A ca125 antigen binding protein and uses thereof

Abstract

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

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biopharmaceuticals, and particularly relates to a CA125 antigen binding protein and application thereof. BACKGROUND

[0002] CA125 antigen is a mucin, which was first obtained by using tumor cells OVCA433 expressing MUC16 as an immunogen to obtain monoclonal antibody OC125 antibody, and the antigen recognized by the 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 3000 O-glycosylation sites. At the same time, the protein has many different isoforms with a molecular weight of 200-2000 kDa. CA125 protein is composed of a short intracellular region, a transmembrane region and a long extracellular region. The extracellular segment is composed of a repeat domain and an N-terminal domain, and the repeat domain contains 56 SEA modules, which contain disulfide bond-connected cysteine loops between the modules, which are the recognition sites of OC125 and M11 class antibodies. A potential cleavage site was found near the SEA module near the transmembrane region. Protein cleavage can cause the shedding of most of the extracellular part of MUC16 into the bloodstream.

[0004] Under normal circumstances, CA125 is at a low expression level and is in a stable combination with the cell membrane and cannot enter the human body, so the content of CA125 in the human body is very small when the human body is healthy or suffers from some benign diseases. When the body grows and metastasizes malignant tumors, and when some non-tumor diseases worsen, CA125 is at a high expression level in the cell and falls off from the cell membrane into the human body fluid and then into the blood circulation and thus is in a high content state. CA125 is one of the earliest discovered tumor markers, and is still the most widely used early marker for ovarian cancer, and is used for monitoring the progress of treatment of epithelial ovarian cancer. The gene MUC16 encoding CA125 was discovered and cloned in 2001, and exists in a type I transmembrane or secreted form, either of which has severe glycosylation.

[0005] The development process of the CA125 detection antibody mainly includes the following steps: main immunogen preparation: generally using recombinant protein, polypeptide or purchasing CA125 natural antigen cells and the like; immunizing animals: selecting appropriate immunized animals, usually using mice, rabbits and the like for immunization. The immunogen is injected into the immunized animals to stimulate the production of CA125 specific antibodies; screening and identification: through screening methods such as phage display and single cell sorting, CA125 specific antibodies with specificity and affinity are screened out. Further identification and evaluation are carried out on the candidate antibodies, including affinity determination, flow cytometry analysis and the like; cloning, antibody optimization and improvement.

[0006] At present, the anti-CA125 protein antibody is mostly polyclonal antibody or mouse monoclonal antibody, and the detection threshold of the CA125 antigen is 35U / mL, and the sensitivity of the paired antibody pair is high. Therefore, a monoclonal antibody capable of specifically binding to the CA125 protein is provided, which has important application value in the diagnosis of ovarian cancer. SUMMARY

[0007] In view of the deficiencies in the prior art, the purpose of the present application is to provide a CA125 antigen binding protein and application thereof. The antibody is a rabbit monoclonal antibody, which has higher affinity and stronger specificity than mouse antibodies and polyclonal antibodies, and the antibody pair obtained by combination has strong specificity and high accuracy.

[0008] To achieve the purpose of the present application, the following technical solutions are adopted:

[0009] In a first aspect, the present application provides a CA125 antigen binding protein, which comprises 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 comprises a 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 the sequence as set forth in SEQ ID NO. 9 or SEQ ID NO. 15.

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

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

[0017] Preferably, the heavy chain variable region comprises a sequence that is at least 90% identical to the sequence as set forth in SEQ ID NO. 1 or SEQ ID NO. 3; and / or,

[0018] the light chain variable region comprises a sequence that is at least 90% identical to the sequence as set forth in SEQ ID NO. 2 or SEQ ID NO. 4.

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

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

[0021] Preferably, the sequence of the heavy chain variable region comprises the sequence as set forth in SEQ ID NO. 1, and the sequence of the light chain variable region comprises the sequence as set forth in SEQ ID NO. 2; or,

[0022] the sequence of the heavy chain variable region comprises the sequence as set forth in SEQ ID NO. 3, and the sequence of the light chain variable region comprises the sequence as set forth in SEQ ID NO. 4.

