CN116715774A - Rabbit monoclonal antibody for human AXL, and preparation method and application thereof - Google Patents
Rabbit monoclonal antibody for human AXL, and preparation method and application thereof Download PDFInfo
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- CN116715774A CN116715774A CN202310549488.2A CN202310549488A CN116715774A CN 116715774 A CN116715774 A CN 116715774A CN 202310549488 A CN202310549488 A CN 202310549488A CN 116715774 A CN116715774 A CN 116715774A
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Abstract
The invention provides a rabbit monoclonal antibody aiming at human AXL, a preparation method and application thereof, in particular to a high-affinity rabbit monoclonal antibody pair aiming at human receptor tyrosine kinase AXL, and a double-antibody sandwich ELISA detection method is developed by utilizing the rabbit monoclonal antibody pair, and the method has the advantages of high specificity, strong anti-interference capability, high detection sensitivity, good stability and the like.
Description
Technical Field
The invention relates to the technical field of immunodetection, in particular to a rabbit monoclonal antibody aiming at human receptor tyrosine kinase AXL, and a preparation method and application thereof.
Background
AXL is one of the TAM family of receptor tyrosine kinases that can transduce signals of the extracellular matrix to the cytoplasm and regulate many physiological processes, playing an important role in proliferation, invasion, migration, epithelial-mesenchymal transition (EMT) of cancer cells, maintenance of stem cell properties, tumor angiogenesis, and immune regulation in the tumor microenvironment.
The GAS6-AXL signal pathway plays a key role in promoting tumor growth and metastasis, tumor immune escape and drug tolerance, plays an important role in the progress and malignancy process of tumors, and can be used as a biomarker for clinical diagnosis of tumors; AXL and its ligand GAS6 are highly expressed in many malignant tumors, such as acute myeloid leukemia, renal cancer, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, etc. Upon binding of AXL to GAS6, multiple pathways can be activated and involved in multiple processes of tumorigenesis and development. AXL is also expressed in a variety of immune cells, mainly mediating the clearance of apoptotic cells and the regulation of innate immune responses, and activation of its downstream signaling pathways, particularly on antigen-presenting cells, can inhibit the secretion of inflammatory cytokines such as type I interferons and tnfα.
Meanwhile, as an important drug target, the lead drug targeting the AXL-Gas6 signal pathway mainly comprises the following components: small molecule inhibitors of AXL, nucleic acid aptamers to AXL, therapeutic antibodies to AXL and AXL decoy proteins, and the majority of clinical studies entered into are AXL small molecule inhibitors such as BGB324, SKI-606, foretinb, LY2801653 and MP-470, etc.
By measuring AXL levels in human body fluids and plasma, a diagnostic assessment of prognosis of cancer and cancer treatment can be made. In serum of normal people, the concentration range of the AXL protein is 21-55 mug/mL, and the concentration range in saliva is about 100pg/mL, so that a high-sensitivity AXL protein detection methodology is developed, and the method has very important clinical significance.
In addition, conventional ELISA detection kits all use mouse anti-human AXL monoclonal antibodies, and the affinity and the specificity of the monoclonal antibodies to human AXL are generally low.
Disclosure of Invention
Based on the above, it is necessary to provide a rabbit monoclonal antibody against human receptor tyrosine kinase AXL and application thereof, specifically, to provide a rabbit monoclonal antibody with high affinity to a and B and human receptor tyrosine kinase AXL, and a double antibody sandwich ELISA detection method established by using the high-affinity rabbit monoclonal antibody has high sensitivity and strong specificity.
The invention adopts the following technical scheme:
the invention provides a rabbit monoclonal antibody aiming at human AXL, wherein the rabbit monoclonal antibody is a rabbit monoclonal antibody A and a rabbit monoclonal antibody B, and the rabbit monoclonal antibody B are respectively combined with different antigenic determinants on the surface of the human AXL.
Wherein, the sequence of the complementarity determining region of the rabbit monoclonal antibody A is respectively shown as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO. 10; the sequence of the light chain variable region of the rabbit monoclonal antibody A is shown as SEQ ID NO.2, and the sequence of the heavy chain variable region is shown as SEQ ID NO. 7. The full-length sequence of the light chain of the rabbit monoclonal antibody A is shown as SEQ ID NO.1, and the full-length sequence of the heavy chain is shown as SEQ ID NO. 6.
