 and 5.5 nanograms per milliliter in serum or plasma of healthy humans. In the acute myocardial infarction process, myocardial cells are damaged, the myocardial cells can be rapidly released into blood plasma, the myocardial cells can be detected after 1-3h, the myocardial cells can be recovered to be normal after 6-8h, and the characteristics of HFABP are beneficial to more comprehensively diagnosing and treating AMI, so that the myocardial cells become an emerging biomarker for detecting AMI.
Currently, common indexes for clinical diagnosis of AMI are myoglobin, troponin, creatine kinase, isozymes thereof, and the like. However, these indicators only increased after several hours of myocardial necrosis, whereas in the early diagnosis of AMI, HFABP was abnormally sensitive within the first 6 hours after myocardial injury, and could even be detected at 1 hour. The sensitivity and the specificity of the strain are better than those of cTnI and CK-MB, but after 6 hours, the accuracy is gradually reduced. Therefore, the HFABP is combined with the detection of other markers such as cTnI and the like, and the accuracy of clinical diagnosis can be effectively improved. Furthermore, HFABP is also associated with other clinical parameters such as leukocytosis in AMI patients. Thus, HFABP can be used as a better biochemical index for early diagnosis, risk assessment and prognosis of AMI.
Clinically, the detection methods of HFABP include Radioimmunoassay (RIA), latex Turbidimetric Immunoassay (LTIA), enzyme-linked immunosorbent assay (ELISA), colloidal gold immunochromatography, and the like. These methods have the major disadvantages of high cost, false positives, low sensitivity, and the like.
The present study was aimed at developing high affinity and high specificity monoclonal antibodies (mAbs) to HFABP and then creating a magnetic particle-based chemiluminescent enzyme immunoassay (CLEIA) that can be used in a low cost, low cost platform for rapid and accurate quantification of HFABP. In order to evaluate the application prospects of the developed monoclonal antibodies and CLEIA, serum HFABP values measured by CLEIA were compared with clinical values detected in hospitals.
Disclosure of Invention
The invention provides an anti-HFABP monoclonal antibody, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of a heavy chain variable region of the monoclonal antibody are shown as SEQ ID NO.1, 2 and 3 respectively; and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region of said monoclonal antibody are shown in SEQ ID NOS 4, 5 and 6, respectively.
Preferably, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO. 14; and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID NO. 16.
Preferably, the nucleotide sequence of the heavy chain of the monoclonal antibody is shown as SEQ ID NO. 13, and the nucleotide sequence of the light chain of the monoclonal antibody is shown as SEQ ID NO. 15.
The invention also provides an anti-HFABP monoclonal antibody, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of the monoclonal antibody are shown as SEQ ID NO. 7, 8 and 9 respectively; and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region of said monoclonal antibody are shown in SEQ ID NOS 10, 11 and 12, respectively.
Preferably, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO. 18; and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID NO. 20.
Preferably, the monoclonal antibody further comprises a constant region, and the constant region of the heavy chain of the monoclonal antibody is any one of IgG1, igG2, igG3 or IgG 4; the constant region of the light chain of the monoclonal antibody is kappa type or lambda type.
Preferably, the invention discloses a nucleotide molecule encoding said monoclonal antibody.
Preferably, the nucleotide sequence of the heavy chain of the monoclonal antibody is shown as SEQ ID NO. 17, and the nucleotide sequence of the light chain of the monoclonal antibody is shown as SEQ ID NO. 19.
The invention discloses an expression vector, which comprises the nucleotide molecule.
The invention discloses a cell, which comprises the vector.
The invention discloses a monoclonal antibody, which is obtained by the expression of cells.
The invention discloses application of a monoclonal antibody in preparing a kit for detecting HFABP.
Preferably, the kit is a chemiluminescent enzyme immunoassay kit.
The invention discloses a chemiluminescent enzyme immunoassay kit for detecting HFABP, which comprises the monoclonal antibody.
In order to achieve the above technical solution, the present inventors utilized prokaryotic expression technology to express and purify human HFABP protein, which is against the shortcomings of the prior art. The purified human HFABP protein has high purity, is used for immunizing BALB/c mice, obtains 17 hybridoma cells which stably secrete anti-human HFABP protein monoclonal antibodies through four times of screening by a hybridoma technology, marks and pairs magnetic beads and alkaline phosphatase, and establishes a chemiluminescent enzyme immunoassay (CLEIA) which is safe, can rapidly and accurately detect the content of the central fatty acid binding protein of a sample to be detected.
The technical scheme adopted by the invention is as follows: a chemiluminescent enzyme immunoassay (CLEIA) method for detecting HFABP was established comprising: the monoclonal antibody of the anti-human heart-type fatty acid binding protein, the magnetic bead coupled antibody for detecting the heart-type fatty acid binding protein and the alkaline phosphatase labeled antibody are prepared.