[0023] Preferably, the antigen binding protein specifically binds human CA125 with an affinity stronger than or equal to 10 -9 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 application, New Zealand white rabbits are immunized with CA125 natural antigen. After 4 immunizations, the titer of the antisera is detected by enzyme-linked immunosorbent assay (ELISA). After the titer reaches a certain dilution, the rabbits are subjected to booster immunization. The serum is collected and purified, and the spleen is collected and ground. Total RNA is extracted from the spleen, and first-strand cDNA is synthesized by reverse transcription. The light and heavy chain variable region fragments (VH / VK) of the immunized rabbits are amplified by a set of primers matching the FR1 and FR4 of the light and heavy chain variable regions of rabbit antibodies. The light and heavy chain variable fragments are given a linker sequence with a complementary overlapping region by secondary amplification with another set of primers. The VH fragments and VK fragments are assembled into single-chain antibody fragments (scFv) by overlap extension PCR (SOE PCR). After the scFv fragments and phage display vectors are digested with the same restriction enzymes, the products are recovered by gel recovery and recombination vector ligation. After the ligation products are cleaned, they are electroporated into E. coli TG1, thereby obtaining a scFv phage display bacterial library. The bacterial library is cultured, and helper phage M13K07 is added for superinfection. The helper phage packages and rescues the phage library, and the scFv fragments are displayed on the surface of the phage. The CA125 natural antigen is coated in an immunoplate, and after the blocking solution is blocked, the phage library is added. The phage displaying specific scFv fragments is captured due to its ability to bind to the CA125 protein, and the unbound phage is removed by rinsing. The phage is eluted from the immunoplate, and logarithmically growing TG1 hosts are infected. After culture, the helper phage is used again for superinfection and packaging rescue. Through several rounds of screening, the TG1 bacterial liquid infected with the screened phage is gradient-diluted, plated, and single colonies are picked and cultured in 96-well plates. The helper phage is used to package the phage of the single colonies, and the binding activity of the phage in the supernatant of the single colonies to CA125 is detected by ELISA, thereby obtaining positive single colonies that specifically bind to CA125.

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

[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 (CDR1 of the heavy chain of M1-4): SYAMG.

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

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

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

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

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

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

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

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

[0040] QSLEESGGRLVTPGTSLTLTCTVSGFSLS SYYMN WVRQAPGKGLEWIG IIYASGSTYYA TWAKG RFTISKTSTTVDLKITSPTTEDTATYFCAG 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 light chain variable region of S1-1)

[0045] AAVLNQTPSPVSPAVGGTVSISC QSSQSVYNNNYLA WYQQKPGQPPKLLIY RASKLAS GVPSRFSGSGSGTQFTLTISGVQCDDAATYYC LGAYDDDTDTA 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 described in the present application can also be prepared into a single-chain antibody (scFv), an antigen-binding fragment Fab, (Fab)2, a bispecific antibody, an added fusion protein, etc., and applied to the field of detection, clinical treatment, etc.

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

[0051] In a third aspect, the present application provides an expression vector containing the nucleic acid molecule of the second aspect.

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

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

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

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

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

[0057] The numerical ranges described in the present application include not only the point values listed, but also any intervening point values between the listed point values, and the present application does not exhaustively list the specific point values included in the ranges for the sake of brevity and simplicity.

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

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

[0060] Figure 1 is the CA125 immune serum titer detection result.

[0061] Figure 2 is the positive antibody purification result graph.

[0062] Figure 3 is the positive clone affinity ELISA assay.

[0063] Figure 4 is the antibody pairing range test. DETAILED DESCRIPTION

[0064] The technical solutions of the present application are further illustrated below by specific embodiments. Those skilled in the art should understand that the embodiments are only used to help understand the present application and should not be regarded as specific limitations on the present application.

