CA125 antigen binding protein and application thereof

Abstract

The invention provides a CA125 antigen binding protein and application thereof. The CA125 antigen binding protein comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region comprises CDR3 with a sequence as shown in SEQ ID NO.7 or SEQ ID NO.13; and the light chain variable region comprises a CDR3 with a sequence as shown in SEQ ID NO. 10 or SEQ ID NO. 16. The CA125 antigen binding protein is a rabbit source monoclonal antibody, has high specificity and affinity to CA125, can be specifically bound with CA125 protein, and has important application value in diagnosis of ovarian cancer.

Description

Technical Field

[0001] The invention belongs to the technical field of biopharmaceuticals, and specifically relates to a CA125 antigen binding protein and an application thereof. Background Art

[0002] CA125 antigen is a mucin. It was originally obtained by using the tumor cell OVCA433 expressing MUC16 as an immunogen to obtain the monoclonal antibody OC125 antibody. The antigen recognized by this antibody was named CA125 antigen. The gene MUC16 encoding CA125 was discovered and cloned in 2001.

[0003] Studies have shown that CA125 contains more than 249 N-glycosylation sites and more than 3,000 O-glycosylation sites. At the same time, the protein has many different isomers with a molecular weight of 200-2000 kDa. The CA125 protein consists of a shorter intracellular region, a transmembrane region, and a longer extracellular region. The extracellular segment consists of a repeating domain and an N-terminal domain. The repeating domain contains 56 SEA modules, which contain disulfide-bonded cysteine-loops, which are the sites recognized by OC125 and M11 antibodies. A potential cleavage site was found near the SEA module close to the transmembrane region. Protein cleavage can cause most of the extracellular part of MUC16 to fall off into the bloodstream.

[0004] Under normal circumstances, CA125 is expressed at a low level and is stably bound to the cell membrane and cannot enter the human blood. Therefore, the content of CA125 in healthy people or those suffering from some benign diseases is very low. When the body is experiencing the growth and metastasis of malignant tumors, and when some non-tumor diseases worsen, CA125 is expressed at a high level in the cells, and falls off the cell membrane into the human body fluids, and then enters the blood circulation and is in a high content state. CA125 is one of the earliest tumor markers discovered, and it is still the most widely used early marker for ovarian cancer, and is used to monitor the progress of epithelial ovarian cancer treatment. The gene MUC16 encoding CA125 was discovered and cloned in 2001. It exists in type I transmembrane or secretory forms, and both forms are severely glycosylated.

[0005] The development process of CA125 detection antibodies mainly includes the following steps: Preparation of main immunogens: generally using recombinant proteins, peptides or cells that purchase CA125 natural antigens; Immunization of immunized animals: selecting suitable immunized animals, usually mice, rabbits, etc. for immunization. Injecting the immunogen into the immunized animal to stimulate it to produce CA125-specific antibodies; Screening and identification: Screening methods such as phage display and single cell sorting to screen out CA125-specific antibodies with specificity and affinity. Further identification and evaluation of candidate antibodies, including affinity determination, flow cytometric analysis, etc.; Cloning, antibody optimization and improvement.

[0006] Currently, most anti-CA125 protein antibodies are polyclonal antibodies or mouse monoclonal antibodies, and the CA125 antigen detection threshold is 35U / mL, requiring a paired antibody pair with higher sensitivity. Therefore, providing a monoclonal antibody that can specifically bind to the CA125 protein has important application value in the diagnosis of ovarian cancer. Summary of the invention

[0007] In view of the shortcomings of the prior art, the present invention aims to provide a CA125 antigen binding protein and its application. The antibody is a rabbit monoclonal antibody. Compared with mouse antibodies and polyclonal antibodies, rabbit antibodies naturally have higher affinity and stronger specificity, and the antibody obtained by combination has strong specificity and high accuracy.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a CA125 antigen binding protein, the CA125 antigen binding protein comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a CDR3 having a sequence as shown in SEQ ID NO.7 or SEQ ID NO.13;

[0010] The light chain variable region includes CDR3 having a sequence as shown in SEQ ID NO.10 or SEQ ID NO.16.

[0011] Preferably, the heavy chain variable region further comprises a CDR1 having a sequence as shown in SEQ ID NO.5 or SEQ ID NO.11.

