阅读说明：本技术 一种bsa单抗制备方法 (Preparation method of BSA (bovine serum albumin) monoclonal antibody ) 是由 韩之波 于 2020-03-30 设计创作，主要内容包括：本发明提供一种BSA单抗制备方法,所述制备方法为：制备多肽：首先制备10mg纯度为90％的多肽,并取其中5mg多肽与KLH偶联；动物免疫：选取五只小白鼠,首先进行初次免疫,接着进行第二次免疫,并重复第二次免疫步骤对小白鼠进行第三次免疫,接着进行第四次免疫；细胞融合；筛选：选择阳性值高且单克隆的孔；培养扩增所选择的单克隆杂交瘤细胞并冻存细胞；得到纯化的单克隆抗体。本发明能够制备出大量的单克隆抗体,将制备的抗体分装后在-20℃温度下保存,短期内使用在2-8℃存放,使用时,仅需按所需浓度将此溶液进行稀释,现配现用,大大提高了抗体的生产量,并通过检验发现单克隆抗体抗体能够与BSA结合,不能够与HAS结合,为未来医学发展奠定了基础。(The invention provides a preparation method of a BSA (bovine serum albumin) monoclonal antibody, which comprises the following steps: preparing polypeptide: firstly, 10mg of polypeptide with the purity of 90% is prepared, and 5mg of polypeptide is coupled with KLH; animal immunization: selecting five mice, firstly carrying out primary immunization, then carrying out secondary immunization, repeating the step of the secondary immunization, carrying out third immunization on the mice, and then carrying out fourth immunization; fusing cells; screening: selecting a hole with a high positive value and a single clone; culturing and amplifying the selected monoclonal hybridoma cells and freezing and storing the cells; obtaining the purified monoclonal antibody. The invention can prepare a large amount of monoclonal antibodies, the prepared antibodies are stored at the temperature of 20 ℃ below zero after being subpackaged, and are stored at the temperature of 2 ℃ to 8 ℃ in a short time, when in use, the solution is diluted according to the required concentration, and is prepared for use, so that the production of the antibodies is greatly improved, and the monoclonal antibodies can be combined with BSA and cannot be combined with HAS through detection, thereby laying the foundation for the future medical development.)

1. a preparation method of a BSA (bovine serum albumin) monoclonal antibody is characterized by comprising the following steps:

s1: preparing polypeptide: firstly, 10mg of polypeptide with the purity of 90 percent is prepared, wherein the peptide sequence of the polypeptide is KEACFAVEGPKLVVSTQT, and 5mg of the polypeptide is coupled with KLH;

s2: animal immunization: selecting five mice, firstly carrying out primary immunization, mixing KLH coupled polypeptide with an equal volume of Freund's complete adjuvant, carrying out subcutaneous immunization on the mice after emulsification is finished, and injecting 400ul of emulsion subcutaneously into each mouse; then carrying out second immunization, mixing and emulsifying 1:1 KLH antigen of 1mg/ml and an equal volume Freund's incomplete adjuvant, injecting 200ul of emulsion into the abdominal cavity of each mouse, repeating the second immunization step to carry out third immunization on the mice, after 10 days of the third immunization, respectively taking blood from the eye vein of each mouse, carrying out centrifugal treatment, taking serum for ELISA detection, and storing the serum of the mice after the third immunization; then, carrying out fourth immunization, mixing and emulsifying 1:1 KLH antigen of 1mg/ml and an equal volume of Freund's incomplete adjuvant, injecting 200ul of emulsion into the abdominal cavity of each white mouse, after 10 days of the fourth immunization, respectively taking blood from the eye vein of each white mouse, carrying out centrifugation treatment, taking serum for ELISA detection, and storing the serum of the white mouse after the fourth immunization;

s3: cell fusion: recovering and culturing mouse myeloma cells, taking spleen of quadruplicate immunized white mouse, preparing single cell suspension by using culture medium, and culturing according to the ratio of 1: 5, myeloma cells and splenocytes of the mice are mixed according to the proportion, PEG is added for cell fusion to prepare fused cell liquid, 100 ul/hole of 2 XHAT complete culture medium is paved into a 96-hole plate, 100ul of fused cell liquid is added into each hole, and half-amount liquid replacement is carried out by 1 XHAT in the process of culturing the fused cells;

