CN114989303B - anti-CD 56 recombinant rabbit monoclonal antibody and application thereof - Google Patents

anti-CD 56 recombinant rabbit monoclonal antibody and application thereof Download PDF

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CN114989303B
CN114989303B CN202210614768.2A CN202210614768A CN114989303B CN 114989303 B CN114989303 B CN 114989303B CN 202210614768 A CN202210614768 A CN 202210614768A CN 114989303 B CN114989303 B CN 114989303B
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monoclonal antibody
rabbit monoclonal
variable region
chain variable
recombinant rabbit
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CN114989303A (en
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刘杨
张伟
巩丽
李艳红
封兰兰
富金
王圆圆
张富琴
宋砚明
吴纯
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Suzhou Baidao Medical Technology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3

Abstract

The invention relates to an anti-CD 56 recombinant rabbit monoclonal antibody, which comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 4; the amino acid sequence of the light chain variable region is shown as SEQ ID No. 5. Compared with the commercially available anti-CD 56 antibody, the anti-CD 56 recombinant rabbit monoclonal antibody provided by the invention has higher affinity with CD56 protein, can recognize and detect the expression of CD56 protein on tumor cells or immune cells with high specificity and high sensitivity, can be applied to the detection and screening fields of Immunohistochemistry (IHC), indirect ELISA, immunoblotting (Westernblotting), antibody chip preparation, flow cytometry and the like, is beneficial to obtaining more accurate detection and evaluation results, and reduces detection cost and interference of background signals.

Description

anti-CD 56 recombinant rabbit monoclonal antibody and application thereof
Technical Field
The invention belongs to the technical field of immunochemistry, and particularly relates to an anti-CD 56 antibody and application thereof, in particular to application in immunohistochemical detection.
Background
CD56 is a group of related cell surface glycoproteins that play an important role in embryogenic development and in the interconnection of neural cells. CD56 antigen is expressed predominantly in neurons, astrocytes, schwann cells, NK cells and small activated T lymphocytes. The antibody mainly determines the diagnosis of tumor derived from neuroectodermal, lung small cell cancer and NK cell lymphoma. CD56 is highly expressed in normal thyroid tissues, nodular goiter, follicular adenoma and papillary hyperplasia, but is not expressed or is weakly expressed in papillary thyroid cancers, so that diagnosis of the papillary thyroid cancers can be assisted by detecting molecular markers such as CD56, and the sensitivity, specificity and accuracy of diagnosis are improved. Therefore, the screening of a high-sensitivity and high-specificity anti-CD 56 antibody plays a very important role in identifying and detecting the expression of CD56 protein on tumor cells or immune cells.
Disclosure of Invention
First, the technical problem to be solved
In view of the above-mentioned shortcomings and disadvantages of the prior art, the invention provides an anti-CD 56 recombinant rabbit monoclonal antibody which has wide application and can accurately recognize CD56 expression, and through immunohistochemical detection of various different tissues, the antibody can well detect the expression of CD56 protein on tumor cells or immune cells, and can be applied to the detection and screening fields of Immunohistochemistry (IHC), indirect ELISA, immunoblotting (Western blotting), antibody chip preparation, flow cytometry and the like. The invention also relates to a nucleotide sequence, a recombinant plasmid or an expression vector for encoding the anti-CD 56 recombinant rabbit monoclonal antibody, a preparation method, application of the anti-CD 56 recombinant rabbit monoclonal antibody in a CD56 protein detection method or device and the like.
(II) technical scheme
In order to achieve the above purpose, the main technical scheme adopted by the invention comprises the following steps:
in a first aspect, the present invention provides an anti-CD 56 recombinant rabbit monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 4; the amino acid sequence of the light chain variable region is shown as SEQ ID No. 5.
The anti-CD 56 recombinant rabbit monoclonal antibody (CD 56 rabbit antibody) can be used for immunohistochemical detection, and can recognize and detect the expression of CD56 protein on tumor cells or immune cells with high specificity and high sensitivity.