The heart type fatty acid binding protein immunodetection reagent comprises: an anti-human HFABP monoclonal antibody; magnetic bead conjugate and enzyme-labeled conjugate; the magnetic bead conjugate is formed by coupling magnetic beads with monoclonal antibodies of anti-human heart-shaped fatty acid binding proteins; the enzyme-labeled conjugate is formed by an alkaline phosphatase-labeled monoclonal antibody against human heart-type fatty acid binding protein.
The invention discloses a preparation method of a heart-type fatty acid binding protein immunodetection reagent.
The invention discloses a preparation method of a monoclonal antibody of an anti-human heart-shaped fatty acid binding protein, which comprises the following steps:
(1) Fully emulsifying the purified recombinant HFABP protein and Freund's complete adjuvant, and performing primary immunization on 5 BALB/c female mice of 4-6 weeks of age at a dose of 100 mu g/mouse respectively by adopting a subcutaneous immunization method;
(2) Two weeks later, immunization 2, fully emulsifying the human HFABP protein and Freund's incomplete adjuvant, and enhancing the immunity of the BALB/c mice by adopting the same dosage and method as in the step (1);
(3) At 2 week intervals, BALB/c mice were boosted with the same dose and method as in step (1), for a total of four immunizations;
(4) Pre-fusion 3 d spleen boost with human HFABP antigen without adjuvant, at 50 μg per dose;
(5) Collecting blood from the canthus of the mice, centrifuging at 4 ℃ and 4000 g for 5min, collecting supernatant, detecting serum titer by an indirect ELISA method, and selecting the mice with the serum titers of more than 1, 000 and 000 to prepare for cell fusion test;
(6) Selecting SP2/0 myeloma cells with good growth state, mixing the SP2/0 myeloma cells with spleen cells of an immunized mouse at a ratio of 1:5-1:10, centrifuging for 5min at 1500 g, collecting cell sediment, and discarding supernatant;
(7) Shaking the centrifuge tube gently to disperse the cells, slowly adding 1mL of preheated PEG 1500 at 37 ℃ and gently mixing for 1.5 min, then adding 20 mL of 1640 culture medium, centrifuging for 5min at 800 g, collecting cell precipitate, discarding the supernatant, adding HAT-1640 culture medium containing 20% serum into the cells and mixing uniformly;
(8) Uniformly spreading the uniformly mixed cells in a 96-well plate, culturing in a cell culture box with the CO2 concentration of 5% at 37 ℃ for 5-7 d, observing the fusion effect, changing the liquid, detecting the supernatant of the hybridoma cells by an indirect ELISA (enzyme-linked immunosorbent assay) method at the 4 th d after the liquid change, selecting holes with high positive values and small cell colony numbers, and subcloning the holes by a limiting dilution method, wherein the theoretical number of cells in each hole is 1-2;
(9) After 3-4 subcloning, the hybridoma cell strain capable of stably secreting the antibody is obtained.
(10) BALB/c mice over 6 weeks were selected, and before 1-2 weeks of hybridoma inoculation, 0.5. 0.5 ml liquid paraffin was injected into the peritoneal fluid of the mice.
(11) Well-grown hybridoma cells were collected and cell densities were adjusted to 1X 106-2X 106 cells/mL, and each mouse was intraperitoneally injected with 0.5. 0.5 mL cell suspension.
(12) 7-12 and d, the abdominal cavity is pierced by a syringe to extract ascites. Centrifuging to absorb pale yellow ascites, subpackaging, and storing at-20deg.C.
(13) The extracted ascites fluid was slowly added to a saturated ammonium sulfate solution at a ratio of 1:1, and the obtained precipitate was dissolved in a volume of PBS (pH 7.45) solution, and dialyzed and equilibrated to 20 volumes of binding buffer to remove ions at a high concentration.
(14) The dialyzed antibody is purified by affinity column chromatography, dialyzed and balanced again into PBS (pH 7.45), and the antibody is packaged and stored for standby. Through four rounds of immunization and screening, the monoclonal antibody 17 strain capable of stably secreting the anti-human HFABP is obtained.
The invention discloses preparation of magnetic beads and monoclonal antibody conjugates of each strain, which comprises the following specific processes:
(1) Firstly, suspending magnetic beads, taking 1ml of suspension into an EP tube, placing the tube on a magnetic rack for 2-3 minutes, and carefully taking out supernatant;
(2) Add 900. Mu.l of binding buffer (0.1M borate buffer pH 9.5) and vortex the beads;
(3) Add 100 μg monoclonal antibody and vortex the beads; 50 μl of catalytic solution (3M ammonium sulfate/0.1M borate buffer, pH 9.8) was added and the reaction was rotated at 37deg.C for 18 hours;
(4) Adding 10 μl of blocking solution (10% bovine serum albumin) below 10deg.C, reacting at 37deg.C for 6h;
(5) After 2-3 minutes of placement on the magnetic rack, carefully suck the supernatant; an appropriate amount of TBS-T washing buffer (25 mM Tris-HCl, pH7.2,0.15M NaCl,0.05% Tween 20)) was added and the beads were vortexed; repeating the washing 3 times; the beads were suspended in PBS (pH 7.2) and stored at 2-8.