The sequence of the complementarity determining region of the rabbit monoclonal antibody B is shown as SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20 respectively. The sequence of the light chain variable region of the rabbit monoclonal antibody B is shown as SEQ ID NO.12, and the sequence of the heavy chain variable region is shown as SEQ ID NO. 17. The full-length sequence of the light chain of the rabbit monoclonal antibody B is shown as SEQ ID NO.11, and the full-length sequence of the heavy chain is shown as SEQ ID NO. 16.
The invention also provides a gene sequence for encoding the rabbit monoclonal antibody against human AXL, or an expression vector or host comprising the gene sequence.
The invention also provides a labeled antibody conjugate which is mainly prepared by reacting the rabbit monoclonal antibody aiming at human AXL with fluorescent labeling molecules.
The invention provides application of a high-affinity rabbit monoclonal antibody pair aiming at human AXL in establishing an enzyme-linked immunosorbent assay method of high-sensitivity human AXL. The enzyme-linked immunosorbent assay method is a double-antibody sandwich method enzyme-linked immunosorbent assay method. In the double-antibody sandwich method ELISA detection method, the capture antibody is a rabbit monoclonal antibody A, and the detection antibody is a biotin-labeled rabbit monoclonal antibody B.
The invention also provides a reagent or a kit for detecting human AXL, comprising rabbit monoclonal antibodies a and B to human AXL. The human AXL includes recombinantly expressed human AXL, or human AXL secreted by cells, or human AXL in human serum.
The invention also provides a preparation method of the human AXL rabbit monoclonal antibody, which comprises the following steps: loading heavy chain genes and light chain genes of rabbit monoclonal antibodies aiming at human AXL on expression vectors respectively, and transfecting engineering cells; culturing to obtain a supernatant containing the rabbit monoclonal antibody pair A and B; purifying to obtain the final product.
Compared with the prior art, the invention has the beneficial effects that:
the immunogen for preparing the human AXL rabbit monoclonal antibody is a human AXL truncated protein which is expressed in a recombination way in vitro by using a lactation expression system and has biological activity after verification; the preparation method is monoclonal antibody development technology based on single B lymphocyte screening and culture.
The invention successfully develops high-affinity anti-human AXL rabbit monoclonal antibodies A and B, and the affinity constant of the combination of the rabbit monoclonal antibody A and recombinant expression human AXL is 5.55X10 -10 RabbitAffinity constant for binding of monoclonal antibody B to recombinant expressed human AXL was 1.14X10 -9 . The invention also proves that the two antibodies can recognize different antigenic determinants on the surface of human AXL, and a double-antibody sandwich method ELISA kit is developed; the double-antibody sandwich method ELISA kit developed by the antibody has the advantages of high specificity, strong anti-interference capability, high detection sensitivity, good stability and the like, and the detection sensitivity is 0.11ng/mL. The establishment of the methodology provides a kit capable of stably detecting trace human AXL in serum and cell culture medium samples, and has important significance in clinical diagnosis and scientific research application.
Drawings
FIG. 1 is a schematic diagram of construction of an expression vector containing a heavy chain constant region of a rabbit monoclonal antibody.
FIG. 2 is a schematic diagram of construction of an expression vector containing a light chain constant region of a rabbit monoclonal antibody.
FIG. 3 is a graph showing the results of purification assays for rabbit monoclonal antibodies A and B directed against the human AXL protein.
FIG. 4 is a graph showing the results of affinity assays for rabbit monoclonal antibodies A and B to the human AXL protein.
FIG. 5 is a graph showing the results of epitope measurement of rabbit monoclonal antibodies A and B against the human AXL protein.
FIG. 6 is a graph showing the results of a double-antibody sandwich ELISA assay for recombinant human AXL using rabbit monoclonal antibody A and rabbit monoclonal antibody B in example 2 of the present invention.
Detailed Description
The technical conception of the invention is to provide a rabbit monoclonal antibody pair aiming at human receptor tyrosine kinase AXL, and a preparation method and application thereof. The developed rabbit monoclonal antibodies (rabbit monoclonal antibody A and rabbit monoclonal antibody B) have high affinity with human receptor tyrosine kinase AXL, and the high-affinity rabbit monoclonal antibodies have high sensitivity and high specificity to the established double-antibody sandwich ELISA detection method, so that the method is convenient for detecting the AXL at a trace level in serum and cell culture medium samples.