s4: screening: preparing feeder cells from Balb/c healthy mice, performing limited dilution on selected fused well cells, diluting the cells with 1 XHAT culture solution, adding cell suspension into 96 wells containing the feeder cells respectively, wherein the concentration of the cell suspension is 100 ul/well, and the wells contain 0.7 ul/well respectively5. 1.5 and 3 feeder cells, and 5% by weight of CO₂Culturing the feeder cells at a temperature of 37 ℃; observing the growth condition of the feeder cell clone under a microscope after 10 days, detecting cell clone holes by using ELISA, selecting holes with high positive values and monoclone, and screening antibodies which can be combined with BSA and not with HAS;

s5: cell expansion: culturing and amplifying the selected monoclonal hybridoma cells and freezing and storing the cells;

s6: and (3) antibody production and purification: after a white mouse is sensitized by paraffin, hybridoma cells are injected into the abdominal cavity of the white mouse; observing that the abdomen of the mouse is obviously enlarged after 7-10 days, and when the mouse is touched by hands, the skin is tense, so that ascites can be collected; incubating and eluting the pretreated ascites by using a Protein G column to finally obtain a purified monoclonal antibody;

s7: monoclonal hybridoma cell sample testing: firstly, extracting total RNA of hybridoma cells by using a Trizol method, and then reversely transcribing the RNA of a sample into cDNA by using a SMART Race technology; by usingAmplifying and obtaining heavy chain and light chain variable regions by a FastPfu Fly DNA Polymerase kit; the target fragment was ligated into the pUC57 vector using T4 ligase and the ligation products were transformed into E.coli competent cells, after which the single clones were picked for sequencing and finally the sequencing results were aligned using the IMGT/V-QUEST database for further analysis and to obtain the test results.

2. The method of claim 1, wherein the ELISA detection step comprises:

(1) the coating amount of the antigen is 100 ng/well, wherein the antigen is HAS and BSA standard substance, the concentration of each well is 100ul, the antigen is diluted by PBS, and the antigen is stored for 17-19h at the temperature of 4 ℃;

(2) pouring out the supernatant, adding 200ul of the protein-free sealant into each hole, and shaking at 37 ℃ for 2 h;

(3) pouring out the supernatant, patting the liquid dry, and freezing at-20 deg.C for use;

(4) after freezing, the coated plate was taken out and placed at room temperature, 100ul of serum diluent was added to each well, and the plate was left at 37 ℃ for 1 hour.

(5) Wash plate 3 times with 0.1% PBST;

(6) labeling goat anti-mouse with horseradish enzyme, diluting with PBS (1 ul concentration and 10ml volume), and standing at 37 deg.C for 45 min;

(7) wash plate 3 times with PBST;

(8) adding 100ul of TMB into each well, and standing at 37 deg.C for 15 min;

(9) the reaction was stopped by adding 2M hydrochloric acid stop solution and the OD was measured at a wavelength of 450 nm.

3. The method of claim 1, wherein a second limiting dilution is required if the assay is not determined to be monoclonal during the ELISA assay of the cell clone wells.

Technical Field

The invention belongs to the field of monoclonal antibody preparation methods, and particularly relates to a BSA monoclonal antibody preparation method.

Background

Monoclonal antibodies are highly homogeneous antibodies produced by a single B cell clone that are directed against only a particular epitope. Usually, hybridoma is prepared by fusing a sensitized B cell having the ability to secrete a specific antibody and a myeloma cell having an unlimited reproductive capacity into a B cell hybridoma based on a cell fusion technique.

The preparation process of the monoclonal antibody mainly comprises the following steps: 1. immunizing animals: the immune animal is a process for immunizing a mouse by using a target antigen to enable the mouse to generate sensitized B lymphocytes, generally, a female BALB/c mouse with the age of 6-8 weeks is selected, immune injection is carried out according to a preset immune scheme, the antigen enters a peripheral immune organ through blood circulation or lymphatic circulation, corresponding B lymphocyte clone is stimulated, and the B lymphocyte is activated, proliferated and differentiated into the sensitized B lymphocytes.