The invention discloses preparation of Alkaline Phosphatase (AP) -labeled monoclonal antibodies of various strains, which comprises the following steps:
(1) Dissolving the AP enzyme with PBS (pH 7.4), centrifuging twice, and removing insoluble precipitate;
(2) EDC and sulfoNHS solution (prepared by MES buffer pH 4.5) are added and activated for 1h;
(3) Adding monoclonal antibody (with PBS buffer pH 4.5) and centrifuging twice; adding PBS buffer solution, and activating for 3 hours;
(4) 1% BSA (PBS buffer pH7.4 configuration) was added; washing twice with PBS (pH 7.4); finally, suspending the precipitate with PBS (pH 7.4), and adding equal volume of glycerol; -20 ° preservation.
The invention discloses a chemiluminescent enzyme immunoassay (CLEIA) method for establishing a heart-type fatty acid binding protein by using the reagent, which comprises the following steps:
(1) Contacting a sample to be tested with the magnetic bead coated antibody;
(2) The mixed reaction solution is contacted with an alkaline phosphatase-labeled antibody;
(3) The chemiluminescent substrate solution was added and the total Relative Luminescence Units (RLU) were read in 10 seconds.
(4) And (3) performing pairing detection on the 17 monoclonal antibodies by using a chessboard method, and screening out the optimal pairing antibody.
(5) And detecting the sample to be detected by using the optimal pairing antibody 2B8/6B 3-AP.
Judging the content of the central fatty acid binding protein of the sample by utilizing the reaction luminescence value; the sample to be tested is clinical serum.
The inventor uses purified human HFABP protein as antigen to immunize BALB/c mice, and obtains 17 monoclonal antibodies of the anti-human HFABP protein through four times of cell fusion and subcloning screening, which have good specificity, stability and sensitivity; and the magnetic beads and alkaline phosphatase are labeled and paired for each monoclonal antibody, so that a chemiluminescent enzyme immunoassay (CLEIA) method which has independent intellectual property rights and can safely, rapidly and accurately detect the content of the central fatty acid binding protein of the sample to be detected is established.
The chemiluminescent enzyme immunoassay method (CLEIA) for detecting the HFABP content can accurately and quantitatively detect the HFABP content of a patient sample clock, and can be used for early detection and monitoring of myocardial ischemic injury. Has the advantages of strong specificity, high sensitivity, good accuracy, rapidness, simplicity and convenience, and the like. And the same standard of the chemiluminescent enzyme immunoassay method CLEIA and the latex enhanced immunoturbidimetry of the hospital are calibrated, and the results show excellent correlation and consistency. And the analysis sensitivity of the chemiluminescent enzyme immunoassay method CLEIA (0.001 mug/L) is equivalent to that of a latex enhanced immunonephelometry method of a hospital. Preliminary shows that the chemiluminescent enzyme immunoassay method (CLEIA) which is independently developed and established is feasible for clinical detection, and provides a good new material and detection method for developing an HFABP diagnostic kit.
Drawings
FIG. 1 shows a partial monoclonal antibody immunoreactivity assay. The results show that each monoclonal antibody has good immunoreactivity with HFABP protein.
FIG. 2 shows another part of the monoclonal antibody immunoreactivity assay of the screen. The results show that each monoclonal antibody has good immunoreactivity with HFABP protein.
FIG. 3 shows the results of pairing assay after labeling of a portion of the monoclonal antibody beads with APase.
FIG. 4 shows the results of pairing assay after labeling of a portion of the monoclonal antibody beads with APase.
FIG. 5 shows the results of pairing assay after labeling of a portion of the monoclonal antibody beads with APase.
FIG. 6 shows the results of pairing assay after labeling of a portion of the monoclonal antibody beads with APase.
FIG. 7 shows the detection efficiency of monoclonal antibodies against 2B8/6B3-AP and 7E7/10D5-AP in a chemiluminescent enzyme immunoassay. The results show that the magnetic bead coupled 2B8 antibody and the AP marked 6B3 antibody have better detection efficiency.
FIG. 8 shows the precision detection of paired monoclonal antibodies. HFABP calibrators at different concentrations were measured 3 times a day with precision CVs of less than 6% for each measurement.
FIG. 9 is a paired antibody 2B8/6B3-AP sensitivity assay. The lower detection limit (LoD) of the paired antibodies is measured, and the LoD of the chemiluminescent enzyme immunoassay method is calculated to be 0.001 mu g/L.
Fig. 10 is a correlation analysis of serum HFABP values obtained by two assays. 106 serum samples were measured by latex enhanced immunoturbidimetry and the chemiluminescent enzyme immunoassay (CLEIA) method of our established 2B8/6B3-AP monoclonal antibody pair.
