Monoclonal antibody for resisting CA125 protein and application thereof

Abstract

The invention discloses an anti-CA125 protein monoclonal antibody and application thereof, and relates to the technical field of preparation and synthesis of monoclonal antibodies. Complementary determining region sequences of a heavy chain and a light chain of the monoclonal antibody are respectively as shown in SEQ ID NO.1-5, or have at least 95% of homology with the sequences as shown in SEQ ID NO.1-5. The invention also provides a substance for coding and expressing the monoclonal antibody, and application of the monoclonal antibody in detection of CA125 protein. The monoclonal antibody provided by the invention can well recognize natural CA125 tumor cells, the supernatant titer of B cell culture can reach 1: 10000, the titer of transfected 293F cells is greater than 1: 100000, and the monoclonal antibody can be completely applied to research of CA125 protein. The chemiluminescence reagent prepared by using the monoclonal antibody has the advantages of high specificity, strong anti-interference capability, high detection sensitivity, good stability and the like, the lowest detection limit reaches 1U / ml, and the chemiluminescence reagent can replace an imported reagent.

Description

Technical Field

[0001] The present invention relates to the technical field of monoclonal antibody preparation and synthesis, and in particular to a monoclonal antibody against CA125 protein and application thereof. Background Art

[0002] CA125 is a glycoprotein with a relative molecular mass of 200kD. It originates from the fetal coelomic epithelial tissue and was discovered and named by Bast et al. in 1981. CA125 is a membrane protein molecule expressed on the surface of epithelial cells. It consists of an intracellular region, a transmembrane region, and an extracellular region. Its coding gene is muc16, so it is also called MUC16.

[0003] The intracellular region of CA125 is composed of non-tandem repeat sequences, and the C segment contains phosphorylation sites; the extracellular region is composed of multiple tandem repeat sequences, each of which is a polypeptide chain composed of 156 amino acids, including antigen sites recognized by CA125 monoclonal antibodies including OC125 and M11. When a tumor occurs, the expression of CA125 increases, glycosylation is abnormal, membrane CA125 is hydrolyzed, the extracellular segment enters the blood, and the intracellular segment enters the nucleus, regulating tumor-related effects such as cell proliferation and invasion.

[0004] CA125 is abnormally elevated in the serum of ovarian cancer patients. Since its discovery, it has been used as a tumor marker for routine screening, clinical auxiliary diagnosis and prognosis of ovarian cancer. The abnormal increase of serum CA125 mainly occurs in patients with non-mucinous ovarian cancer such as ovarian serous adenocarcinoma and endometrioid adenocarcinoma. About 82% of ovarian cancer patients have elevated serum CA125 levels, while only about 50% of early ovarian cancer patients have elevated serum CA125.

[0005] In actual work, CA125 is mainly used for preoperative differential diagnosis of suspected ovarian cancer (pelvic mass, ascites, etc.). Although CA125 is not the "gold standard" for ovarian cancer diagnosis, it plays an important role in the efficacy and prognosis monitoring of ovarian cancer. At present, the CA125 quantitative detection kits used in clinical practice in China mainly rely on imports, which are costly, limiting the further application of CA125 as a routine clinical screening item.

[0006] Therefore, it is necessary to develop a highly sensitive and highly active anti-CA125 protein monoclonal antibody that can be used by downstream reagent manufacturers. Summary of the invention

[0007] In order to prepare an anti-CA125 monoclonal antibody with high specificity, strong anti-interference ability, high detection sensitivity and good stability, the present invention provides an anti-CA125 protein monoclonal antibody and its application, which is specifically achieved through the following technologies.

[0008] A monoclonal antibody against CA125 protein, wherein in the heavy chain variable region of the monoclonal antibody, the amino acid sequence of the complementary determining region CDR1 is as shown in SEQ ID NO.1 or has at least 95% homology with the sequence shown in SEQ ID NO.1, the amino acid sequence of the complementary determining region CDR2 is as shown in SEQ ID NO.2 or has at least 95% homology with the sequence shown in SEQ ID NO.2, and the amino acid sequence of the complementary determining region CDR3 is as shown in SEQ ID NO.3 or has at least 95% homology with the sequence shown in SEQ ID NO.3;

[0009] In the light chain variable region of the monoclonal antibody, the amino acid sequence of the complementarity determining region CDR1 is as shown in SEQ ID NO.4 or has at least 95% homology with the sequence shown in SEQ ID NO.4, the amino acid sequence of the complementarity determining region CDR2 is as shown in SEQ ID NO.5 or has at least 95% homology with the sequence shown in SEQ ID NO.5, and the amino acid sequence of the complementarity determining region CDR3 is as shown in SEQ ID NO.6 or has at least 95% homology with the sequence shown in SEQ ID NO.6.