2. Cell fusion: the mice were sacrificed with carbon dioxide gas, spleens were removed by aseptic technique and crushed and ground in a flat vessel to prepare a splenocyte suspension. Mixing prepared homologous myeloma cells and mouse spleen cells according to a certain proportion, and adding fusion promoter polyethylene glycol. Under the action of polyethylene glycol, various lymphocytes can be fused with myeloma cells to form hybridoma cells.

3. Selective culture: the purpose of selective culture is to select fused hybridoma cells, typically using HAT selective medium. In HAT medium, unfused myeloma cells die because they lack hypoxanthine-guanine-phosphoribosyltransferase and cannot synthesize DNA using a salvage pathway, and unfused lymphocytes, which have hypoxanthine-guanine-phosphoribosyltransferase but cannot survive in vitro for a long period of time, die gradually. Only fused hybridoma cells survive and proliferate in HAT medium because they obtain hypoxanthine-guanine-phosphoribosyltransferase from spleen cells and have the property that myeloma cells can proliferate indefinitely.

4. Screening and cloning of hybridoma positive clones: hybridoma cells grown in HAT medium have only a few cells secreting monoclonal antibodies of the desired specificity, and therefore must be screened and cloned. Usually, a limiting dilution method is adopted to carry out cloning culture on hybridoma cells, a sensitive, rapid and specific immunological method is adopted to screen out positive hybridoma cells capable of producing a required monoclonal antibody, cloning and amplification are carried out, and after the immunoglobulin type, subclass, specificity, affinity, epitope for recognizing antigen and molecular weight of the secreted monoclonal antibody are comprehensively identified, cryopreservation is carried out in time.

5. Mass preparation of monoclonal antibody A large amount of monoclonal antibodies are prepared mainly by in vivo animal induction and in vitro culture.

(1) In vivo induction method: BALB/c mice were first pretreated by intraperitoneal injection of 0.5ml liquid paraffin or pristane. After 1-2 weeks, hybridoma cells were inoculated intraperitoneally. The hybridoma cells proliferate in the abdominal cavity of the mouse and produce and secrete monoclonal antibodies, and the abdominal distension of the mouse is observed for about 1-2 weeks. The ascites is extracted by a syringe, and a large amount of monoclonal antibodies can be obtained.

(2) In vitro culture method: and (3) placing the hybridoma cells into a culture bottle for culture, wherein the hybridoma cells generate and secrete the monoclonal antibody in the culture process, collecting culture supernatant, and centrifuging to remove cells and fragments thereof to obtain the required monoclonal antibody. However, this method has a limited amount of antibodies produced, and various novel culture techniques and devices have been developed, which greatly increase the amount of antibody production.

Disclosure of Invention

In order to solve the technical problems, the invention provides a preparation method of a BSA (bovine serum albumin) monoclonal antibody, which comprises the following steps:

s1: preparing polypeptide: firstly, 10mg of polypeptide with the purity of 90 percent is prepared, wherein the peptide sequence of the polypeptide is KEACFAVEGPKLVVSTQT, and 5mg of the polypeptide is coupled with KLH;

s2: animal immunization: selecting five mice, firstly carrying out primary immunization, mixing KLH coupled polypeptide with an equal volume of Freund's complete adjuvant, carrying out subcutaneous immunization on the mice after emulsification is finished, and injecting 400ul of emulsion subcutaneously into each mouse; then carrying out second immunization, mixing and emulsifying 1:1 KLH antigen of 1mg/ml and an equal volume Freund's incomplete adjuvant, injecting 200ul of emulsion into the abdominal cavity of each mouse, repeating the second immunization step to carry out third immunization on the mice, after 10 days of the third immunization, respectively taking blood from the eye vein of each mouse, carrying out centrifugal treatment, taking serum for ELISA detection, and storing the serum of the mice after the third immunization; then, carrying out fourth immunization, mixing and emulsifying 1:1 KLH antigen of 1mg/ml and an equal volume of Freund's incomplete adjuvant, injecting 200ul of emulsion into the abdominal cavity of each white mouse, after 10 days of the fourth immunization, respectively taking blood from the eye vein of each white mouse, carrying out centrifugation treatment, taking serum for ELISA detection, and storing the serum of the white mouse after the fourth immunization;