Detailed Description
The following examples further illustrate the invention but are not to be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
EXAMPLE 1 selection of animal immunization and hybridoma cell lines
(1) Expression and purification of cardiac fatty acid binding proteins: extracting total RNA of human cardiac muscle cells by adopting a Trizol method, reversely transcribing the extracted total RNA into cDNA by using a reverse transcription kit, and amplifying an HFABP coding sequence by using the cDNA as a template through PCR. Connecting the target gene with an amplification vector to construct a recombinant plasmid, and transforming E.coli cells (Top 10F') to carry out plasmid amplification. After plasmid extraction, the HFABP gene fragment was recovered by double digestion. Cloning it into expression plasmid, transforming colibacillus cell (ER 2566), adding IPTG to induce recombinant HFABP expression of fusion his-tag. After a large amount of recombinant HFABP expression strain is expressed and then is subjected to ultrasonic crushing, protein supernatant is subjected to saturated ammonium sulfate precipitation, ni-IDA column purification and molecular sieve chromatographic column purification, and the protein purity of the recombinant HFABP reaches the monoclonal antibody preparation requirement (the specific purification method is described in the following documents: zheng Jiao, yilan, liaojing, li Xiaodan, zhang Jiansong, wu Yi, what is known as Sai, liang Xianghui, guo Xiangrong, liu Rudan. Procalcitonin prokaryotic expression and monoclonal antibody preparation [ J ]. Laser biology report, 2016, v.25; no.126 (02): 147-155).
Wherein the nucleotide sequence of the encoding gene of the HFABP protein used in the experimental process is as follows (SEQ ID NNO: 23): atggtggacgctttcctgggcacctggaagctagtggacagcaagaatttcgatgactacatgaagtcactcggtgtgggttttgctaccaggcaggtggccagcatgaccaagcctaccacaatcatcgaaaagaatggggacattctcaccctaaaaacacacagcaccttcaagaacacagagatcagctttaagttgggggtggagttcgatgagacaacagcagatgacaggaaggtcaagtccattgtgacactggatggagggaaacttgttcacctgcagaaatgggacgggcaagagaccacacttgtgcgggagctaattgatggaaaactcatcctgacactcacccacggcactgcagtttgcactcgcacttatgagaaagaggcatga.
(2) Purified HFABP was diluted with PBS buffer to a final concentration of 0.5 mg/mL, and 1:1 was fully emulsified with Freund's complete adjuvant, 3 mL total. Taking 5 BALB/c female mice with the age of 4-6 weeks for marking, and performing primary immunization on the 5 mice at the dosage of 100 mu g/mouse respectively by adopting a subcutaneous immunization method; after two weeks, the protein solution and Freund's incomplete adjuvant were thoroughly emulsified 1:1 and the mice were immunized again in the same manner at the same dose. The immunization protocol was identical to the second immunization, with a total of four immunizations at 2 week intervals. Mice with the immunization titer were subjected to spleen booster immunization at a dose of 50 μg/mouse prior to 3 d for cell fusion. 5 mice were subjected to venous blood collection before each immunization, and changes in the titer of antibodies in serum were detected.
(3) Washing SP2/0 myeloma cells with good growth state and spleen cells of immunized mice respectively with a serum-free 1640 culture medium until no large particle precipitation exists, mixing at a ratio of 1:5-1:10, centrifuging at a ratio of 1 g for 1 min, and discarding the supernatant. After the cells were uniformly dispersed by shaking the centrifuge tube gently, 1mL of PEG 1500 preheated at 37℃was slowly added and gently blown and mixed, 60 mL of 1640 medium was immediately added to the mixture to terminate the fusion after 60 mL of s, and then 20% FBS-HAT-1640 medium was added to suspend the cells and mixed.
(4) Uniformly spreading the culture medium in 96-well plate, and placing in 5% CO 2 Culturing in an incubator at 37 ℃, carrying out tracking observation every day, replacing 7-10 d with 10% FBS-HT-1640 culture solution after fusion, detecting the antibody titer of the supernatant of the hybridoma cells by an indirect ELISA method when clones grow to 1/3-1/2 of the bottom area of the holes, and cloning the holes with high positive values and small cell colony numbers by a limiting dilution method. After 4 times of subcloning, a positive monoclonal cell strain capable of stably secreting specificity is obtained, and then the positive monoclonal cell strain is subjected to expansion culture, and 17 cell strains capable of stably secreting monoclonal antibodies are obtained through four times of immunization and cell hybridization screening in the research.
EXAMPLE 2 production and purification of monoclonal antibodies
(1) Selecting BALB/c mice with more than 6 weeks, before inoculating hybridoma cells for 1-2 weeks,the mice were injected with 0.5. 0.5 ml liquid paraffin in the abdominal cavity. Collecting well-grown hybridoma cells, centrifuging and washing for 1 time, and re-suspending in serum-free culture solution to adjust cell density to 1×10 6 -2×10 6 Each mouse was injected intraperitoneally with 0.5 mL cell suspension per mL. After 7-12 and d, the abdomen of the mice is obviously enlarged, the skin of the abdomen is disinfected, and the ascites is extracted by penetrating the abdominal cavity with a syringe. 3000 g, centrifuging for 10 min, removing upper layer oil, sucking pale yellow ascites, subpackaging, and preserving at-20deg.C for use.