Wherein, the full-length amino acid sequence of the light chain of the rabbit monoclonal antibody A is as follows:
MDTRAPTQLLGLLLLWLPGARCADIVMTQTPASVEAAVGDTVTIKCQASQNIYSNLAWYQQKPGQPPKLLIYFASNLPSGVPSRFKGSGSGTEYTLTISGVQCDDAATYYCQNYYINEIVFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC(SEQ ID No.1)；
the amino acid sequence of the light chain variable region of rabbit monoclonal antibody a is:
ADIVMTQTPASVEAAVGDTVTIKCQASQNIYSNLAWYQQKPGQPPK LLIYFASNLPSGVPSRFKGSGSGTEYTLTISGVQCDDAATYYCQNYYINEIV FGGGTEVVVK(SEQ ID No.2)；
the amino acid sequences of the three complementarity determining regions (CDR regions) of the light chain variable region of rabbit monoclonal antibody a are respectively:
QNIYSNLAW(SEQ ID No.3)，
LIYFASNLPSGV(SEQ ID No.4)，
QNYYINEIVF(SEQ ID No.5)。
the full-length amino acid sequence of the heavy chain of rabbit monoclonal antibody a is:
METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGIDLSTYAMIWVRQAPGEGLEWIGTIYTDGATAYARWAKGRFTISKTSTTVDLKMTSLTTEDTATYFCARGFGISKFVFSLWGPGTLVTVSLGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELPGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID No.6)；
the amino acid sequence of the heavy chain variable region of rabbit monoclonal antibody a is:
QSVEESGGRLVTPGTPLTLTCTVSGIDLSTYAMIWVRQAPGEGLEWIG TIYTDGATAYARWAKGRFTISKTSTTVDLKMTSLTTEDTATYFCARGFGIS KFVFSLWGPGTLVTVSL(SEQ ID No.7)；
the amino acid sequences of the three complementarity determining regions of the heavy chain variable region of the rabbit monoclonal antibody a are:
IDLSTYAMI(SEQ ID No.8)，
WIGTIYTDGATAYARWAK(SEQ ID No.9)，
YFCARGFGISKFVFSL(SEQ ID No.10)。
wherein, the full-length amino acid sequence of the light chain of the rabbit monoclonal antibody B is as follows:
MDTRAPTQLLGLLLLWLPGARCAIDMTQTPSSVSAAVGDTVTINCQASEDIYSFLAWYQQKPGHSPKLLIYDASKLASGVSSRFKGSGSGTQFTLTISGVQCDDAATYYCQQTYSVSNADTAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC(SEQ IDNo.11)；
the amino acid sequence of the light chain variable region of rabbit monoclonal antibody B is shown as:
AIDMTQTPSSVSAAVGDTVTINCQASEDIYSFLAWYQQKPGHSPKLLI YDASKLASGVSSRFKGSGSGTQFTLTISGVQCDDAATYYCQQTYSVSNAD TAFGGGTEVVVK(SEQ ID No.12)；
the amino acid sequences of the three complementarity determining regions (CDR regions) of the light chain variable region of the rabbit monoclonal antibody B are respectively:
EDIYSFLAW(SEQ ID No.13)，
LIYDASKLASGV(SEQ ID No.14)，
QQTYSVSNADTAF(SEQ ID No.15)；
the full-length amino acid sequence of the heavy chain of rabbit monoclonal antibody B is:
METGLRWLLLVAVLKGVQCQEQLKESGGGLVQPGGSLKLSCKASGFSLSYYGVNWVRQAPGKGLEWIACIDPVFGATYYASWVNDRFTISSHNAQNTLYLQLSSLTPADTATYFCARGRYYRYGYTYATGMDIWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELPGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID No.16)；
the amino acid sequence of the heavy chain variable region of rabbit monoclonal antibody B is: QEQLKESGGGLVQPGGSLKLSCKASGFSLSYYGVNWVRQAPGKGLEWIA CIDPVFGATYYASWVNDRFTISSHNAQNTLYLQLSSLTPADTATYFCARGR YYRYGYTYATGMDIWGPGTLVTVSS (SEQ ID No. 17);
the amino acid sequences of the three complementarity determining regions of the heavy chain variable region of the rabbit monoclonal antibody B are:
FSLSYYGVN(SEQ ID No.18)，
WIACIDPVFGATYYASWVN(SEQ ID No.19)，
YFCARGRYYRYGYTYATGMDI(SEQ ID No.20)。
the present invention will be described in further detail with reference to specific examples so as to more clearly understand the present invention by those skilled in the art. The following examples are given for illustration of the invention only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art without creative efforts are within the protection scope of the present invention based on the specific embodiments of the present invention. In the examples of the present invention, all raw material components are commercially available products well known to those skilled in the art unless specified otherwise; in the embodiments of the present invention, unless specifically indicated, all technical means used are conventional means well known to those skilled in the art.