[0065] If a specific technique or condition is not specified in the embodiments, it is performed according to the technique or condition described in the literature in the art or according to the product manual. If the reagent or instrument used is not specified by the manufacturer, it is a conventional product that can be commercially available through a regular channel.

[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.). Two rabbits (purchased from Guangdong Medical Experimental Animal Center, name: ordinary New Zealand rabbits, specification: about 1.8 kg) were immunized with each polypeptide.

[0068] 1. One mL of blood was taken from the ears of two rabbits, and the serum was collected for use as a control. Each rabbit was immunized with 1 mg of antigen (the antigen type was human natural antigen, purchased from XEMA, item number R222), emulsified with complete adjuvant, 2 mg of antigen was diluted in 1.5 mL of PBS, mixed with 1.5 mL of complete adjuvant, and oscillated on a shaker for more than 15 minutes until the water-in-oil state was achieved; 1.5 mL was immunized on the back of each rabbit.

[0069] 2. Two weeks later, the second immunization was performed, each rabbit was immunized with 0.5 mg of antigen, emulsified with incomplete adjuvant, 1 mg of antigen was diluted in 1 mL of PBS, mixed with 1 mL of incomplete adjuvant, and oscillated on a shaker for more than 15 minutes until the water-in-oil state was achieved; 1 mL was immunized on the back of each rabbit.

[0070] 3. One week later, each rabbit was immunized with 0.5 mg of antigen, emulsified with incomplete adjuvant, 1 mg of antigen was diluted in 1 mL of PBS, mixed with 1 mL of incomplete adjuvant, and oscillated on a shaker for more than 15 minutes until the water-in-oil state was achieved; 1 mL was immunized on the back of each rabbit.

[0071] 4. One week later, each rabbit was immunized with 0.5 mg of antigen, emulsified with incomplete adjuvant, 1 mg of antigen was diluted in 1 mL of PBS, mixed with 1 mL of incomplete adjuvant, and oscillated on a shaker for more than 15 minutes until the water-in-oil state was achieved; 1 mL was immunized on the back of each rabbit.

[0072] 5. One week later, 1 mL of blood was taken from the ears, and the titer was detected by ELISA. If it reached more than 1:100,000, impact immunization was performed.

[0073] 6. Impact immunity, each rabbit immunized with 1 mg antigen, emulsified with complete adjuvant, 2 mg antigen diluted in 1.5 mL PBS, mixed with 1.5 mL complete adjuvant, shaken on the oscillator for more than 15 minutes, until the oil-in-water state; each rabbit immunized with 1.5 mL on the back.

[0074] 7. Three days later, ear blood sampling, 5 mL of blood was collected from each rabbit to measure the titer, and the titer met the expected value. The whole blood was collected in an anticoagulant tube, and the serum was separated.

[0075] Note: The materials involved in the above operation are common reagent consumables, not limited to brand suppliers.

[0076] Example 2: Titer ELISA detection of antiserum

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

[0078] 2. Wash the enzyme-labeled plate with PBST 3 times, 300 μL per well, and the last time, tap the residual liquid on the paper towel.

[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 enzyme-labeled plate with PBST 3 times, 300 μL per well, and the last time, tap the residual liquid on the paper towel.

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

[0082] 6. Wash the enzyme-labeled plate with PBST 6 times, 300 μL per well, and the last time, tap the residual liquid on the paper towel.

[0083] 5. Add 100 μL of PBS-diluted Goat Anti-rabbit IgG-Fc Secondary Antibody (HRP) (anti-rabbit secondary antibody, Beijing Yiqiao God, item number SSA003) to each well, and incubate at room temperature for 1 hour.

[0084] 6. Wash the enzyme-labeled plate with PBST 6 times, 300 μL per well, and the last time, tap the residual liquid on the paper towel.

[0085] 7. Take the A and B liquids of the TMB substrate developing solution from 4°C, restore to room temperature, and mix in equal proportions to prepare an appropriate amount of developing solution. Add 100 μL to each well of the enzyme-labeled plate, and react in the dark for 10 minutes.