s3: cell fusion: recovering and culturing mouse myeloma cells, taking spleen of quadruplicate immunized white mouse, preparing single cell suspension by using culture medium, and culturing according to the ratio of 1: 5, myeloma cells and splenocytes of the mice are mixed according to the proportion, PEG is added for cell fusion to prepare fused cell liquid, 100 ul/hole of 2 XHAT complete culture medium is paved into a 96-hole plate, 100ul of fused cell liquid is added into each hole, and half-amount liquid replacement is carried out by 1 XHAT in the process of culturing the fused cells;

s4: screening: preparing feeder cells by Balb/c healthy mice, carrying out limited dilution on the selected fused well cells, diluting the cells by using 1 XHAT culture solution, respectively adding cell suspensions into 96 wells containing the feeder cells, wherein the concentration is 100 ul/well, the wells respectively contain 0.75, 1.5 and 3 feeder cells, and adding CO2 with the mass fraction of 5%, and culturing the feeder cells at the temperature of 37 ℃; observing the growth condition of the feeder cell clone under a microscope after 10 days, detecting cell clone holes by using ELISA, selecting holes with high positive values and monoclone, and screening antibodies which can be combined with BSA and not with HAS;

s5: cell expansion: culturing and amplifying the selected monoclonal hybridoma cells and freezing and storing the cells;

s6: and (3) antibody production and purification: after a white mouse is sensitized by paraffin, hybridoma cells are injected into the abdominal cavity of the white mouse; observing that the abdomen of the mouse is obviously enlarged after 7-10 days, and when the mouse is touched by hands, the skin is tense, so that ascites can be collected; incubating and eluting the pretreated ascites by using a Protein G column to finally obtain a purified monoclonal antibody;

s7: monoclonal hybridoma cell sample testing: firstly, extracting total RNA of hybridoma cells by using Trizol method, and then using SMART Race technology to extract RNA of sampleReverse transcription into cDNA; using TransAmplifying and obtaining heavy chain and light chain variable regions by a FastPFFly DNA Polymerase kit; the target fragment was ligated into the pUC57 vector using T4 ligase and the ligation products were transformed into E.coli competent cells, after which the single clones were picked for sequencing and finally the sequencing results were aligned using the IMGT/V-QUEST database for further analysis and to obtain the test results.

Preferably, the ELISA detecting step is:

(1) the coating amount of the antigen is 100 ng/well, wherein the antigen is HAS and BSA standard substance, the concentration of each well is 100ul, the antigen is diluted by PBS, and the antigen is stored for 17-19h at the temperature of 4 ℃;

(2) pouring out the supernatant, adding 200ul of the protein-free sealant into each hole, and shaking at 37 ℃ for 2 h;

(3) pouring out the supernatant, patting the liquid dry, and freezing at-20 deg.C for use;

(4) after freezing, the coated plate was taken out and placed at room temperature, 100ul of serum diluent was added to each well, and the plate was left at 37 ℃ for 1 hour.

(5) Wash plate 3 times with 0.1% PBST;

(6) labeling goat anti-mouse with horseradish enzyme, diluting with PBS (1 ul concentration and 10ml volume), and standing at 37 deg.C for 45 min;

(7) wash plate 3 times with PBST;

(8) adding 100ul of TMB into each well, and standing at 37 deg.C for 15 min;

(9) the reaction was stopped by adding 2M hydrochloric acid stop solution and the OD was measured at a wavelength of 450 nm.

Preferably, in the ELISA process of detecting cell clone holes, if the detection is not determined to be monoclonal, a second limiting dilution is needed.

Compared with the prior art, the invention has the beneficial effects that: the monoclonal antibody is prepared by seven steps of preparing polypeptide, animal immunization, cell fusion, screening, cell amplification, antibody production and purification and monoclonal hybridoma cell sample inspection, a large amount of monoclonal antibodies can be prepared, the prepared antibodies are subpackaged and stored at the temperature of 20 ℃ below zero and stored at the temperature of 2-8 ℃ in a short time, when the monoclonal antibody is used, the solution is diluted according to the required concentration, the monoclonal antibody can be prepared and used at the moment, the production of the antibodies is greatly improved, and the monoclonal antibody can be combined with BSA and cannot be combined with HAS through inspection, so that the foundation is laid for the future medical development.