Example 1
The embodiment provides a preparation method of a monoclonal antibody against human AXL, which is a monoclonal antibody development technology based on single B lymphocyte screening and culturing, and specifically comprises the following steps:
1.1, animal immunization: 4 New Zealand white rabbits were immunized with recombinant human AXL truncated protein (human AXL truncated protein which is expressed recombinantly in vitro by a mammalian expression system and has been verified to have biological activity, commercially available, accession number: RP 00087) as an immunogen; each white rabbit was immunized 300 μg of immunogen for the first time, and the immunogen was mixed with an equivalent amount of complete Freund's adjuvant (purchased from Sigma company) to prepare an emulsifier before the first immunization, and injected subcutaneously in the abdomen and back of the rabbits at multiple points; 150 μg of immunogen was mixed with an equal amount of incomplete Freund's adjuvant (purchased from Sigma) at 2 weeks intervals after the first immunization to prepare an emulsifier, which was injected subcutaneously in the abdomen and back of rabbits in multiple spots to boost the immunization twice. Serum samples of rabbits were collected after three immunizations, titers against human AXL were determined by ELISA, rabbits with highest serum titers were boosted by subcutaneous multipoint injection with 300 μg immunogen for one time, and spleens were obtained three days later.
1.2, spleen cells were isolated: taking out a culture dish in a safe cabinet in a sterile operation mode, adding 30-40 mL of basic culture medium, placing a cell screen, taking out spleen, placing the spleen in the cell screen, shearing superfluous connective tissue and fat on rabbit spleen tissue, shearing spleen tissue, placing the spleen tissue into the cell screen for grinding, taking a clean grinding rod, and grinding the tissue by using the tail end of the pressed part of the grinding rod. The cells in the membrane slowly come out and are suspended in the culture dish solution after passing through a cell sieve; the washed cell screen was washed with 10mL of basal medium and the basal medium outside the cell screen was collected. Centrifuging at room temperature for 5min by using a centrifugal force of 400g, removing supernatant, reserving cells, adding 13mL of RBC erythrocyte lysate at room temperature (purchased from BioGems company), gently blowing off cell clusters by using a pipettor, timing for 1min, performing erythrocyte lysis, adding 37mL of basal medium, uniformly mixing, stopping erythrocyte lysis, centrifuging at room temperature for 5min by using a centrifugal force of 400g, removing supernatant, reserving cells, adding 40mL of basal medium placed at room temperature, gently blowing off cell clusters by using a pipettor, resuspending cells, completing the first cleaning, centrifuging at room temperature for 5min by using a centrifugal force of 400g, removing supernatant, reserving cells, adding 20mL of basal medium placed at room temperature, gently blowing off cell clusters by using a pipettor, and resuspending cells; the resuspended cells were filtered again through a cell screen to remove agglomerated cells, after which the cells were counted.
1.3, B lymphocyte sorting, see patent 201910125091.4, method for efficient isolation of individual antigen-specific B lymphocytes from spleen cells.
1.4 cloning of the Gene encoding Rabbit monoclonal antibody:
the cultured B cell supernatants were used to identify positive clones by antigen coated ELISA. Cell collection of Positive clones after lysis with Quick-RNA TM The MicroPrep kit (available from ZYMO corporation) extracts RNA and reverse transcribes it into cDNA. The naturally paired rabbit monoclonal antibody light and heavy chain variable region genes (VH and VL) were amplified from the cDNA of the corresponding positive clone using PCR and sequenced.
The cDNA is used as a template, a PCR method is adopted to amplify the light chain variable region (VL) and heavy chain variable region (VH) genes of a naturally paired rabbit monoclonal antibody from the cDNA of the corresponding positive clone, and a plurality of clones are selected for sequencing, and the sequencing work is completed by Jin Kairui biotechnology limited company.