(2) The extracted ascites is slowly added into a saturated ammonium sulfate solution in a ratio of 1:1, and the mixture is shaken and mixed uniformly while adding until white precipitation appears. 12000 g, centrifugation for 10 min, removal of supernatant, dissolution of the pellet with a volume of PBS (pH 7.45) solution, transfer to a dialysis bag equilibrated to 20 volumes of binding buffer (provided in Protein A Sefinose Kit) to remove high concentrations of ions. The affinity column in the kit was mounted, stored upright, and then pre-equilibrated with 5 mL volumes of binding buffer. Sample 1mL was applied and the affinity column was continuously penetrated with 30 mL of binding buffer and the flow rate was controlled at 1 mL/min. The affinity column was washed with 10-15-mL volumes of elution buffer while protein peaks were collected and then identified by SDS-PAGE. The antibody collected after affinity chromatography purification is put into a dialysis bag, dialyzed and balanced into PBS (pH 7.45), dialyzed for 12 h at 4 ℃, and sub-packaged and preserved for standby.
EXAMPLE 3 screening of the best paired antibodies
1) Preparation of magnetic beads and monoclonal antibody conjugates of each strain
Firstly, suspending magnetic beads, taking 1ml of suspension into an EP tube, placing the tube on a magnetic rack for 2-3 minutes, and carefully taking out supernatant; add 900. Mu.l of binding buffer (0.1M borate buffer pH 9.5) and vortex the beads; add 100 μg of antibody and vortex the beads; 50 μl of catalytic solution (3M ammonium sulfate/0.1M borate buffer, pH 9.8) was added and the reaction was rotated at 37deg.C for 18 hours; adding 10 μl of blocking solution (10% bovine serum albumin) below 10deg.C, and reacting at 37deg.C for 6 hr; after 2-3 minutes of placement on the magnetic rack, carefully suck the supernatant; an appropriate amount of TBS-T washing buffer (25 mM TRIs-HCl, pH7.2,0.15M NaCl,0.05% Tween 20) was added and the beads were vortexed; repeating the washing 3 times; the beads were suspended in PBS (pH 7.2) and stored at 2-8deg.C.
2) Preparation of Alkaline Phosphatase (AP) -labeled monoclonal antibodies of the respective strains
Dissolving the AP enzyme with PBS (pH 7.4), centrifuging twice, and removing insoluble precipitate; EDC and sulfoNHS solution (prepared by MES buffer pH 4.5) are added and activated for 1h; adding antibody (with PBS buffer pH 4.5) and centrifuging twice; adding PBS buffer solution, and activating for 3 hours; 1% BSA (PBS buffer pH7.4 configuration) was added; washing twice with PBS (pH 7.4); finally, suspending the precipitate with PBS (pH 7.4), and adding equal volume of glycerol; preserving at-20 ℃.
3) Screening of optimal pairing antibody pairs
Adding an HFABP antigen standard substance and a magnetic bead coated antibody with the concentration of 0.05ng/ml into a reaction tube, incubating for 5min, and then cleaning; and adding an AP labeled antibody of 1:2000, incubating for 5min, washing magnetism, adding a chemiluminescent substrate, reading the total relative luminous intensity within 10s, and screening out the optimal pairing antibody. (see FIGS. 3-7 for pairing check results). The result shows that the antibody pair 2B8/6B3-AP antibody has higher sensitivity than the antibody pair 7E7/10D5-AP antibody pair, and has better detection effect on the HFABP calibrator.
Example 4 optimal paired antibody precision detection
Adding 4 HFABP antigen standard substances with different concentrations (the concentrations are respectively S1:0ng/ml, S2:0.2ng/ml, S3:10 ng/ml, S4:50 ng/ml and S5:100 ng/ml) and 2B8 magnetic bead coated antibodies with the concentration of 0.05ng/ml into a reaction tube, incubating for 5min, and then washing; after further addition of 1:2000 of 6B3-AP antibody, incubation for 5min, washing with magnetism, addition of chemiluminescent substrate, reading of total relative luminescence intensity within 10s, and three independent experiments were performed to determine CVs. (for the detection results, see FIG. 8)
Table 1 best paired monoclonal antibody precision detection
Example 5 determination of the optimal paired antibody pair detection lower limit
Heart type fatty acid binding protein assay kit (latex enhanced immunoturbidimetry) (REF H-FABP 7080, LOT H-FABP 191101) was used as a calibration material and diluted with dilution buffer to six gradient concentrations (S1: 0ng/ml, S2:4.06 ng/ml, S3:8.12 ng/ml, S4:16.25 ng/ml, S5:32.16 ng/ml, S6:65 ng/ml). The concentration of calibrator is specified by the kit. HFABP measurements of the calibrator were then performed. S0 (HFABP antigen content is 0) and 2B8 magnetic bead coated antibody with concentration of 0.05ng/ml are added into a reaction tube, and the reaction tube is washed after being incubated for 5 min; adding a 6B3-AP antibody diluted by 1:2000, incubating for 5min, and then washing the magnet; chemiluminescent substrate was added and the total relative luminous intensity was read for 10S and the average of 20S 0 was determined. LoD was defined as the concentration converted from the average RLU plus three SDs of S1 (n=20) and calculated from the calibration curve in the same batch, giving a LoD of 0.001 μg/L for CLEIA (see fig. 9 for detection results).