[0012] Preferably, the heavy chain variable region further comprises a CDR2 having a sequence as shown in SEQ ID NO.6 or SEQ ID NO.12.

[0013] Preferably, the light chain variable region further comprises a CDR1 having a sequence as shown in SEQ ID NO.8 or SEQ ID NO.14.

[0014] Preferably, the light chain variable region further comprises a CDR2 having a sequence as shown in SEQ ID NO.9 or SEQ ID NO.15.

[0015] Preferably, the heavy chain variable region comprises a CDR1 having a sequence as shown in SEQ ID NO.5, a CDR2 having a sequence as shown in SEQ ID NO.6 and a CDR3 having a sequence as shown in SEQ ID NO.7, and the light chain variable region comprises a CDR1 having a sequence as shown in SEQ ID NO.8, a CDR2 having a sequence as shown in SEQ ID NO.9 and a CDR3 having a sequence as shown in SEQ ID NO.10; or,

[0016] The heavy chain variable region includes a CDR1 having a sequence as shown in SEQ ID NO.11, a CDR2 having a sequence as shown in SEQ ID NO.12, and a CDR3 having a sequence as shown in SEQ ID NO.13, and the light chain variable region includes a CDR1 having a sequence as shown in SEQ ID NO.14, a CDR2 having a sequence as shown in SEQ ID NO.15, and a CDR3 having a sequence as shown in SEQ ID NO.16.

[0017] Preferably, the heavy chain variable region comprises a sequence having at least 90% identity with the sequence shown in SEQ ID NO.1 or SEQ ID NO.3; and / or,

[0018] The light chain variable region comprises a sequence having at least 90% identity with the sequence shown in SEQ ID NO.2 or SEQ ID NO.4.

[0019] Preferably, the sequence of the heavy chain variable region includes SEQ ID NO.1 or SEQ ID NO.3.

[0020] Preferably, the sequence of the light chain variable region includes SEQ ID NO.2 or SEQ ID NO.4.

[0021] Preferably, the sequence of the heavy chain variable region includes the sequence shown in SEQ ID NO.1, and the sequence of the light chain variable region includes the sequence shown in SEQ ID NO.2; or,

[0022] The sequence of the heavy chain variable region includes the sequence shown in SEQ ID NO.3, and the sequence of the light chain variable region includes the sequence shown in SEQ ID NO.4.

[0023] Preferably, the antigen binding protein has a strength greater than or equal to 10 -9 It specifically binds to human CA125 with an affinity of nmol / L.

[0024] Preferably, the antigen binding protein is an antibody.

[0025] Preferably, the antibody is a monoclonal antibody.

[0026] Preferably, the constant region of the monoclonal antibody is a rabbit-derived constant region.

[0027] In the present invention, CA125 natural antigen is used to immunize New Zealand white rabbits. After 4 immunizations, the antiserum titer is detected by enzyme-linked immunosorbent assay (ELISA). After the serum titer reaches a certain dilution, impact immunization is performed, serum is collected for polyclonal antibody purification, and spleen is collected for grinding. Total RNA is extracted from the spleen, and the first-chain cDNA is synthesized by reverse transcription. The light and heavy chain variable region fragments (VH / Vκ) of the immunized rabbit are amplified by a set of primers matching the light and heavy chain variable regions FR1 and FR4 of the rabbit antibody, and secondary amplification is performed by another set of primers, and the antibody light and heavy chain variable fragments are added with a linker sequence having a complementary overlapping region, and the VH fragment and the Vκ fragment are assembled into a single-chain antibody fragment (scFv) by overlap extension PCR (SOE PCR). After the scFv fragment and the phage display vector are digested with the same restriction endonuclease, they are recovered by gel and connected with the recombinant vector, and after the connection product is cleaned, it is electrotransferred to Escherichia coli TG1, thereby obtaining the scFv phage display bacterial library. By culturing the bacterial library, adding the helper phage M13K07 for superinfection, the phage library is rescued by auxiliary packaging, and the scFv fragment is displayed on the phage surface. The CA125 natural antigen is coated in the immunoplate, and the phage library is added after blocking with the blocking solution. The phage displaying the specific scFv fragment is captured because it can bind to the CA125 protein, and the unbound phage is removed by rinsing. The phage is eluted from the immunoplate and infected with the TG1 host in the logarithmic growth phase. After cultivation, the helper phage can be used again for superinfection and packaging rescue. After several rounds of screening, the TG1 bacterial solution infected by the screened phage is gradiently diluted and spread on the plate, and then a single clone is picked and cultured in a 96-well plate. The helper phage is added to package the monoclonal phage, and the binding activity of the phage and CA125 in the monoclonal supernatant is detected by ELISA to obtain a positive monoclonal clone that specifically binds to CA125.