The anti-CD 56 monoclonal antibody is obtained by recombinant expression of mammalian cells. Specifically, the anti-CD 56 recombinant rabbit monoclonal antibody provided by the invention is produced by eukaryotic expression of 293 cells through rabbit hybridoma fusion screening. In the preparation of the anti-CD 56 monoclonal antibody, an antigen for immunizing rabbits (New Zealand white rabbits) is a synthetic polypeptide, the amino acid sequence of which is shown as SEQ ID No.1, and the synthetic polypeptide is obtained by artificial chemical synthesis. After immunization, a positive hybridoma cell line capable of efficiently secreting monoclonal antibodies is obtained through cell fusion, clone screening, a molecular cloning technology is used for obtaining nucleotide sequences of heavy chain amino acid sequences and light chain amino acid sequences of the antibodies, the nucleotide sequences are constructed on eukaryotic expression vectors, the eukaryotic expression vectors are transfected into 293 cell lines through transfection reagents, cell supernatants are collected, and the cell supernatants are purified through Protein A column affinity chromatography, so that rabbit monoclonal antibodies are obtained. Immunohistochemical detection showed that the antibody specifically recognized CD56 protein.
The anti-CD 56 monoclonal antibody can recognize the recombinant CD56 antigen protein and CD56 molecules on tumor cells and immune cells; the anti-CD 56 monoclonal antibody can also be applied to an immunohistochemical pathological diagnosis reagent.
In a second aspect, the present invention provides a coding gene for encoding the above anti-CD 56 recombinant rabbit monoclonal antibody.
Preferably, the coding gene comprises a DNA sequence shown as SEQ ID No.2 for coding the heavy chain variable region of the anti-CD 56 recombinant rabbit monoclonal antibody; and the DNA sequence shown as SEQ ID No.3 is used for encoding the light chain variable region of the anti-CD 56 recombinant rabbit monoclonal antibody.
In a third aspect, the invention relates to a nucleic acid molecule comprising a coding gene for said anti-CD 56 recombinant rabbit monoclonal antibody.
In a fourth aspect, the invention provides an expression vector or recombinant plasmid comprising a nucleic acid molecule as described above.
In a fifth aspect, the present invention provides a method for preparing an anti-CD 56 recombinant rabbit monoclonal antibody, transfecting cells with the above expression vector, culturing the transfected cells, collecting cell supernatant and purifying to obtain the anti-CD 56 recombinant rabbit monoclonal antibody.
More preferably, the preparation method comprises the steps of:
(1) Immunization of rabbits: analyzing the molecular sequence of CD56 protein, selecting and using proper polypeptide sequence as immunogen according to the structure, antigenicity, hydrophilicity and hydrophobicity of constituent amino acid and secondary structure of CD56 on cell membrane, coupling via KLH or OVA, and immunizing rabbit; the polypeptide is an artificially synthesized polypeptide shown in SEQ ID No. 1;
(2) Preparation of hybridoma cell lines: obtaining a positive hybridoma stable cell line capable of efficiently secreting antibodies through cell fusion, cloning and screening, and separating total RNA from the hybridoma cell line;
(3) Obtaining an antibody sequence: obtaining nucleotide sequences of an antibody heavy chain variable region and an antibody light chain variable region by using a specific primer through a PCR amplification technology;
(4) Antibody expression and purification: cloning the nucleotide sequence into an expression vector, transiently transfecting the cultured cells by using a transfection method, collecting the supernatant after the culture, and purifying the supernatant by using Protein A to obtain the antibody with the purity of more than 95 percent.
In a sixth aspect, the anti-CD 56 recombinant rabbit monoclonal antibody, the coding gene, the nucleic acid molecule, the expression vector or the recombinant plasmid is applied to the preparation of a CD56 protein molecule detection device. The detection device includes, but is not limited to, a kit, an antibody chip, and the like.
In a seventh aspect, the present invention also provides a CD56 assay kit comprising the anti-CD 56 recombinant rabbit monoclonal antibody described above and an immunohistochemical assay reagent.
Preferably, the CD56 detection kit comprises: anti-CD 56 recombinant rabbit monoclonal antibody, HRP enzyme-labeled secondary antibody, EDTA repair liquid, catalase blocking liquid, DAB concentrated liquid, DAB buffer liquid, hematoxylin and bluing liquid.
In the case of immunohistochemical detection, the detection steps comprise dewaxing, antigen retrieval, endogenous peroxidase inactivation, blocking, primary antibody incubation, secondary antibody incubation, DAB color development, counterstaining, dehydration, sealing and microscopic examination, and the like.