Wherein, the PCR reaction system is as follows: 4. Mu.L of cDNA, 1. Mu.L of forward primer (10 mM), 1. Mu.L of reverse primer (10 mM), 12.5. Mu.L of 2 XGloria HiFi (Botek, wuhan), 6.5. Mu.L of N.F H 2 O。
Wherein, the light chain variable region primer pair is:
VL-Primer-F：
5'-tgaattcgagctcggtacccatggacacgagggcccccac-3'；
VL-Primer-R：
5'-cacacacacgatggtgactgttccagttgccacctgatcag-3'。
the heavy chain variable region primer pair is:
VH-Primer-F：
5'-tgaattcgagctcggtacccatggagactgggctgcgctg-3'；
VH-Primer-R：
5'-gtagcctttgaccaggcagcccagggtcaccgtggagctg-3'。
PCR amplification procedure: the reaction mixture was subjected to preliminary denaturation at 98℃for 30s, followed by 40 cycles at 98℃for 10s,64℃for 30s, and 72℃for 30s, and finally kept at 72℃for 5min, and the resulting reaction mixture was kept at 4 ℃.
1.5 preparation and purification of monoclonal antibodies
The heavy chain and light chain genes of the plurality of rabbit monoclonal antibodies selected in step 1.4 are respectively loaded on an expression vector (see fig. 1 and 2). In FIGS. 1 and 2, pBR322 origin and f1 origin are replication promoters in E.coli (E.Coli), ampcilin is a plasmid resistance gene, CMV immearly promotor is a promoter in eukaryotes, SV40 PA terminator is a tailing signal, heavy chain constant is the nucleotide sequence of the heavy chain constant region of a rabbit monoclonal antibody in FIG. 1, and Light chain constant is the nucleotide sequence of the light chain constant region of a rabbit monoclonal antibody in FIG. 2.
The specific method comprises the following steps:
mammalian cell expression vectors containing the heavy chain constant region (FIG. 1) and the light chain constant region (FIG. 2) of the rabbit monoclonal antibodies were routinely linearized with NheI and XbaI restriction enzymes, respectively.
Purifying the PCR product amplified in the step 1.4, and respectively constructing a heavy chain variable region gene and a light chain variable region gene into corresponding mammal expression vectors by adopting a homologous recombination mode; after sequencing verification, the expression vectors containing the light chain genes and the heavy chain genes of the corresponding rabbit monoclonal antibodies are transfected into 293F cells together; and (5) carrying out transfection for 72-96 hours and centrifuging to obtain a culture supernatant.
Purifying recombinant rabbit monoclonal antibody recognizing human AXL protein from the supernatant of the transfected culture medium by using protein A affinity gel resin, wherein the affinity chromatography experimental steps are as follows:
the culture supernatant was transferred to a sterile 50ml centrifuge tube, centrifuged at 1000g, at 4℃or at room temperature for 10 minutes, and the supernatant was collected. The pretreated Protein AAgarose (brand: tiandi manhe; cat. SA 023100) suspension was added to the centrifuged cell supernatant and incubated at room temperature for 3-4 hours or overnight at 4 ℃. After incubation, 1000g was centrifuged for 10min, the Protein A Agarose suspension was transferred to an adsorption column, and centrifuged at room temperature for 1min with a palm centrifuge to separate the solid from the liquid and collect the flow-through. Ten times the Protein AAgarose volume of wash buffer (phosphate buffer pH 7.0) was added and the particles resuspended, centrifuged in a centrifuge, the wash solution collected and the wash repeated twice more. Adding elution buffer (pH of citrate buffer is 3.0) into adsorption column, centrifuging with centrifuge to obtain antibody supernatant, loading into dialysis bag (Soy Bao YA 1070), dialyzing overnight, collecting antibody, packaging after the antibody is qualified, and preserving at-20deg.C.
The result of SDS-PAGE running gel detection of purified rabbit monoclonal antibody is shown in FIG. 3, and the result of subsequent verification test shows that: the obtained monoclonal antibodies A and B have correct sizes, single bands and high antibody purity.
1.6 monoclonal antibody screening and identification
After a plurality of human AXL rabbit monoclonal antibodies are obtained, the rabbit monoclonal antibodies are firstly subjected to preliminary identification and screening, including identification of antibody affinity and identification of antigen recognition epitopes, and the specific method is as follows:
screening of monoclonal antibodies: gator biological separation Using Probe Life CoThe affinity of the obtained rabbit monoclonal antibody was preliminarily determined by a molecular interaction analyzer. Wherein the material used is recombinant human AXL protein, the concentration is 150nM and 75nM, and the concentration of the rabbit monoclonal antibody is 2 mug/mL; by comparing the affinities of the respective antibodies, an affinity constant of 1X 10 or less is selected from the above -9 Is a human antibody.