EXAMPLE 6 variable region sequencing of monoclonal antibodies 2B8 and 6B3
1) mRNA of the prepared hybridoma cells 2B8 and 6B3 was extracted by Trizol method, and cDNA was synthesized according to BDSMART company reverse transcription kit.
2) The heavy chain fragment and the light chain fragment of the monoclonal antibody secreted by the 2B8 and 6B3 hybridoma cells are obtained by PCR (polymerase chain reaction) amplification by using a designed amplification primer (5 '-CTCAGGGAARTARCCYTTGAC-3') of the heavy chain constant region sequence of the mouse antibody, a designed amplification primer (5'-TCACTGCCATCAATCTTCCAC-3') of the light chain constant region sequence of the mouse antibody and a joint primer in a kit, and are sequenced after pGEM-T vector construction. And translating the obtained sequence into an amino acid sequence of a protein.
Wherein the full length nucleotide sequence of the 2B8 heavy chain is as follows (429 bp): ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCCACTCCCAGGGCCAACTGCAGCAGCCTGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTAAAACTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTATTGGATGAACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAATGGATTGGCATGATTGATCCTTCAGAAAGTGAAACTCATTACAATCAAATATTCAAGGACAAGGCCACATTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGGCCCATATCCCCAGGGTATGTTGGCTACGTTTCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO: 13).
The full length of the amino acid sequence encoded by the 2B8 heavy chain is as follows (143 aa):
MGWSCIILFLVATATGVHSQGQLQQPGAELVRPGASVKLSCKASGYTFTSYWMNWVKQRPGQGLEWIGMIDPSESETHYNQIFKDKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARPISPGYVGYVSWFAYWG QGTLVTVSA（SEQ ID NO:14）。
identification was performed by conventional methods, and it was found that, in the heavy chain: the CDR1 sequence is: SYWMN (SEQ ID NO: 1); the CDR2 sequence is: MIDPSESETHYNQIFKD (SEQ ID NO: 2); the CDR3 sequence is: PISPGYVGYVSWFAY (SEQ ID NO: 3).
Wherein, the full-length nucleotide sequence of the 2B8 light chain is as follows (381 bp):
ATGGAGTCACAGACTCAGGTCTTTGTATACATGTTGCTGTGGTTGTCTGGTGTTGATGGAGACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTTGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGGGTACTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACTGGTTTACTCGGCATCCTTCCGGCACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACTGATTTCACACTCAGCATCATCAATGTGCAGTCTGAAGACTTGGCAGAGCTTTTCTGTCAGCAATATAACAGCTTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATGAAA（SEQ ID NO:15）。
the full length of the amino acid sequence encoded by the 2B8 light chain is as follows (127 aa):
MESQTQVFVYMLLWLSGVDGDIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALVYSASFRHSGVPDRFTGSGSGTDFTLSIINVQSEDLAELFCQQYNSFPYTFGGGTKLEMK（SEQ ID NO:16）。
identification was performed by conventional methods, and it was found that in the light chain, the CDR1 sequence was: KASQNVGTNVA (SEQ ID NO: 4); the CDR2 sequence is: SASFRHS (SEQ ID NO: 5); the CDR3 sequence is: QQYNSFPYT (SEQ ID NO: 6).
Wherein, the full-length nucleotide sequence of the 6B3 heavy chain is as follows (405 bp):
ATGGTGTTAAGTCTTCTGTACCTGTTGACAGCCCTTCCGGGTATCCTGTCAGAGGTGCAGCTTCAGGAGTCAGGACCTAGCCTCGTGAAACCTTCTCAGACTCTGTCCCTCACCTGTTCTGTCACTGGCGACTCCATCACCAGTGGTTACTGGAACTGGATCCGGAAATTCCCAGGGAATAAACTTGAGTACATGGGGTACATAACCTACAGTGATATCACTTACTACAATCCATCTCTCAAAAGTCGAATCTCCATCACTCGAGACACATCCAAGAACCAGTACTATCTGCAGTTGATTTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGATTCCGGGACATGGGGAACTACTTTGACTACTGGGGCCAAGGCACCGCTCTCACTGTCTCCTCA（SEQ ID NO:17）。
the full-length amino acid sequence of the 6B3 heavy chain is as follows (135 aa):
MVLSLLYLLTALPGILSEVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGNKLEYMGYITYSDITYYNPSLKSRISITRDTSKNQYYLQLISVTTEDTATYYCARFRDMGNYFDYWGQGTALTVSS（SEQ ID NO:18）。
identification was performed by conventional methods, and it was found that, in the heavy chain: the CDR1 sequence is: SGYWN (SEQ ID NO: 7); the CDR2 sequence is: YITYSDITYYNPSLKS (SEQ ID NO: 8); the CDR3 sequence is: FRDMGNYFDY (SEQ ID NO: 9).