(III) beneficial effects
The anti-CD 56 recombinant rabbit monoclonal antibody provided by the invention has high specificity and high sensitivity in combination with CD56 protein molecules, can specifically identify and detect the expression of CD56 protein on tumor cells or immune cells, and has positive high expression when detecting CD56 protein, so that the antibody can be applied to the detection and screening fields of Immunohistochemistry (IHC), indirect ELISA, immunoblotting (Western blotting), antibody chip preparation, flow cytometry and the like, and is favorable for obtaining accurate evaluation and detection results. The CD56 recombinant rabbit monoclonal antibody cloned by 165A3F9 is easy to score in IHC staining due to the characteristics of good specificity, strong positive signals and the like, and is favorable for accurately detecting and distinguishing cancers.
Drawings
FIG. 1 shows the result of immunohistochemical detection of the 165A3F9 anti-CD 56 monoclonal antibody prepared in the present invention and the commercial anti-CD 56 clone MRQ-42 antibody in human appendiceal tissue, wherein the primary antibody is used at a concentration of 0.6. Mu.g/mL.
FIG. 2 is a statistical plot of potency detection of 165A3F9 anti-CD 56 monoclonal antibodies of the invention at 7 gradient concentrations.
FIG. 3 is a graph comparing fluorescence signal intensities (MFI) of a blank, 165A3F9 anti-CD 56 monoclonal antibody sample obtained by flow cytometry staining analysis after addition of a quantitative 165A3F9 anti-CD 56 monoclonal antibody to cells expressing CD56 protein.
Detailed Description
The technical means adopted by the invention and the effects thereof are further described below with reference to the examples and the attached drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting thereof.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted. The human tissue samples are formalin-fixed and paraffin-embedded human tissue samples, and are subjected to pathological confirmation and informed consent of patients.
Example 1
This example is the preparation and screening of anti-CD 56 recombinant rabbit monoclonal antibodies, comprising the steps of:
(1) Antigen preparation
The specific sequence of the CD56 antigen is shown as SEQ ID NO. 1.
SEQ ID No.1：VKTVPNDATQTKENESKA。
The polypeptide sequence is selected by analyzing the sequence of the CD56 molecule according to the structure, antigenicity, hydrophilicity and hydrophobicity of constituent amino acids and secondary structure of the CD56 protein molecule on a cell membrane. The polypeptide with the sequence shown in SEQ ID NO.1 is synthesized artificially, and the synthesized polypeptide is used as an antigen for immunizing rabbits. In immunization, the polypeptide with the sequence shown in SEQ ID NO.1 is coupled through KLH or OVA and then used as an immunogen to immunize rabbits.
(2) Immunization
The polypeptide sequence (CD 56 antigen) of SEQ ID NO.1 is respectively mixed and emulsified with complete Freund's adjuvant (1:1), a plurality of New Zealand white rabbits are respectively immunized by subcutaneous injection, and after two weeks, the CD56 antigen containing the polypeptide shown in SEQ ID NO.1 is emulsified with incomplete Freund's adjuvant (1:1) for the second immunization and the third immunization. Blood is taken after three immunization, and serum titer is measured by ELISA method gradient dilution; and selecting the rabbit with the highest immune antibody titer with the SEQ ID NO.1 antigen for the next cell fusion.
(3) Cell fusion
The sp2/0 myeloma cells derived from the mice were prepared in advance, and the sp2/0 myeloma cells were allowed to be in the logarithmic growth phase at the time of fusion. Taking immunized rabbit spleenPreparing a lymphocyte single-cell suspension by using the viscera; mixing rabbit spleen lymphocytes with myeloma cells, dripping 50% PEG1500, adding IMDM culture medium, centrifuging, removing supernatant, adding HAT culture medium, suspending gently, mixing, fixing volume to 800mL, packaging in 96-well plate, placing at 37deg.C, and 5% CO 2 Culturing in a constant temperature incubator. After 6-9 days of fusion, the state of the fused cells in the 96-well plate is observed, and the solution is replaced by HT and is continuously placed at 37 ℃ and 5% CO 2 Culturing in a constant temperature incubator.
(4) Screening and cloning
The cloned cells were screened 7-10 days after fusion by ELISA test using CD56 antigen (SEQ ID NO: 1). And labeling corresponding cell strain numbers, and carrying out limiting dilution on positive hole cells until the result of ELISA measurement of the whole 96-well plate is positive. And selecting a monoclonal stable strain with high positive value to obtain a hybridoma cell strain secreting the specific monoclonal antibody, and recording the hybridoma cell strain as 165A3F9.