[0028] The candidate positive clones were sequenced and analyzed, and constructed into the eukaryotic expression vector pcDNA3.1(+) containing the constant regions of the heavy and light chains of rabbit antibodies to obtain complete light and heavy chain expression vectors. The light and heavy chain vector plasmids were co-transfected into HEK293E cells for expression, and the positive clone antibody protein was purified from the culture supernatant using Protein A. The binding specificity of the positive clone antibody protein to CA125 was verified by ELISA and the affinity was determined, and finally two antibodies M1-4 and S1-1 that specifically bind to CA125 were obtained. The sequences are shown below, and the underlined sequences in the sequences are the antibody CDR regions.

[0029] SEQ ID NO.1 (amino acid sequence of the heavy chain variable region of M1-4)

[0030] QSLEESGGRLVTPGTPLTLTCTVSGFSLS SYAMG WVRQAPGKGLEWIG IIYAGSGSTYY ASWAKG RFTVSATSTTVDLKITSPTTEDTATYFCAR ALVSGWGHGMDL WGPGTLVTVSS.

[0031] SEQ ID NO.5 (heavy chain CDR1 of M1-4): SYAMG.

[0032] SEQ ID NO.6 (heavy chain CDR2 of M1-4): IIYAGSGSTYYASWAKG.

[0033] SEQ ID NO. 7 (heavy chain CDR3 of M1-4): ALVSGWGHGMDL.

[0034] SEQ ID NO.2 (amino acid sequence of light chain variable region of M1-4)

[0035] YVMMTQTPASMSEPVGGTVTIKC QASQRISSSYLS WYQQKPGQPPKLLIY STSTLAS G VSSRFKGSGSGTQFTLTISDLECADAATYYC LGGYFGGSYVGA FGGGTKVEIK.

[0036] SEQ ID NO. 8 (light chain CDR1 of M1-4): QASQRISSSYLS.

[0037] SEQ ID NO.9 (light chain CDR2 of M1-4): STSTLAS.

[0038] SEQ ID NO.10 (light chain CDR3 of M1-4): LGGYFGGSYVGA.

[0039] SEQ ID NO.3 (amino acid sequence of the heavy chain variable region of S1-1)

[0040] QSLEESGGRLVTPGTSLTLTCTVSGFSLS SYYMN WVRQAPGKGLEWIG IIYASGSTYYA TWAKG RFTISKTSTTTVDLKITSPTTEDTATYFCAG VYSSGSIYYPDL WGQGTLVTVSS.

[0041] SEQ ID NO.11 (heavy chain CDR1 of S1-1): SYYMN.

[0042] SEQ ID NO.12 (heavy chain CDR2 of S1-1): IIYASGSTYYATWAKG.

[0043] SEQ ID NO.13 (heavy chain CDR3 of S1-1): VYSSGSIYYPDL.

[0044] SEQ ID NO.4 (amino acid sequence of the light chain variable region of S1-1)

[0045] AAVLNQTPSPVSPAVGGTVSISC QSSQSVYNNNYLA WYQQKPGQPPKLLIY RASKLAS GVPSRFSGSGSGTQFTLTISGVQCDDAATYYC LGAYDDTDTA FGGGTEVVVK.

[0046] SEQ ID NO.14 (light chain CDR1 of S1-1): QSSQSVYNNNYLA.

[0047] SEQ ID NO.15 (light chain CDR2 of S1-1): RASKLAS.

[0048] SEQ ID NO.16 (light chain CDR3 of S1-1): LGAYDDDTDTA.