Drawings

FIG. 1 is a graph comparing the serum titers of mice after a third and fourth immunization according to the present invention;

FIG. 2 is a diagram showing the results of whole plate ELISA detection after the fusion of mouse spleen cells and myeloma cells to grow clones according to the present invention;

FIG. 3 is a graph of the retest results of the positive fusion wells of the present invention;

FIG. 4 is a graphical representation of the results of a limiting dilution positive well retest of the present invention;

FIG. 5 is a graph showing the results of the second limiting dilution cloning wells and the detection of the primary amplified cells according to the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention is further described below:

example (b):

a preparation method of a BSA (bovine serum albumin) monoclonal antibody comprises the following steps:

(1) preparing polypeptide: firstly, 10mg of polypeptide with the purity of 90 percent is prepared, wherein the peptide sequence of the polypeptide is KEACFAVEGPKLVVSTQT, and 5mg of the polypeptide is coupled with KLH;

(2) animal immunization: selecting five mice, firstly carrying out primary immunization, mixing KLH coupled polypeptide with an equal volume of Freund's complete adjuvant, carrying out subcutaneous immunization on the mice after emulsification is finished, and injecting 400ul of emulsion subcutaneously into each mouse; then carrying out second immunization, mixing and emulsifying 1:1 KLH antigen of 1mg/ml and an equal volume Freund's incomplete adjuvant, injecting 200ul of emulsion into the abdominal cavity of each mouse, repeating the second immunization step to carry out third immunization on the mice, after 10 days of the third immunization, respectively taking blood from the eye vein of each mouse, carrying out centrifugal treatment, taking serum for ELISA detection, and storing the serum of the mice after the third immunization; and then carrying out a fourth immunization, mixing and emulsifying 1:1 KLH antigen of 1mg/ml and an equal volume of Freund's incomplete adjuvant, injecting 200ul of emulsion into the abdominal cavity of each white mouse, taking blood from the eye vein of each white mouse after 10 days of the fourth immunization, centrifuging, taking serum for ELISA detection, storing the serum of the white mouse after the fourth immunization, and comparing the titer of the serum of the mouse after the third immunization and the serum of the mouse after the fourth immunization as shown in the attached figure 1, wherein the ELISA detection step is as follows:

1. the coating amount of the antigen is 100 ng/well, wherein the antigen is HSA and BSA standard substance, the concentration of each well is 100ul, the antigen is diluted by PBS, and the antigen is stored for 17-19h at the temperature of 4 ℃;

2. pouring out the supernatant, adding 200ul of the protein-free sealant into each hole, and shaking at 37 ℃ for 2 h;

3. pouring out the supernatant, patting the liquid dry, and freezing at-20 deg.C for use;

4. after freezing, the coated plate was taken out and placed at room temperature, 100ul of serum diluent was added to each well, and the plate was left at 37 ℃ for 1 hour.

5. Wash plate 3 times with 0.1% PBST;

6. labeling goat anti-mouse with horseradish enzyme, diluting with PBS (1 ul concentration and 10ml volume), and standing at 37 deg.C for 45 min;

7. wash plate 3 times with PBST;

8. adding 100ul of TMB into each well, and standing at 37 deg.C for 15 min;

9. adding 2M hydrochloric acid stop solution to terminate the reaction, and measuring the OD value by using the wavelength of 450 nm;

(3) cell fusion: recovering and culturing mouse myeloma cells, taking spleen of quadruplicate immunized white mouse, preparing single cell suspension by using culture medium, and culturing according to the ratio of 1: 5, myeloma cells and splenocytes of the mice are mixed according to the proportion, PEG is added for cell fusion to prepare fused cell liquid, 100 ul/hole of 2 XHAT complete culture medium is paved into a 96-hole plate, 100ul of fused cell liquid is added into each hole, and half-amount liquid replacement is carried out by 1 XHAT in the process of culturing the fused cells;