(5) Antibody sequencing is carried out on the hybridoma cell strains which are screened
Total RNA was isolated from 165A3F9 hybridoma cells according to the reagent TriZol instructions, reverse transcribed into cDNA according to TIANScript first strand cDNA synthesis kit instructions, amplified using specific primers (heavy chain variable region primer, VH-F: AGACTGGGCTGCGCTGGCTTC, VH-R GTGAGGGTGCCCGAG; light chain variable region primer: VK-F ATGGACAYGAGGGCCCCCACTC, VK-R: GGTGGGAAGATGAGGACAGTAGG) to obtain nucleotide sequences of the antibody heavy chain variable region and antibody light chain variable region, and then cloned into eukaryotic expression vectors (InvivoGen, pfuse-rchg, pfuse2-rclk 1) in preparation for cell transfection.
(6) Cell transfection and screening
293 cells to be transfected are prepared in advance, fresh culture medium is centrifugally replaced and then is respectively put into 24-well plates, 1.5ml of culture medium is added into each well according to the required quantity, and the density is 3 multiplied by 10 6 And each ml.
Mixing the eukaryotic expression vector and PEI according to the proportion of 1:6, adding the mixture into prepared 293 cells, and placing the mixture at 37 ℃ and 5% CO 2 Is cultured in a shaker. After 3-5 days of culture, the transfected cell supernatant and the pair were subjected toScreening positive holes by ELISA detection on antigens, and continuing to perform immunohistochemical detection on cell supernatants of the positive holes, wherein if the immunohistochemical detection is positive, the detected antibody sequences are correct.
(7) Preparation and purification of cell-on-list antibodies
And (3) carrying out a large number of cell transfection on the expression vector with positive confirmation, continuously culturing for 3-5 days, collecting cell suspension, centrifuging, taking supernatant, and purifying by using an affinity chromatography method. Measuring the concentration of the purified monoclonal antibody, sub-packaging, and storing in a refrigerator at 4-8 ℃.
Finally, the heavy chain variable region amino acid sequence of the 165A3F9 anti-CD 56 recombinant rabbit monoclonal antibody is encoded by the DNA sequence shown in SEQ ID No.2, and the light chain variable region amino acid sequence of the anti-CD 56 recombinant rabbit monoclonal antibody is encoded by the DNA sequence shown in SEQ ID No. 3.
The specific sequence of SEQ ID No.2-3 is as follows:
SEQ ID No.2：
cagtcggtggaggagtccgggggtcgcctggtcacgcctgggacacccctgacactcacctgcacagtctctggattctccctcagtagctatggaatgaactgggtccgccaggctccagggaaggggctggaatacatcggaatcattagtattagtggtaacacattctacgcgagctgggcgaaaggccgattcaccatctccagaacctcgaccacggtggatctgaaaatcaccagtccgacaaccgaggacacggccacctatttctgtgccagatcttatactggtagtagtacttatggttttgatccctggggcccaggcaccctggtcaccgtctcctca。
SEQ ID No.3：
gcctatgatatgacccagactccagcctctgtggaggtagctgtgggaggcacagtcaccatcaagtgccaggccagtcagagcattagtagctacttagcctggtatcagcagaaaccagggcagcctcccaagctcctgatctacagggcatccactctggcatctggggtcccatcgcggttcaaaggcagtggatctgggacagagttcactctcaccattagcgacctggagtgtgccgatgctgccacttattactgtcaacagacttatgctaatggtgatgttgataatagtttcggcggagggaccgaggtggtggtcaaa。
and translating the obtained base sequence into an amino acid sequence, and analyzing to obtain the heavy chain variable region amino acid sequence of the 165A3F9 anti-CD 56 recombinant rabbit monoclonal antibody shown in SEQ ID No.4, wherein the light chain variable region amino acid sequence of the anti-CD 56 recombinant rabbit monoclonal antibody is shown in SEQ ID No. 5.