[0049] The antibody of the present invention can also be prepared into single-chain antibody (scFv), antigen-binding fragment Fab, (Fab)2, bispecific antibody, added fusion protein, etc., and applied in detection, clinical and other fields.

[0050] In a second aspect, the present invention provides a nucleic acid molecule encoding the CA125 antigen binding protein described in the first aspect.

[0051] In a third aspect, the present invention provides an expression vector, wherein the expression vector contains the nucleic acid molecule described in the second aspect.

[0052] In a fourth aspect, the present invention provides a host cell, wherein the host cell contains at least one copy of the expression vector described in the third aspect; or the nucleic acid molecule described in the second aspect is integrated into the genome of the host cell.

[0053] In a fifth aspect, the present invention provides a reagent for detecting CA125 protein in a sample, wherein the reagent comprises the CA125 antigen binding protein described in the first aspect.

[0054] In a sixth aspect, the present invention provides a kit, wherein the kit comprises the CA125 antigen binding protein described in the first aspect; or the kit comprises the reagent for detecting CA125 protein in a sample described in the fifth aspect.

[0055] In a seventh aspect, the present invention provides use of the anti-CA125 protein monoclonal antibody described in the first aspect in the preparation of a tumor detection product.

[0056] The anti-CA125 protein monoclonal antibody of the present invention can be applied to the diagnosis of diseases that may cause changes in CA125 concentration, such as the early diagnosis of ovarian cancer.

[0057] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0058] Compared with the prior art, the present invention has the following beneficial effects:

[0059] The monoclonal antibody obtained by the present invention can specifically bind to the CA125 target, and the antibody can be used for the diagnosis of diseases related to changes in CA125 concentration, such as early diagnosis of ovarian cancer; it has great potential in immunological research and the development of new treatment strategies. The antibody obtained by the present invention can be used directly or by various labeling and packaging into a kit for use. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 It is the result of CA125 immune serum titer test.

[0061] Figure 2 This is a diagram of the positive antibody purification results.

[0062] Figure 3 The positive clones were determined by affinity ELISA.

[0063] Figure 4 It is an antibody pair matching range test. DETAILED DESCRIPTION

[0064] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0065] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0066] Example 1 Animal Immunization and Serum Titer Test

[0067] The immunization in this example was completed by a CRO company (Guangzhou Laidemeng Biotechnology Co., Ltd.), and 2 rabbits (purchased from Guangdong Medical Experimental Animal Center, name: ordinary New Zealand rabbit, specification: about 1.8 kg) were immunized with each polypeptide.

[0068] 1. Take 1 mL of blood from the ears of two rabbits respectively, and collect serum for use as a control. Immunize each rabbit with 1 mg of antigen (antigen type is human natural antigen, purchased from XEMA, item number R222), emulsify with complete adjuvant, dilute 2 mg of antigen into 1.5 mL of PBS, mix 1.5 mL of antigen + 1.5 mL of complete adjuvant, and shake on an oscillator for more than 15 minutes until the oil-in-water state; immunize each rabbit with 1.5 mL on the back.

[0069] 2. Two weeks later, the second immunization was carried out. Each rabbit was immunized with 0.5 mg of antigen. It was emulsified with incomplete adjuvant. 1 mg of antigen was diluted into 1 mL of PBS. The mixture was 1 mL of antigen + 1 mL of incomplete adjuvant and oscillated on an oscillator for more than 15 minutes until it reached the oil-in-water state. Each rabbit was immunized with 1 mL on the back.

[0070] 3. One week later, each rabbit was immunized with 0.5 mg of antigen, which was emulsified with incomplete adjuvant. 1 mg of antigen was diluted into 1 mL of PBS, and mixed as 1 mL of antigen + 1 mL of incomplete adjuvant. The mixture was shaken on an oscillator for more than 15 minutes until the mixture reached the oil-in-water state. Each rabbit was immunized with 1 mL on its back.

[0071] 4. One week later, each rabbit was immunized with 0.5 mg of antigen, which was emulsified with incomplete adjuvant. 1 mg of antigen was diluted into 1 mL of PBS, and mixed as 1 mL of antigen + 1 mL of incomplete adjuvant. The mixture was shaken on an oscillator for more than 15 minutes until it reached the oil-in-water state. Each rabbit was immunized with 1 mL on its back.