(4) screening: as shown in figure 2, fusion cloning wells were tested by ELISA, according to the results of figure 2, selected and selected to be 1#1a4, 1#3G2, 1#3F4 for limiting dilution, as shown in figure 3, positive fusion wells were retested using BSA, HSA standards, after retesting feeder cells were prepared from Balb/c healthy mice, the selected fusion well cells were limited diluted, the cells were diluted with 1 xhat medium, the cell suspensions were added to 96 wells containing feeder cells at concentrations of 100 ul/well, the wells contained 0.75, 1.5 and 3 feeder cells, respectively, and CO2 at 5% mass, and the feeder cells were cultured at 37 ℃; observing the growth condition of the feeder cell clone under a microscope after 10 days, detecting the cell clone hole by using ELISA, selecting a hole with high positive value and monoclone, as shown in figure 4, if the detection is uncertain to be monoclone, carrying out secondary limited dilution, as shown in figure 5, detecting the second limited diluted clone hole by using an ELISA method, wherein the results show that the two limited diluted clone holes are all positive values, selecting a cell strain with high positive value and good cell state for amplification, and the screened antibody can be combined with BSA and can not be combined with HAS;

(5) cell expansion: culturing and amplifying the selected monoclonal hybridoma cells and freezing the cells to obtain a frozen fine cell strain: JN-1033-1N-F3F4-D6-E5, JN-1033-1N-F3F4-D6-C8, JN-1033-1N-F3G 2-B5-B5;

(6) and (3) antibody production and purification: after a white mouse is sensitized by paraffin, hybridoma cells are injected into the abdominal cavity of the white mouse; observing that the abdomen of the mouse is obviously enlarged after 7-10 days, and when the mouse is touched by hands, the skin is tense, so that ascites can be collected; and incubating the pretreated ascites by using a Protein G column, and eluting to finally obtain the purified monoclonal antibody.

(7) As shown in fig. 2, monoclonal hybridoma cell samples were tested: firstly, extracting total RNA of hybridoma cells by using a Trizol method, and then reversely transcribing the RNA of a sample into cDNA by using a SMART Race technology; using TransAmplifying and obtaining heavy chain and light chain variable regions by a FastPfu Fly DNA Polymerase kit; the target fragment was ligated into the pUC57 vector using T4 ligase and the ligation products were transformed into E.coli competent cells, after which single clones were picked for sequencing and finally aligned for further analysis using the IMGT/V-QUEST database.

Specifically, the sequencing results for the heavy and light chain variable regions are:

heavy chain variable region base sequence:

GATGTGCAGTTGGTGGAGTCTGGGGGAGACTTAGTGCAGCCTGGAGGGTCCCGGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTTAGTAGCTTTGGAATGCACTGGGTTCGTCAGGCTCCAGAGAAGGGGCTGGAGTGGGTCGCATATATTAGTACTGACAGTAGTTTGATCTACTATGGAGACACAGTGAAGGGCCGATTCACCATCTCCAGAGACAATCCCAAGAACACCCTCTTCCTGCAAATGACCAGTCTAAGGTCTGAGGACACGGCCATGTATTACTGTGCAAGATTCTTTCTGCGTTCCGGGTTTGCTGATTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGATGTGCAGTTGGTGGAGTCTGGGGGAGACTTAGTGCAGCCTGGAGGGTCCCGGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTTAGTAGCTTTGGAATGCACTGGGTTCGTCAGGCTCCAGAGAAGGGGCTGGAGTGGGTCGCATATATTAGTACTGACAGTAGTTTGATCTACTATGGAGACACAGTGAAGGGCCGATTCACCATCTCCAGAGACAATCCCAAGAACACCCTCTTCCTGCAAATGACCAGTCTAAGGTCTGAGGACACGGCCATGTATTACTGTGCAAGATTCTTTCTGCGTTCCGGGTTTGCTGATTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA；

heavy chain variable region amino acid sequence:

DVQLVESGGDLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISTDSSLIYYGDTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARFFLRSGFADWGQGTLVTVSADVQLVESGGDLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISTDSSLIYYGDTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARFFLRSGFADWGQGTLVTVSA；

light chain variable region base sequence:

GACATCAAGATGACCCAGTCTCCATCTTCCATGTATGCATCTCTAGGAGAGAGAGTCACTATCACTTGCAAGGCGAGTCAGGACATTAATAGCTATTTAAGTTGGTTCCAGCAGAAACCAGGGaAATCTCCTAAGCCCCTGATCTATCGTGCAAAAAGATTGGTAGATGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGCAAGATTATTCTCTCACCATCAGCAGCCTGGAGTATGAAGATATGGGAATTTATTATTGTCTACAGTATGATGCGTTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAAGACATCAAGATGACCCAGTCTCCATCTTCCATGTATGCATCTCTAGGAGAGAGAGTCACTATCACTTGCAAGGCGAGTCAGGACATTAATAGCTATTTAAGTTGGTTCCAGCAGAAACCAGGGAAATCTCCTAAGCCCCTGATCTATCGTGCAAAAAGATTGGTAGATGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGCAAGATTATTCTCTCACCATCAGCAGCCTGGAGTATGAAGATATGGGAATTTATTATTGTCTACAGTATGATGCGTTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA；

light chain variable region amino acid sequence:

DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKPLIYRAKRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDAFPLTFGAGTKLELKDIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKPLIYRAKRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDAFPLTFGAGTKLELK。

it should be noted that, in this document, moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Preparation method of BSA (bovine serum albumin) monoclonal antibody

The applicant: tianjin Oncai cell Gene engineering Co Ltd

Heavy chain variable region base sequence:

gatgtgcagttggtggagtctgggggagacttagtgcagcctggagggtcccggaaactctcctgtgcagcctctggattcacttttagtagctttggaatgcactgggttcgtcaggctccagagaaggggctggagtgggtcgcatatattagtactgacagtagtttgatctactatggagacacagtgaagggccgattcaccatctccagagacaatcccaagaacaccctcttcctgcaaatgaccagtctaaggtctgaggacacggccatgtattactgtgcaagattctttctgcgttccgggtttgctgattggggccaagggactctggtcactgtctctgcagatgtgcagttggtggagtctgggggagacttagtgcagcctggagggtcccggaaactctcctgtgcagcctctggattcacttttagtagctttggaatgcactgggttcgtcaggctccagagaaggggctggagtgggtcgcatatattagtactgacagtagtttgatctactatggagacacagtgaagggccgattcaccatctccagagacaatcccaagaacaccctcttcctgcaaatgaccagtctaaggtctgaggacacggccatgtattactgtgcaagattctttctgcgttccgggtttgctgattggggccaagggactctggtcactgtctctgca；

heavy chain variable region amino acid sequence:

dvqlvesggdlvqpggsrklscaasgftfssfgmhwvrqapekglewvayistdssliyygdtvkgrftisrdnpkntlflqmtslrsedtamyycarfflrsgfadwgqgtlvtvsadvqlvesggdlvqpggsrklscaasgftfssfgmhwvrqapekglewvayistdssliyygdtvkgrftisrdnpkntlflqmtslrsedtamyycarfflrsgfadwgqgtlvtvsa；

light chain variable region base sequence:

gacatcaagatgacccagtctccatcttccatgtatgcatctctaggagagagagtcactatcacttgcaaggcgagtcaggacattaatagctatttaagttggttccagcagaaaccagggaaatctcctaagcccctgatctatcgtgcaaaaagattggtagatggggtcccatcaaggttcagtggcagtggatctgggcaagattattctctcaccatcagcagcctggagtatgaagatatgggaatttattattgtctacagtatgatgcgtttccgctcacgttcggtgctgggaccaagctggagctgaaagacatcaagatgacccagtctccatcttccatgtatgcatctctaggagagagagtcactatcacttgcaaggcgagtcaggacattaatagctatttaagttggttccagcagaaaccagggaaatctcctaagcccctgatctatcgtgcaaaaagattggtagatggggtcccatcaaggttcagtggcagtggatctgggcaagattattctctcaccatcagcagcctggagtatgaagatatgggaatttattattgtctacagtatgatgcgtttccgctcacgttcggtgctgggaccaagctggagctgaaa；

light chain variable region amino acid sequence:

dikmtqspssmyaslgervtitckasqdinsylswfqqkpgkspkpliyrakrlvdgvpsrfsgsgsgqdysltissleyedmgiyyclqydafpltfgagtklelkdikmtqspssmyaslgervtitckasqdinsylswfqqkpgkspkpliyrakrlvdgvpsrfsgsgsgqdysltissleyedmgiyyclqydafpltfgagtklelk。