The specific sequence of SEQ ID No.4-5 is as follows:
SEQ ID No.4：
QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYGMNWVRQAPGKGLE YIGIISISGNTFYASWAKGRFTISRTSTTVDLKITSPTTEDTATYFCARSY TGSSTYGFDPWGPGTLVTVSS。
SEQ ID No.5:
AYDMTQTPASVEVAVGGTVTIKCQASQSISSYLAWYQQKPGQPP KLLIYRASTLASGVPSRFKGSGSGTEFTLTISDLECADAATYYCQQTYA NGDVDNSFGGGTEVVVK。
example 2
The present example is an immunohistochemical detection of anti-CD 56 recombinant rabbit monoclonal antibody as primary antibody, the method is as follows:
(1) Sample slice preparation: and (3) baking slices of the human appendiceal tissue after formalin-fixed paraffin embedding in a constant temperature oven at 60 ℃ for 1-2 hours, and preserving for later use.
(2) Slice dewaxing: paraffin sections were first dewaxed in fresh xylene and soaked 2 times for 10min each.
(3) Slice hydration: sequentially soaking in absolute ethanol, 95% ethanol, 85% ethanol and 70% ethanol for 5min for hydration, and washing with purified water for 2 times each for 3min.
(4) Antigen retrieval: repairing for 3min by using a high-temperature thermal repairing method (if an automatic repairing instrument is used, the repairing can be carried out for 20min at the high temperature of 98 ℃), naturally cooling the slice to room temperature, then looping the tissue to be detected by using an immunohistochemical pen, and flushing with purified water for 2 times for 3min each time.
(5) Inactivation of endogenous peroxidases: proper amount of endogenous peroxidase blocking agent is dripped to completely cover the tissue, after incubation for 10min at room temperature, purified water is washed 2 times for 3min each time, and PBST is washed once.
(6) Incubation resistance: the experiments were divided into two groups, one group was added with 100. Mu.L of 0.6. Mu.g/mL 165A3F9 anti-CD 56 recombinant rabbit monoclonal antibody and the other group was added with commercially available anti-CD 56 cloned MRQ-42 antibody to completely cover the tissue, and the tissue was incubated in an incubator at 37℃for 1h, and PBST was rinsed 3 times for 5min each.
(7) Secondary antibody incubation: the secondary antibody incubation was performed according to the instructions of the DAB staining solution kit of the secondary antibody staining system used, and after incubation, the PBST was washed 3 times, 5min each time, with purified water 1 time.
(8) DAB color development: preparing DAB color development liquid according to the instruction of the DAB color development liquid kit, dripping a proper amount of prepared DAB color development liquid until the DAB color development liquid completely covers tissues, stopping dyeing when the color is not deepened, and flushing with purified water for 3 times.
(9) Hematoxylin counterstain: counterstaining the sections according to hematoxylin manufacturer instructions and advice, PBST or tap water washing to return to blue.
(10) And (3) dehydration and transparency: sequentially soaking 70%,85%,95%,100% gradient alcohol and 3min each time; 2 times the xylene is transparent, 5min each time.
(11) Sealing piece: the samples were flaked with neutral gum.
As can be seen from the results of FIG. 1, CD56 protein is stained with specific cell membranes in human appendiceal tissue covered with the 165A3F9 clone antibody and MRQ-42 antibody, and the former is more intense (shows darker color after gray scale treatment) and the range of staining is greater. Therefore, the affinity between the CD56 recombinant rabbit monoclonal antibody cloned by 165A3F9 and the CD56 protein is superior to that of a commercial MRQ-42 antibody, and the monoclonal antibody has the characteristics of better specificity, stronger positive signals and the like, and is more accurate for detecting and distinguishing cancers.
Example 3
This example is an assay for affinity for a 165A3F9 anti-CD 56 recombinant rabbit monoclonal antibody, as follows:
(1) The labeled CD56 polypeptide was removed from 4℃and returned to room temperature. Diluted to a concentration of 1. Mu.g/ml, added to a 96-well microplate at 100. Mu.L/well and incubated overnight at 4℃followed by blocking overnight at 4℃with 2% BSA.
(2) The CD56 recombinant rabbit monoclonal antibody cloned by 165A3F9 is diluted to an initial concentration of 0.5 mug/mL, and is subjected to 2-time gradient dilution in sequence, and 7 concentration gradients are set for comparison.
(3) The diluted anti-CD 56 recombinant rabbit monoclonal antibody is added to a 96-well ELISA plate with polypeptide according to 100 mu L/well, a sealing plate film is covered, and the temperature is kept constant at 37 ℃ for 1h, so that the reaction reaches equilibrium.
(4) And taking out the ELISA plate after the reaction is finished, discarding the liquid, washing with purified water for 5 times, and drying the water by beating.