[0072] 5. One week later, collect 1 mL of blood from the ear and test the titer by ELISA. If it reaches 1:100,000 or above, perform shock immunization.

[0073] 6. For shock immunization, 1 mg of antigen was immunized per rabbit. Emulsified with complete adjuvant, 2 mg of antigen was diluted into 1.5 mL of PBS, and mixed as 1.5 mL of antigen + 1.5 mL of complete adjuvant. Oscillate on an oscillator for more than 15 minutes until the mixture reaches the oil-in-water state. 1.5 mL was applied to the back of each rabbit for immunization.

[0074] 7. Three days later, blood was collected from the ears. 5 mL of blood was collected from each rabbit for titer measurement. If the titer was in line with expectations, whole blood was collected in anticoagulant tubes and serum was separated.

[0075] Note: The materials involved in the above operations are all commonly used reagents and consumables, regardless of brand suppliers.

[0076] Example 2 Antiserum titer ELISA test

[0077] 1. Dilute CA125 natural antigen (antigen type is human natural antigen, purchased from XEMA, item number R222) with PBS to 1 μg / mL, add 100 μL to each well of the immunoplate, and incubate in a 4°C refrigerator overnight.

[0078] 2. Wash the ELISA plate 3 times with PBST, 300 μL per well. Pat off any residual liquid on a paper towel the last time.

[0079] 3. Add 300 μL of 3% BSA blocking solution to each well and incubate at 37°C for 1 hour.

[0080] 4. Wash the ELISA plate 3 times with PBST, 300 μL per well. Pat off any residual liquid on a paper towel for the last wash.

[0081] 5. Dilute the antiserum with PBS, add 100 μL to each well of the immunoplate, and incubate at 37°C for 1 hour.

[0082] 6. Wash the ELISA plate 6 times with PBST, 300 μL per well. Pat off any residual liquid on a paper towel for the last wash.

[0083] 5. Add 100 μL of Goat Anti-rabbit IgG-Fc Secondary Antibody (HRP) (anti-rabbit secondary antibody, Beijing Sino Biological, Cat. No. SSA003) diluted in PBS to each well and incubate at room temperature for 1 hour.

[0084] 6. Wash the ELISA plate 6 times with PBST, 300 μL per well. Pat off any residual liquid on a paper towel for the last wash.

[0085] 7. Take out Solution A and Solution B of TMB substrate colorimetric solution from 4°C, return to room temperature, mix in equal proportions to prepare an appropriate amount of colorimetric solution, add 100 μL to each well of the ELISA plate, and react for 10 minutes in the dark.

[0086] 8. Add 50 μL 1M HCl to each well to terminate the color development reaction and read the OD value on a microplate reader. 450 value.

[0087] The titer test results are as attached. Figure 1 , the titer is greater than 1:102400, and the immunization is successful.

[0088] Example 3 Screening of immune rabbit antibody scFv display library

[0089] Rabbit spleen cells were ground and RNA was extracted to establish a cDNA library. The antibody variable region sequences in the library were amplified by literature methods and a phage display library was constructed for antibody screening. After three rounds of panning, 96 monoclones were picked and 10 positive clones were obtained, of which the preferred two were M1-4 and S1-1.

[0090] The amino acid sequence of the heavy chain variable region of M1-4 is shown in SEQ ID NO.1; the amino acid sequence of the light chain variable region of M1-4 is shown in SEQ ID NO.2; the amino acid sequence of the heavy chain variable region of S1-1 is shown in SEQ ID NO.3; and the amino acid sequence of the light chain variable region of S1-1 is shown in SEQ ID NO.4.

[0091] Example 4 Expression and purification of positive cloned antibodies

[0092] Recombinant antibody expression: The M1-4 and S1-1 light and heavy chain sequences were constructed into the pCDNA3.1(+) vector after the Guangzhou Liuhe gene synthesis and codon sequence optimization. After obtaining the vector, the plasmid was extracted, and then the light and heavy chain vectors were mixed at a molar ratio of 1:1. Take 300μg of plasmid, add 12.5mL OPM 293CD05 culture medium and 800μL 1mg / mL PEI (polyethyleneimine, POLYSCIENCE) solution and mix well. Let it stand at room temperature for half an hour, add it to 250mL of HEK293F cells being cultured, put it in a shaker, 37℃, 8% CO 2 , cultured at 110 rpm, added OPM 293 cell feed every other day, cultured for 7 days, and collected the cell supernatant by centrifugation.