[0086] 8. Add 50 μL 1M HCl to each well to terminate the color reaction, and read OD 450 values.

[0087] The titer detection results are shown in the following table: Figure 1 The titer is greater than 1:102400, and the immunization is successful.

[0088] Example 3 Screening of the Rabbit Antibody scFv Display Library

[0089] The rabbit spleen cells were taken for grinding and RNA extraction to establish a cDNA library. The antibody variable region sequences in the library were amplified by the literature method, and a phage display library was constructed for antibody screening. After three rounds of screening, 96 monoclonal colonies were picked, and 10 positive clones were obtained, of which two preferred clones 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 Clone Antibodies

[0092] Recombinant antibody expression: The light and heavy chain sequences of M1-4 and S1-1 were synthesized by Guangzhou Lihe and the codon sequences were optimized to construct a pCDNA3.1(+) vector. After obtaining the vector, the plasmid was extracted, and then the light and heavy chain vectors were mixed in a molar ratio of 1:1. 300 μg of plasmid was added to 12.5 mL of OPM 293CD05 culture medium and 800 μL of 1 mg / mL PEI (polyethyleneimine, POLYSCIENCE) solution, mixed, and incubated at room temperature for half an hour. Then it was added to 250 mL of HEK293F cells being cultured, and placed in a shaker, incubated at 37°C, 8% CO2, 110 rpm. OPM 293 cell feed was added every other day, and the cells were cultured for 7 days. The supernatant was collected by centrifugation.

[0093] Recombinant antibody purification: The received supernatant was added to Protein A filler (Yiqiao God State) 3 mL, incubated at room temperature for half an hour, and then the filler was removed and loaded into a purification column. 20 mL of PBS solution was added for washing, and then 10 mL of 100 mM glycine solution at pH 3.0 was added for elution. The eluate was neutralized to pH 7.0 with 1M Tris solution. The neutralized eluate was concentrated to 1 mL, and the protein concentration was determined. Representative antibodies were run on SDS-PAGE for purity identification. Figure 2 is a positive antibody purification result graph, Figure 2Lane M is molecular weight marker, lane 1 is recombinant antibody S1-1, lane 2 is recombinant antibody M1-4, lane 3 is recombinant antibody S1-1 and M1-4 mixed together, lane 4 is negative control. Figure 2 It can be seen that the antibodies reached the electrophoretic purity standard, and no obvious other bands were seen in addition to the main band. Figure 2 .

[0094] Example 5 Affinity ELISA assay of positive clones

[0095] 1. CA125 natural antigen (purchased from XEMA, item number R222) was coated on the immunoplate overnight. The enzyme-labeled plate was washed with PBST 3 times, 300 μL per well, and the last time the residual liquid was tapped on the paper towel. 2. 3% BSA-PBS 300 μL was added, and the plate was blocked at 37°C for 2 hours. The enzyme-labeled plate was washed with PBST 3 times, 300 μL per well, and the last time the residual liquid was tapped on the paper towel. 3. The antibody was diluted at different concentrations, 100 μL was added, and the plate was incubated at 37°C for 1 hour. The enzyme-labeled plate was washed with PBST 6 times, 300 μL per well, and the last time the residual liquid was tapped on the paper towel. 4. Diluted commercial horseradish peroxidase-conjugated Goat Anti-rabbit IgG-Fc Secondary Antibody (HRP) 100 μL was added, and the plate was incubated at 37°C for 1 hour. The enzyme-labeled plate was washed with PBST 6 times, 300 μL per well, and the last time the residual liquid was tapped on the paper towel. 5. Tetramethylbenzidine microwell peroxidase substrate (TMB) was used as a substrate for color development for 10 minutes; 6. After adding 50 μL of 0.1 mol / L sulfuric acid to terminate the reaction, the absorbance at 450 nm was measured.

[0096] The affinity determination results are shown in the following table: Figure 3 According to the ELISA results, the affinities of the two antibodies were calculated by origin software to be about 10 -9 .