[0010] Furthermore, in the heavy chain variable region of the monoclonal antibody, the amino acid sequence of the framework region FR1 is as shown in SEQ ID NO.7 or has at least 95% identity with the sequence shown in SEQ ID NO.7, the amino acid sequence of the framework region FR2 is as shown in SEQ ID NO.8 or has at least 95% identity with the sequence shown in SEQ ID NO.8, the amino acid sequence of the framework region FR3 is as shown in SEQ ID NO.9 or has at least 95% identity with the sequence shown in SEQ ID NO.9, and the amino acid sequence of the framework region FR4 is as shown in SEQ ID NO.10 or has at least 95% identity with the sequence shown in SEQ ID NO.10;

[0011] In the light chain variable region, the amino acid sequence of the framework region FR1 is as shown in SEQ ID NO.11 or has at least 95% identity with the sequence shown in SEQ ID NO.11, the amino acid sequence of the framework region FR2 is as shown in SEQ ID NO.12 or has at least 95% identity with the sequence shown in SEQ ID NO.12, the amino acid sequence of the framework region FR3 is as shown in SEQ ID NO.13 or has at least 95% identity with the sequence shown in SEQ ID NO.13, and the amino acid sequence of the framework region FR4 is as shown in SEQ ID NO.14 or has at least 95% identity with the sequence shown in SEQ ID NO.14.

[0012] Furthermore, the amino acid sequence of the heavy chain of the monoclonal antibody is as shown in SEQ ID NO.15, or has at least 95% homology with the sequence shown in SEQ ID NO.15; the amino acid sequence of the light chain of the monoclonal antibody is as shown in SEQ ID NO.16, or has at least 95% homology with the sequence shown in SEQ ID NO.16. The light chain constant region of the above monoclonal antibody is a κ chain, and the heavy chain constant region is of IgG1 type.

[0013] Furthermore, the gene sequence encoding the heavy chain of the monoclonal antibody is as shown in SEQ ID NO.16 or has at least 95% homology with the sequence shown in SEQ ID NO.16; the gene sequence encoding the light chain of the monoclonal antibody is as shown in SEQ ID NO.17 or has at least 95% homology with the sequence shown in SEQ ID NO.17.

[0014] The present invention also provides and claims a substance, which is any one of the following:

[0015] (1) A nucleic acid molecule encoding any one of the above-mentioned monoclonal antibodies;

[0016] (2) an expression vector or a vector set, wherein the expression vector or the vector set contains the nucleic acid molecule;

[0017] (3) A host cell comprising the expression vector or vector set.

[0018] The present invention also provides and claims a compound against CA125 protein, comprising any one of the above monoclonal antibodies, and also comprising a conjugate connected to the monoclonal antibody via a covalent bond or a non-covalent bond.

[0019] Furthermore, the conjugate includes at least one of alkaline phosphatase, horseradish peroxidase, and streptavidin.

[0020] The present invention also provides and claims the use of any one of the above monoclonal antibodies or any one of the above anti-CA125 protein compounds, which can be used to prepare products for detecting CA125 protein, or to detect CA125 protein for purposes other than disease diagnosis and treatment.

[0021] Furthermore, the product is a detection kit, a detection test strip or a detection chip.

[0022] Furthermore, the detection kit is a colloidal gold detection kit, an ELISA detection kit, an immunochromatography kit, an immunoturbidimetric detection kit, a magnetic particle detection kit, a chemiluminescence detection kit, an immunofluorescence detection kit or a radioimmunoassay kit; and the detection test strip is a colloidal gold test strip or an ELISA test strip.