[0093] Purification of recombinant antibodies: Add 3 mL of ProteinA filler (Sino Biological) to the supernatant, incubate at room temperature for half an hour, remove the filler and load it into an empty purification column, add 20 mL of PBS solution to rinse, then add 10 mL of 100 mM glycine solution at pH 3.0 for elution, and add 1 M Tris solution to neutralize the eluate to pH 7.0. Concentrate the neutralized eluate to 1 mL, determine the protein concentration, and run SDS-PAGE for purity identification of representative antibodies. Figure 2 This is the result of positive antibody purification. Figure 2In the figure, lane M is the molecular weight standard, lane 1 is the recombinant antibody S1-1, and lane 2 is the recombinant antibody M1-4. Figure 2 It can be seen that the antibody has reached the electrophoresis purity standard, and no other bands are obviously attached except the main band. Figure 2 .

[0094] Example 5 Affinity ELISA determination of positive clones

[0095] 1. Coat the CA125 natural antigen (purchased from XEMA, item number R222) on the immunoplate overnight. Wash the ELISA plate 3 times with PBST, 300μL per well, and tap the residual liquid on a paper towel the last time. 2. Add 3% BSA-PBS 300μL and block at 37℃ for 2 hours. Wash the ELISA plate 3 times with PBST, 300μL per well, and tap the residual liquid on a paper towel the last time. 3. Dilute the antibody at different concentrations, add 100μL, and incubate at 37℃ for 1 hour. Wash the ELISA plate 6 times with PBST, 300μL per well, and tap the residual liquid on a paper towel the last time. 4. Add 100μL of diluted commercial horseradish peroxidase-conjugated Goat Anti-rabbit IgG-FcSecondary Antibody (HRP) and incubate at 37℃ for 1 hour. Wash the ELISA plate 6 times with PBST, 300μL per well, and tap the residual liquid on a paper towel the last time. 5. Use tetramethylbenzidine microporous peroxidase substrate (TMB) as a substrate for color development for 10 minutes; 6. Add 50 μL of 0.1 mol / L sulfuric acid to terminate the reaction, and measure the absorbance at 450 nm.

[0096] The results of affinity determination are attached. Figure 3 According to the ELISA results, the affinity of the two antibodies was calculated by the origin software 10 -9 about.

[0097] Example 6 Antibody Pairing Range Test

[0098] 1. Coat the CA125 antibody on the immunoplate overnight. Wash the ELISA plate 3 times with PBST, 300μL per well, and tap the residual liquid on a paper towel the last time. 2. Add 200μL of 3% BSA-PBS and block at 37℃ for 2 hours. Wash the ELISA plate 3 times with PBST, 300μL per well, and tap the residual liquid on a paper towel the last time. 3. Dilute the CA125 natural antigen at different concentrations, add 100μL, and incubate at 37℃ for 1 hour. Wash the ELISA plate 6 times with PBST, 300μL per well, and tap the residual liquid on a paper towel the last time. 4. Add 100μL of diluted anti-CA125 antibody-HRP and incubate at 37℃ for 1 hour. Wash the ELISA plate 6 times with PBST, 300μL per well, and tap the residual liquid on a paper towel the last time. 5. Use tetramethylbenzidine microporous peroxidase substrate (TMB) as a substrate for color development for 10 minutes; 6. Add 50 μL of 0.1 mol / L sulfuric acid to terminate the reaction, and measure the absorbance at 450 nm.

[0099] The results of the antibody pair test are attached. Figure 4 , its linear range is 1.65-800U / ml (R 2 >0.99).

[0100] In summary, the present invention provides an anti-CA125 monoclonal antibody (CA125 antigen binding protein) and its application. The monoclonal antibody is a rabbit monoclonal antibody. Compared with mouse antibodies and polyclonal antibodies, rabbit antibodies naturally have higher affinity and stronger specificity, and the antibody obtained by combination has strong specificity and high accuracy, and has great potential in immunological research and the development of new treatment strategies.