(5) The HRP-labeled goat anti-rabbit IgG was diluted according to the instructions for secondary antibody use, added to the elisa plate at 100 μl/well and incubated at 37 ℃ for 1h to equilibrate the reaction.
(6) And taking out the ELISA plate after the reaction is finished, discarding the liquid, washing with purified water for 5 times, and drying the water by beating.
(7) TMB color-developing solution was added at 100. Mu.L/well, and the reaction was carried out at room temperature for 6 minutes.
(8) After completion of the reaction, 2M H was added at a concentration of 50. Mu.L/well 2 SO 4 The color development was terminated.
(9) OD values were read at 450nm on a microplate reader, data were collated, and analysis results are shown in fig. 2.
The results show that in 7 concentration gradient tests, the anti-CD 56 recombinant rabbit monoclonal antibody of 165A3F9 clone has strong affinity and high sensitivity to CD56 protein molecules, can still reach a higher OD value under the condition of lower antibody concentration, and can save the experiment and detection cost.
Example 4
This example describes a flow cytometry staining assay for 165A3F9 secreted anti-CD 56 antibodies, the embodiment being as follows:
(1) 1X 10≡6 cells expressing CD56 protein were collected, washed once with 500. Mu.L of PBS, and resuspended with 100. Mu.L of PBS.
(2) 0.5 μg of the anti-CD 56 recombinant rabbit monoclonal antibody of 165A3F9 clone was added into the cells, mixed gently and incubated at room temperature for 15min.
(3) The cells were incubated 2 times with 500. Mu.L PBS wash and resuspended with 100. Mu.L PBS.
(4) 0.5 mug Alexa fluor 488 fluorescence-labeled goat anti-rabbit fluorescence secondary antibody is added into the cells, and after being mixed evenly by light blowing, the cells are incubated for 15min at room temperature and in a dark place.
(5) Cells were washed 2 times with 500 μl PBS to wash residual fluorescent secondary antibodies and resuspended in 500 μl PBS for flow-on-machine detection, and 20000 cells were sampled for analysis of fluorescent signal intensity in both cases (no antibody and 165A3F9 antibody addition). The results of fluorescence signal intensity relative values (MFI) are shown in table 1, and the results of flow analysis and detection are shown in fig. 3.
Table 1:
sample of MFI
Blank control 25.6
165A3F9 antibodies 1732
The experimental results show that the 165A3F9 anti-CD 56 recombinant rabbit monoclonal antibody has higher antibody affinity, compared with a blank control group, under the condition that the cell number is the same, the MFI signal intensity of the 165A3F9 anti-CD 56 antibody group is enhanced by 67.65 times, which indicates that the 165A3F9 anti-CD 56 recombinant rabbit monoclonal antibody can obtain very high fluorescence signal intensity.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Sequence listing
<110> Suzhou Baidao medical science and technology Co., ltd
<120> an anti-CD 56 recombinant rabbit monoclonal antibody and application thereof
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cagtcggtgg aggagtccgg gggtcgcctg gtcacgcctg ggacacccct gacactcacc 60
tgcacagtct ctggattctc cctcagtagc tatggaatga actgggtccg ccaggctcca 120
gggaaggggc tggaatacat cggaatcatt agtattagtg gtaacacatt ctacgcgagc 180
tgggcgaaag gccgattcac catctccaga acctcgacca cggtggatct gaaaatcacc 240
agtccgacaa ccgaggacac ggccacctat ttctgtgcca gatcttatac tggtagtagt 300
acttatggtt ttgatccctg gggcccaggc accctggtca ccgtctcctc a 351
<210> 3
<211> 330
<212> DNA/RNA
<213> Artificial Sequence
<400> 3
gcctatgata tgacccagac tccagcctct gtggaggtag ctgtgggagg cacagtcacc 60
atcaagtgcc aggccagtca gagcattagt agctacttag cctggtatca gcagaaacca 120
gggcagcctc ccaagctcct gatctacagg gcatccactc tggcatctgg ggtcccatcg 180
cggttcaaag gcagtggatc tgggacagag ttcactctca ccattagcga cctggagtgt 240
gccgatgctg ccacttatta ctgtcaacag acttatgcta atggtgatgt tgataatagt 300
ttcggcggag ggaccgaggt ggtggtcaaa 330
<210> 4
<211> 117
<212> PRT
<213> Artificial Sequence
<400> 4
Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro
1 5 10 15
Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr Gly
20 25 30
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly
35 40 45
Ile Ile Ser Ile Ser Gly Asn Thr Phe Tyr Ala Ser Trp Ala Lys Gly
50 55 60
Arg Phe Thr Ile Ser Arg Thr Ser Thr Thr Val Asp Leu Lys Ile Thr
65 70 75 80
Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Ser Tyr
85 90 95
Thr Gly Ser Ser Thr Tyr Gly Phe Asp Pro Trp Gly Pro Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 5
<211> 110
<212> PRT
<213> Artificial Sequence
<400> 5
Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly
1 5 10 15
Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile
35 40 45
Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asp Leu Glu Cys
65 70 75 80
Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Ala Asn Gly Asp
85 90 95
Val Asp Asn Ser Phe Gly Gly Gly Thr Glu Val Val Val Lys
100 105 110

Claims (9)

1. An anti-CD 56 recombinant rabbit monoclonal antibody, which is characterized by comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 4; the amino acid sequence of the light chain variable region is shown as SEQ ID No. 5.