Identification of antigen recognition epitopes: the obtained rabbit monoclonal antibody is subjected to pairing reaction by using a Gator biomolecular interaction analyzer of Probe Life company to test the identified epitope determinant; wherein the material used is recombinant human AXL protein, the concentration is 5 mug/mL, the concentration of the first rabbit monoclonal antibody is 2 mug/mL, and the concentration of the second rabbit monoclonal antibody is 5 mug/mL; two antibodies recognizing different epitope determinants were selected from the two antibodies, named rabbit monoclonal antibody a and rabbit monoclonal antibody B, respectively, by analyzing the pairing data between the two antibodies.
The affinity of the rabbit monoclonal antibody A and the rabbit monoclonal antibody B was further measured, and the measurement results are shown in FIG. 4. The affinity constants calculated from figure 4 are shown in table 1. Further epitope determination was performed on both, and the results are shown in FIG. 5. As can be seen from fig. 5, the rabbit monoclonal antibodies a and B recognize different epitopes, respectively, and thus both can be used for ELISA pairing.
TABLE 1 affinity constants for Rabbit monoclonal antibody A and Rabbit monoclonal antibody B
| Monoclonal antibodies | Kon(1/Ms) | Koff(1/s) | KD(M) |
| A | 2.37×10 5 | 1.32×10 -4 | 5.55×10 -10 |
| B | 3.41×10 5 | 3.87×10 -4 | 1.14×10 -9 |
Nucleotide sequencing was performed on the selected rabbit monoclonal antibody A and rabbit monoclonal antibody B, respectively, and the sequencing work was completed by Jin Kairui Biotechnology Co.
Wherein, the nucleotide sequence of the light chain variable region of the rabbit monoclonal antibody A is:
GCAGATATTGTGATGACCCAGACGCCGGCGAGTGTGGAAGCGGCTGTCGGTGATACCGTGACCATAAAGTGCCAAGCATCCCAGAACATATACTCAAATCTGGCTTGGTACCAGCAGAAACCCGGCCAACCACCTAAATTGCTTATCTACTTTGCTTCCAATTTGCCCAGCGGCGTCCCCTCCAGGTTCAAGGGATCTGGTTCTGGAACCGAGTATACACTGACGATTTCTGGGGTCCAGTGTGATGACGCAGCCACATATTATTGTCAGAACTACTATATCAACGAGATCGTGTTTGGCGGAGGAACCGAAGTCGTAGTCAAG。
the nucleotide sequence of the heavy chain variable region of rabbit monoclonal antibody a is:
CAAAGCGTGGAGGAGTCTGGGGGAAGACTCGTTACTCCCGGCACACCGCTGACACTCACTTGCACAGTCTCAGGGATCGATCTCAGCACCTACGCGATGATTTGGGTCCGCCAGGCACCAGGGGAGGGGCTCGAATGGATTGGTACCATCTATACTGATGGAGCCACTGCCTACGCCCGCTGGGCAAAAGGAAGGTTCACAATTTCTAAGACAAGCACAACAGTCGACCTAAAGATGACTTCTCTCACTACCGAGGACACCGCTACATACTTTTGTGCACGGGGGTTCGGTATCAGCAAATTCGTGTTTTCTCTCTGGGGTCCGGGGACCTTGGTGACAGTCTCTCTG。
the nucleotide sequence of the light chain variable region of rabbit monoclonal antibody B is:
GCGATTGATATGACTCAAACGCCAAGCTCTGTTAGCGCTGCCGTAGGGGATACGGTTACAATCAATTGCCAGGCCTCTGAGGACATATACTCCTTCCTGGCCTGGTACCAGCAGAAGCCTGGTCACTCTCCTAAGCTGTTGATATACGATGCATCCAAGCTGGCTAGCGGAGTGAGCAGTCGATTTAAGGGCTCAGGATCTGGGACCCAATTTACCCTGACCATCTCCGGGGTGCAATGCGATGACGCGGCCACCTATTACTGTCAGCAGACATATTCCGTCTCCAACGCAGACACGGCCTTCGGCGGGGGCACCGAGGTGGTAGTCAAG。
the nucleotide sequence of the heavy chain variable region of rabbit monoclonal antibody B is:
CAGGAGCAGCTGAAGGAGTCCGGGGGGGGTCTAGTACAGCCCGGGGGTTCTCTGAAGCTGAGTTGCAAAGCAAGCGGATTCAGCCTTTCTTACTACGGGGTAAACTGGGTGCGTCAGGCACCTGGTAAAGGTCTAGAATGGATCGCATGCATCGACCCCGTGTTTGGCGCTACGTATTACGCTAGTTGGGTGAATGACCGGTTTACTATTAGCTCTCATAACGCCCAGAATACCCTATACCTGCAACTCTCTAGTCTGACCCCGGCCGACACCGCAACTTACTTCTGTGCCCGTGGCAGATATTATAGGTACGGCTATACCTATGCAACAGGCATGGACATCTGGGGACCCGGGACCCTTGTCACGGTGTCAAGT。
the amino acid sequences of rabbit monoclonal antibodies A and B were deduced from nucleotide sequence translations.