[0097] Example 6 Test of antibody pairing range

[0098] 1. Coating CA125 antibody on the immunoplate overnight. Washing the enzyme plate with PBST for 3 times, 300ul per well, and the last time, tap the residual liquid on the paper towel. 2. Adding 3% BSA-PBS 200ul, 37℃ blocking for 2 hours. Washing the enzyme plate with PBST for 3 times, 300ul per well, and the last time, tap the residual liquid on the paper towel. 3. Diluting CA125 natural antigen with different concentrations, adding 100ul, 37℃ incubating for 1 hour. Washing the enzyme plate with PBST for 6 times, 300ul per well, and the last time, tap the residual liquid on the paper towel. 4. Adding 100ul of diluted anti-CA125 antibody-HRP, 37℃ incubating for 1 hour. Washing the enzyme plate with PBST for 6 times, 300ul per well, and the last time, tap the residual liquid on the paper towel. 5. Using tetramethyl benzidine microperoxidase substrate (TMB) as substrate for color development for 10 minutes; 6. Adding 50ul of 0.1mol / L sulfuric acid to terminate the reaction, and measuring the absorbance at 450nm.

[0099] The results of antibody pair matching assay are shown in the following table Figure 4 , and the linear range is 1.65-800U / ml (R 2 >0.99).

[0100] In conclusion, the application 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 have higher affinity and stronger specificity. The antibody pair obtained has strong specificity and high accuracy, and has great potential in immunological research and development of new treatment strategies.

[0101] The applicant declares that the above is only a specific embodiment of the application, but the protection scope of the application is not limited thereto. It should be understood by those skilled in the art that any changes or replacements within the technical scope disclosed by the application can be easily thought of by those skilled in the art, and all fall within the protection scope and disclosure scope of the application.

Claims

1. An anti-CA125 monoclonal antibody or an antigen-binding fragment thereof, characterized in that, The monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the CDR1 of the heavy chain variable region is as shown in SEQ ID NO. 5, the CDR2 is as shown in SEQ ID NO. 6, and the CDR3 is as shown in SEQ ID NO. 7; the CDR1 of the light chain variable region is as shown in SEQ ID NO. 8, the CDR2 is as shown in SEQ ID NO. 9, and the CDR3 is as shown in SEQ ID NO. 10; Or, the CDR1 of the heavy chain variable region is as shown in SEQ ID NO. 11, the CDR2 is as shown in SEQ ID NO. 12, and the CDR3 is as shown in SEQ ID NO. 13; the CDR1 of the light chain variable region is as shown in SEQ ID NO. 14, the CDR2 is as shown in SEQ ID NO. 15, and the CDR3 is as shown in SEQ ID NO.

16.

2. The anti-CA125 monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that, The sequence of the heavy chain variable region is as shown in SEQ ID NO. 1, and the sequence of the light chain variable region is as shown in SEQ ID NO. 2; or, The sequence of the heavy chain variable region is as shown in SEQ ID NO. 3, and the sequence of the light chain variable region is as shown in SEQ ID NO.

4.

3. The anti-CA125 monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that, The antibody binds specifically to human CA125 with an affinity stronger than or equal to 10 -9 nmol / L.

4. The anti-CA125 monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that, The constant region of the monoclonal antibody is a rabbit-derived constant region.

5. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the anti-CA125 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-4.

6. An expression vector, characterized by, The expression vector comprises the nucleic acid molecule according to claim 5.

7. A host cell, characterized in that, The host cell comprises at least one copy of the expression vector according to claim 6; or the nucleic acid molecule according to claim 5 is integrated into the genome of the host cell.

8. A reagent for detecting CA125 protein in a sample, characterized in that, The reagent comprises the anti-CA125 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-4.

9. A kit characterized in that, The kit comprises the anti-CA125 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-4; or the kit comprises the reagent for detecting CA125 protein in a sample according to claim 8.

10. Use of the anti-CA125 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-4 in the preparation of an ovarian cancer detection product.

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