[0101] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A CA125 antigen binding protein, characterized in that The CA125 antigen binding protein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR3 having a sequence as shown in SEQ ID NO.7 or SEQ ID NO.13; The light chain variable region includes CDR3 having a sequence as shown in SEQ ID NO.10 or SEQ ID NO.

16.

2. The CA125 antigen binding protein according to claim 1, characterized in that The heavy chain variable region also includes a CDR1 having a sequence as shown in SEQ ID NO.5 or SEQ ID NO.11; Preferably, the heavy chain variable region further comprises a CDR2 having a sequence as shown in SEQ ID NO.6 or SEQ ID NO.12; Preferably, the light chain variable region further comprises a CDR1 having a sequence as shown in SEQ ID NO.8 or SEQ ID NO.14; Preferably, the light chain variable region further comprises a CDR2 having a sequence as shown in SEQ ID NO.9 or SEQ ID NO.

15.

3. The CA125 antigen binding protein according to claim 1 or 2, characterized in that The heavy chain variable region comprises a CDR1 having a sequence as shown in SEQ ID NO.5, a CDR2 having a sequence as shown in SEQ ID NO.6, and a CDR3 having a sequence as shown in SEQ ID NO.7, and the light chain variable region comprises a CDR1 having a sequence as shown in SEQ ID NO.8, a CDR2 having a sequence as shown in SEQ ID NO.9, and a CDR3 having a sequence as shown in SEQ ID NO.10; or, The heavy chain variable region includes a CDR1 having a sequence as shown in SEQ ID NO.11, a CDR2 having a sequence as shown in SEQ ID NO.12, and a CDR3 having a sequence as shown in SEQ ID NO.13, and the light chain variable region includes a CDR1 having a sequence as shown in SEQ ID NO.14, a CDR2 having a sequence as shown in SEQ ID NO.15, and a CDR3 having a sequence as shown in SEQ ID NO.

16.

4. The CA125 antigen binding protein according to any one of claims 1 to 3, characterized in that The heavy chain variable region comprises a sequence having at least 90% identity with the sequence shown in SEQ ID NO.1 or SEQ ID NO.3; and / or, The light chain variable region comprises a sequence having at least 90% identity with the sequence shown in SEQ ID NO.2 or SEQ ID NO.

4.

5. The CA125 antigen binding protein according to any one of claims 1 to 4, characterized in that The sequence of the heavy chain variable region includes SEQ ID NO.1 or SEQ ID NO.3; Preferably, the sequence of the light chain variable region includes SEQ ID NO.2 or SEQ ID NO.

4.

6. The CA125 antigen binding protein according to claim 5, characterized in that The sequence of the heavy chain variable region includes the sequence shown in SEQ ID NO.1, and the sequence of the light chain variable region includes the sequence shown in SEQ ID NO.2; or, The sequence of the heavy chain variable region includes the sequence shown in SEQ ID NO.3, and the sequence of the light chain variable region includes the sequence shown in SEQ ID NO.

4.

7. The CA125 antigen binding protein according to any one of claims 1 to 6, characterized in that The antigen binding protein is stronger than or equal to 10 -9 It specifically binds to human CA125 with an affinity of nmol / L.

8. The CA125 antigen binding protein according to claim 7, characterized in that The antigen binding protein is an antibody; Preferably, the antibody is a monoclonal antibody; Preferably, the constant region of the monoclonal antibody is a rabbit-derived constant region.

9. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the CA125 antigen binding protein according to any one of claims 1 to 8.

10. An expression vector, characterized in that: The expression vector contains the nucleic acid molecule according to claim 9.

11. A host cell, characterized in that The host cell contains at least one copy of the expression vector according to claim 10; or the nucleic acid molecule according to claim 9 is integrated into the genome of the host cell.

12. A reagent for detecting CA125 protein in a sample, characterized in that: The reagent comprises the CA125 antigen binding protein according to any one of claims 1-8.

13. A kit, characterized in that: The kit comprises the CA125 antigen binding protein according to any one of claims 1 to 8; or the kit comprises the reagent for detecting CA125 protein in a sample according to claim 12.

14. Use of the anti-CA125 monoclonal antibody according to any one of claims 1 to 8 in the preparation of a tumor detection product.

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