Example 2
The embodiment provides a double-antibody sandwich ELISA detection method established based on an anti-human AXL rabbit monoclonal antibody A and a rabbit monoclonal antibody B, which specifically comprises the following steps:
2.1, coating: the rabbit monoclonal antibody A is diluted to 4 mug/mL by 1 XPBS, and after being mixed evenly by a vortex meter, 100 mug/hole is added into a 96-hole micro-pore plate, covered with a cover plate film and placed in a refrigerator at 4 ℃ for 16-20h.
2.2, washing the plate: after the incubation was completed, the well liquid was discarded, the plate was washed once with 1 XPBST, 300. Mu.L was added, and after 40s of standing, the well liquid was discarded, and the well liquid was dried on a piece of flat paper.
2.3, sealing: adding blocking solution (containing 2% BSA+5% sucrose+0.05% Tween 20+0.1%proclin 300,pH =7.2 in 1×PBS) into the plate hole at 200 μl/hole, covering with cover plate film, blocking at 37deg.C for 2 hr, discarding blocking solution after blocking, drying the ELISA plate, baking at 37deg.C for 0.5-2 hr, and taking out.
2.4, adding protein: the human AXL protein (recombinant human AXL truncated protein, commercially available, cat# RP 00087) was diluted with phosphate buffer containing 2% bovine serum albumin, 0.05% Tween-20, 0.1% proclin300, ph=7.2, at the following concentration: 10,5,2.5,1.25,0.63,0.31,0.16,0ng/mL, and then added to the ELISA plate at 100. Mu.L/well, covered with a cover plate membrane, and incubated at 37℃for 2h.
2.5, washing the plate: after the incubation was completed, the well liquid was discarded, the plate was washed three times with 1 XPBST, 300. Mu.L was added, and after 40s of standing, the well liquid was discarded, and the well liquid was dried on a piece of flat paper.
2.6, adding detection antibody: after dilution of the biotin-labeled rabbit monoclonal antibody B to 0.003. Mu.g/ml, the mixture was sequentially added to the ELISA plate at 100. Mu.L/well, covered with a cover plate membrane, and incubated at 37℃for 1 hour.
In the step, the method for labeling the rabbit monoclonal antibody B by biotin comprises the following steps:
anti-human AXL rabbit monoclonal antibody B was prepared as a 1mg/mL solution, and NHS-LC-biotin (N-succinimidyl 6-biotin aminocaproic acid, available from Thermo corporation) was prepared as a 60mg/mL solution with DMSO (dimethyl sulfoxide);
200 mu L of 1mg/mL of anti-human AXL rabbit monoclonal antibody B solution is taken, and 10 mu L of 60mg/mL of NHS-LC-biotin solution is added; after mixing, standing at room temperature for 30 minutes, 50. Mu.g of 500mM Tris-HCl solution of pH=9.0 was added to terminate the reaction;
finally, 4mL of 1 x PBS buffer at ph=7.4 was added and centrifuged with a 30KDa exclusion limit to remove excess biotin molecules and allow for equilibration of the buffer system.
2.7, washing the plate: after the incubation was completed, the well liquid was discarded, the plate was washed three times with 1 XPBST, 300. Mu.L was added, and after 40s of standing, the well liquid was discarded, and the well liquid was dried on a piece of flat paper.
2.8, adding SA-HRP: 100 XSA-HRP (horseradish peroxidase labeled streptavidin, purchased from Wohan Sanying organism) concentrate was diluted 100-fold, and added sequentially to an ELISA plate at 100. Mu.L/well, covered with a cover plate membrane, and incubated at 37℃for 0.5h.
2.9, washing the plate: after the incubation was completed, the well liquid was discarded, the plate was washed three times with 1 XPBST, 300. Mu.L was added, and after 40s of standing, the well liquid was discarded, and the well liquid was dried on a piece of flat paper.