2. A coding gene for the anti-CD 56 recombinant rabbit monoclonal antibody of claim 1.
3. The coding gene according to claim 2, characterized in that it comprises: a DNA sequence as set forth in SEQ ID No.2 for encoding the heavy chain variable region of said anti-CD 56 recombinant rabbit monoclonal antibody, and a DNA sequence as set forth in SEQ ID No.3 for encoding the light chain variable region of said anti-CD 56 recombinant rabbit monoclonal antibody.
4. A nucleic acid molecule comprising the coding gene of claim 2 or 3.
5. An expression vector or recombinant plasmid comprising the nucleic acid molecule of claim 4.
6. A preparation method of an anti-CD 56 recombinant rabbit monoclonal antibody is characterized in that the expression vector of claim 5 is adopted to transfect cells, the cells are continuously cultured after transfection, and cell supernatant is collected and purified to obtain the anti-CD 56 recombinant rabbit monoclonal antibody.
7. Use of an anti-CD 56 recombinant rabbit monoclonal antibody according to claim 1, a coding gene according to claim 2 or 3, a nucleic acid molecule according to claim 4, an expression vector according to claim 5 or a recombinant plasmid for the preparation of a CD56 detection device.
8. A CD56 assay kit comprising the anti-CD 56 recombinant rabbit monoclonal antibody of claim 1 and an immunohistochemical detection reagent.
9. The CD56 assay kit of claim 8, wherein the immunohistochemical detection reagent comprises HRP enzyme-labeled secondary antibody, EDTA repair solution, catalase blocking solution, DAB concentrate, DAB buffer, hematoxylin, and bluing solution.
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CN116041520B (en) * 2022-12-28 2023-09-22 武汉爱博泰克生物科技有限公司 Rabbit monoclonal antibody aiming at Human CD63, and preparation method and application thereof
CN116355093B (en) * 2023-06-02 2023-08-18 苏州百道医疗科技有限公司 anti-CK 7 recombinant rabbit monoclonal antibody and application thereof
CN116789834B (en) * 2023-08-29 2023-10-27 苏州为度生物技术有限公司天津分公司 Anti-human CD56 engineering antibody and application thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992016563A1 (en) * 1991-03-12 1992-10-01 Biogen, Inc. Monoclonal antibodies recognizing lymphocyte function associated antigen-3
CN107488231A (en) * 2017-09-15 2017-12-19 四川大学 Anti- CD56 antibody and application thereof
CN113831410A (en) * 2021-08-12 2021-12-24 福州迈新生物技术开发有限公司 anti-CD 56 protein monoclonal antibody and cell strain, preparation method and application thereof

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CA2994412A1 (en) * 2015-07-31 2017-02-09 Memorial Sloan-Kettering Cancer Center Antigen-binding proteins targeting cd56 and uses thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992016563A1 (en) * 1991-03-12 1992-10-01 Biogen, Inc. Monoclonal antibodies recognizing lymphocyte function associated antigen-3
CN107488231A (en) * 2017-09-15 2017-12-19 四川大学 Anti- CD56 antibody and application thereof
CN113831410A (en) * 2021-08-12 2021-12-24 福州迈新生物技术开发有限公司 anti-CD 56 protein monoclonal antibody and cell strain, preparation method and application thereof

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