Wherein the full length nucleotide sequence of the 6B3 light chain is as follows (393 bp): ATGGAGACAGACCCACTCCTGCTATGGGTGCTGCTGCTCTGGGTTCCAGGCTCCACTGGTGACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGACAGAGGGCCACCATATCCTGCCAAGCCAGCGAAAGTGTCAGTTTTGCTGGTGCAAGTTTAATGCACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATCGTGCATCCAGCCTAGATTCTGGAGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGAGTCAGACTTCACTCTCACCATCGATCCTGTGGAGGAAGATGATGCTGCAATGTATTACTGTATGCAAAGTATGGAAGGTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA (SEQ ID NO: 19)
The full-length amino acid sequence of the 6B3 light chain is as follows (131 aa):
METDPLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCQASESVSFAGASLMHWYQQKPGQPPKLLIYRASSLDSGVPARFSGSGSESDFTLTIDPVEEDDAAMYYCMQSMEGPLTFGAGTKLELK（SEQ ID NO:20）
by conventional methods, it can be known that, in the light chain, the CDR1 sequence is: QASESVSFAGASLMH (SEQ ID NO: 10); the CDR2 sequence is: RASSLDS (SEQ ID NO: 11); the CDR3 sequence is: MQSMEGPLT (SEQ ID NO: 12).
EXAMPLE 7 establishment of a method for chemiluminescent enzyme immunoassay (CLEIA) for cardiac fatty acid binding proteins
And simultaneously detecting the standard substance and the clinical sample by using the determined optimal magnetic bead labeled antibody and alkaline phosphatase labeled antibody concentration. And then bringing the detection luminescence value of the clinical sample into a standard curve to obtain a clinical detection value. The test values obtained are compared with clinical values in hospitals. Clinical serum samples are collected in Hunan province people's hospitals, the samples are detected by using a heart type fatty acid binding protein assay kit (latex enhanced immunoturbidimetry), and the detection value is obtained by using the CLEIA detection established in the invention as the clinical value of the samples.
The specific operation is as follows: adding a standard substance, a clinical serum sample and a magnetic bead coating 2B8 antibody with the concentration of 0.05ng/ml into a reaction tube, incubating for 5min, and then cleaning; add 50 μl of 1:2000 diluted 6B3-AP antibody, incubating for 5min, and washing; chemiluminescent substrate was added and the total relative luminous intensity was read for 10 s. The correlation of the test values with hospital clinical values is compared.
TABLE 2 determination of clinical values in hospitals and CLEIA method
The actual application value of the paired antibodies was evaluated by comparing the difference between the detected value and the clinical value (see fig. 10 for the results). The detection result shows that: the clinical sample HFABP detection value detected by using the optimal pairing antibody and the working condition has better correlation with the clinical value detected by the kit for the hospital, so the established magnetic particle chemiluminescence method can be used for clinical detection of HFABP.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that it will be apparent to those skilled in the art that several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the scope of the invention.
<110> Liu Rudan
<120> an anti-HFABP monoclonal antibody and use thereof
<130> 2022
<160> 23
<170> PatentIn version 3.5
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Pro Ile Ser Pro Gly Tyr Val Gly Tyr Val Ser Trp Phe Ala Tyr
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atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt ccactcccag 60
ggccaactgc agcagcctgg ggctgagctg gtgaggcctg gggcttcagt aaaactgtcc 120
tgcaaggctt ctggctacac cttcaccagc tattggatga actgggtgaa gcagaggcct 180
ggacaaggcc ttgaatggat tggcatgatt gatccttcag aaagtgaaac tcattacaat 240
caaatattca aggacaaggc cacattgact gtagacaaat cctccagcac agcctacatg 300
caactcagca gcctgacatc tgaggactct gcggtctatt actgtgcaag gcccatatcc 360
ccagggtatg ttggctacgt ttcctggttt gcttactggg gccaagggac tctggtcact 420
gtctctgca 429
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Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
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Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Ser Tyr Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu
Glu Trp Ile Gly Met Ile Asp Pro Ser Glu Ser Glu Thr His Tyr Asn
Gln Ile Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser
Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Tyr Cys Ala Arg Pro Ile Ser Pro Gly Tyr Val Gly Tyr Val Ser
Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala
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atggagtcac agactcaggt ctttgtatac atgttgctgt ggttgtctgg tgttgatgga 60
gacattgtga tgacccagtc tcaaaaattc atgtccacat cagttggaga cagggtcagc 120
gtcacctgca aggccagtca gaatgtgggt actaatgtag cctggtatca acagaaacca 180
gggcaatctc ctaaagcact ggtttactcg gcatccttcc ggcacagtgg agtccctgat 240
cgcttcacag gcagtggatc tgggactgat ttcacactca gcatcatcaa tgtgcagtct 300
gaagacttgg cagagctttt ctgtcagcaa tataacagct ttccgtacac gttcggaggg 360
gggaccaagc tggaaatgaa a 381