2.10, adding TMB color development liquid: TMB (3, 3', 5' -tetramethylbenzidine) color development solution was added to the ELISA plate at 100. Mu.L/well, covered with a cover plate film, and incubated at 37℃for 15min.
2.11, after incubation, the ELISA plate was removed, 50. Mu.L of stop solution (1 mol/L hydrochloric acid) was added to each well, absorbance values were measured immediately with an ELISA reader at 450nm and 630nm, and the absorbance value corrected at the OD630nm position was subtracted from the OD450 nm.
2.12, plotted on the abscissa with recombinant human AXL protein concentration and absorbance value correction Y1 (y1=od450 nm-OD630 nm) on the ordinate, a graph obtained using Logistic curve four-parameter fitting is shown in fig. 6.
In FIG. 6, the absorbance difference ΔOD (OD 450nm -OD 630nm ) The relation with the recombinant human AXL protein concentration X satisfies the equation
Y1＝0.2337+8.55677/[1+(X/15.69706) -1.5281 ](R 2 ＝0.9999)。
Substituting an average value of 200 holes and a SD value of 2 into a standard fitting curve, and back calculating to obtain the sensitivity, wherein the calculated detection sensitivity of the double-antibody sandwich ELISA method established based on the anti-human AXL protein rabbit monoclonal antibodies A and B is 0.11ng/mL.
The foregoing description is only of the preferred embodiments of the present invention and is not intended to limit the scope of the present invention. Various modifications and alterations of this invention will occur to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A rabbit monoclonal antibody aiming at human AXL, which is characterized in that the rabbit monoclonal antibody is a rabbit monoclonal antibody a and a rabbit monoclonal antibody B, which respectively bind different antigenic determinants on the surface of human AXL;
the sequence of the complementarity determining region of the rabbit monoclonal antibody A is respectively shown as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO. 10;
the sequence of the complementarity determining region of the rabbit monoclonal antibody B is shown as SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20 respectively.
2. The rabbit monoclonal antibody directed against human AXL of claim 1, wherein the sequence of the light chain variable region of rabbit monoclonal antibody a is shown in SEQ ID No.2 and the sequence of the heavy chain variable region is shown in SEQ ID No. 7;
the sequence of the light chain variable region of the rabbit monoclonal antibody B is shown as SEQ ID NO.12, and/or the sequence of the heavy chain variable region is shown as SEQ ID NO. 17.
3. The rabbit monoclonal antibody directed against human AXL of claim 2, wherein the full length sequence of the light chain of rabbit monoclonal antibody a is shown in SEQ ID No.1 and/or the full length sequence of the heavy chain is shown in SEQ ID No. 6;
the full-length sequence of the light chain of the rabbit monoclonal antibody B is shown as SEQ ID NO.11, and/or the full-length sequence of the heavy chain is shown as SEQ ID NO. 16.
4. A gene sequence encoding a rabbit monoclonal antibody directed against human AXL according to any one of claims 1 to 3, or an expression vector or host comprising the gene sequence.
5. A labeled antibody conjugate prepared by reacting the rabbit monoclonal antibody directed against human AXL of any one of claims 1-3 with a fluorescent labeling molecule.
6. Use of a rabbit monoclonal antibody directed against human AXL according to any one of claims 1 to 3 for the preparation of a reagent or kit for detecting human AXL.
7. The use according to claim 6, wherein the reagent or kit is used for enzyme-linked immunosorbent assay of human AXL, wherein the rabbit monoclonal antibody a is used as a capture antibody and the rabbit monoclonal antibody B is used as a marker antibody.
8. A kit or kit for detecting human AXL, comprising a rabbit monoclonal antibody a and a rabbit monoclonal antibody B directed against human AXL according to any one of claims 1 to 3.
9. The reagent or kit for detecting human AXL according to claim 8, wherein said human AXL is at least one selected from recombinant human AXL, human AXL secreted by cells, and human AXL in serum.
10. A method of preparing a rabbit monoclonal antibody directed against human AXL according to any one of claims 1 to 3, comprising the steps of:
loading the heavy chain gene and the light chain gene of the rabbit monoclonal antibody against human AXL according to any one of claims 1 to 3 onto expression vectors, respectively, and transfecting engineering cells;
culturing to obtain a supernatant containing the rabbit monoclonal antibody A and the rabbit monoclonal antibody B;
purifying to obtain the final product.
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