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Val Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro
Lys Ala Leu Val Tyr Ser Ala Ser Phe Arg His Ser Gly Val Pro Asp
Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Ile
Asn Val Gln Ser Glu Asp Leu Ala Glu Leu Phe Cys Gln Gln Tyr Asn
Ser Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Met Lys
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atggtgttaa gtcttctgta cctgttgaca gcccttccgg gtatcctgtc agaggtgcag 60
cttcaggagt caggacctag cctcgtgaaa ccttctcaga ctctgtccct cacctgttct 120
gtcactggcg actccatcac cagtggttac tggaactgga tccggaaatt cccagggaat 180
aaacttgagt acatggggta cataacctac agtgatatca cttactacaa tccatctctc 240
aaaagtcgaa tctccatcac tcgagacaca tccaagaacc agtactatct gcagttgatt 300
tctgtgacta ctgaggacac agccacatat tactgtgcaa gattccggga catggggaac 360
tactttgact actggggcca aggcaccgct ctcactgtct cctca 405
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Gln Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Asp Ser Ile Thr Ser
Gly Tyr Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Lys Leu Glu Tyr
Met Gly Tyr Ile Thr Tyr Ser Asp Ile Thr Tyr Tyr Asn Pro Ser Leu
Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Tyr Tyr
Leu Gln Leu Ile Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys
Ala Arg Phe Arg Asp Met Gly Asn Tyr Phe Asp Tyr Trp Gly Gln Gly
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atggagacag acccactcct gctatgggtg ctgctgctct gggttccagg ctccactggt 60
gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctaggaca gagggccacc 120
atatcctgcc aagccagcga aagtgtcagt tttgctggtg caagtttaat gcactggtac 180
caacagaaac caggacagcc acccaaactc ctcatctatc gtgcatccag cctagattct 240
ggagtccctg ccaggttcag tggcagtggg tctgagtcag acttcactct caccatcgat 300
cctgtggagg aagatgatgc tgcaatgtat tactgtatgc aaagtatgga aggtccgctc 360
acgttcggtg ctgggaccaa gctggagctg aaa 393
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Val Ser Phe Ala Gly Ala Ser Leu Met His Trp Tyr Gln Gln Lys Pro
Gly Gln Pro Pro Lys Leu Leu Ile Tyr Arg Ala Ser Ser Leu Asp Ser
Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Glu Ser Asp Phe Thr
Leu Thr Ile Asp Pro Val Glu Glu Asp Asp Ala Ala Met Tyr Tyr Cys
Met Gln Ser Met Glu Gly Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu
Glu Leu Lys
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ctcagggaar tarccyttga c 21
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tcactgccat caatcttcca c 21
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Atggtggacgctttcctgggcacctggaagctagtggacagcaagaatttcgatgactacatgaagtcactcggtgtgggttttgctaccaggcaggtggccagcatgaccaagcctaccacaatcatcgaaaagaatggggacattctcaccctaaaaacacacagcaccttcaagaacacagagatcagctttaagttgggggtggagttcgatgagacaacagcagatgacaggaaggtcaagtccattgtgacactggatggagggaaacttgttcacctgcagaaatgggacgggcaagagaccacacttgtgcgggagctaattgatggaaaactcatcctgacactcacccacggcactgcagtttgcactcgcacttatgagaaagaggcatga 402

Claims (9)

1. The anti-HFABP monoclonal antibody is characterized in that the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of the monoclonal antibody are shown as SEQ ID NO.1, 2 and 3 respectively; and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region of the monoclonal antibody are shown in SEQ ID NOs 4, 5 and 6 respectively; or, the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of the monoclonal antibody are shown in SEQ ID NO. 7, 8 and 9 respectively; and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region of said monoclonal antibody are shown in SEQ ID NOS 10, 11 and 12, respectively.
2. The monoclonal antibody according to claim 1, wherein the heavy chain variable region amino acid sequences of the monoclonal antibody are shown in SEQ ID No. 14, respectively; and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID NO. 16; or the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO. 18; and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID NO. 20.
3. The monoclonal antibody of any one of claims 1-2, further comprising a constant region, the constant region of the heavy chain of the monoclonal antibody being any one of IgG1, igG2, igG3, or IgG 4; the constant region of the light chain of the monoclonal antibody is kappa type or lambda type.
4. A nucleic acid molecule encoding the monoclonal antibody of any one of claims 1-3.
5. The nucleic acid molecule of claim 4, wherein the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO. 13 and the nucleotide sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID NO. 15; or, the nucleotide sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO. 17, and the nucleotide sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID NO. 19.
6. An expression vector comprising the nucleic acid molecule of claim 4 or 5.
7. A cell comprising the vector of claim 6.
8. Use of a monoclonal antibody according to any one of claims 1-3 for the preparation of a kit for detecting HFABP.
9. A chemiluminescent enzyme immunoassay kit for detecting HFABP, said kit comprising the monoclonal antibody of any one of claims 1-3.
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