[0023] Compared with the prior art, the invention is beneficial in that the monoclonal antibody provided by the invention can well identify natural CA125 tumor cells, and the titer of the supernatant of B cell culture can reach 1:10000, and the titer of the transfected 293F cells is greater than 1:100000, which can be fully applied to the research of CA125 protein. The chemiluminescent reagent prepared by using the monoclonal antibody of the invention has the advantages of high specificity, strong anti-interference ability, high detection sensitivity and good stability, and the detection limit is as low as 1U / ml, which can replace imported reagents. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The OD value results of hybridoma cell screening in Example 2;

[0025] Figure 2 This is a gel electrophoresis diagram of the antibody molecular mass identification during the monoclonal antibody property identification in Example 2;

[0026] Figure 3 The ELISA titer determination results of the monoclonal antibody in Example 2;

[0027] Figure 4 The linear range test results of the monoclonal antibody turbidimetric reagent of the present invention are shown in FIG. DETAILED DESCRIPTION

[0028] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the functional differences of components as the criterion for distinction. As mentioned throughout the specification and claims, "including" or "comprising" are open-ended terms and should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present application, but the description is based on the general principles of the specification and is not intended to limit the scope of the present application. The scope of protection of the present application shall be determined by the attached claims.

[0030] The technical terms mentioned in this specification have the same meanings as those generally understood by those skilled in the art. In case of any conflict, the definitions in this specification shall prevail.

[0031] Generally speaking, the terms used in this specification have the following meanings.

[0032] In this specification, the term "monoclonal antibody" means an antibody derived from a colony of substantially homologous antibodies, that is, each antibody constituting the colony is identical and / or binds to the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during the production of monoclonal antibody products), such variants are usually present in trace amounts. Different from polyclonal antibody products that generally include different antibodies for different determinants (epitopes), each monoclonal antibody of a monoclonal antibody product is directed to a single determinant on an antigen. Thus, the modifier "monoclonal" indicates that the antibody is derived from a substantially homologous antibody colony, and should not be construed as requiring the antibody to be produced by any ad hoc method. For example, the monoclonal antibody to be used according to the present invention can be prepared by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods using all or part of transgenic animals comprising human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0033] In the specification of the present application, the terms "heavy chain" ("CH"), "light chain" ("CL"), "heavy chain constant region", "light chain constant region", "light chain variable region" ("VL"), "heavy chain variable region" ("VH"), "framework region" ("Framework Region, FR"), and "complementarity determining region" ("CDR") are components of antibodies, and different types of antibodies can be formed through different combinations. For example, a conventional IgG antibody is a tetramer composed of two light chains and two heavy chains. Among them, VL and VH are both composed of 3 complementary determining regions (CDR) and 4 framework regions (FR). Among them, CDR contains residues that contact the antigen and determine the antigen specificity of VL and VH, and FR is used to maintain the variable region structure and determine the position of the CDR loop. Usually, the heavy chain variable region and the heavy chain constant region constitute a complete heavy chain, while the light chain variable region and the light chain constant region constitute a complete light chain.

[0034] In the specification of the present application, a conjugate refers to a molecule that, after binding to an antibody, can indicate the location or amount of the antibody by color, chemical reaction, excitation light, mass spectrometry, etc.; including but not limited to alkaline phosphatase, peroxidase, luciferase, fluorescein, fluorescent protein, biotin, streptomycin or isotope, etc.

[0035] In the specification of the present application, the terms "polynucleotide", "nucleic acid" and "nucleic acid molecule" are used interchangeably, including but not limited to DNA, RNA, cDNA (complementary DNA), mRNA (messenger RNA), rRNA (ribosomal RNA), shRNA (small hairpin RNA), snRNA (small nuclear RNA), snoRNA (short nucleolar RNA), miRNA (micro RNA), genomic DNA, synthetic DNA, synthetic RNA and / or tRNA.

[0036] In the specification of the present application, the term "vector" (expression vector or vector group) refers to a vector through which a polynucleotide sequence (such as a foreign gene) can be introduced into a host cell to transform the host and promote the expression (such as transcription and translation) of the introduced sequence. Vectors include plasmids, phage vectors, viral vectors, etc. Among them, a "viral vector" is a vector transformed from a viral genome, which introduces foreign genes into a host cell through viral infection. "Plasmid" refers to a DNA molecule other than chromosomes (or nucleoids) in organisms such as bacteria, yeasts and actinomycetes, which exists in the cytoplasm or nucleus and has the ability to replicate autonomously, so that it can also maintain a constant copy number in daughter cells and express the genetic information it carries.

[0037] In the specification of the present application, "enzyme-linked immunosorbent assay (ELISA)" refers to a detection method that utilizes the characteristic that antibody molecules can specifically bind to antigen molecules, combines free impurities with the target protein bound to a solid phase carrier, and uses special markers to conduct qualitative or quantitative analysis thereof. The principle of ELISA is: antigens or antibodies can be physically adsorbed on the solid surface and maintain their immune activity; antigens or antibodies can form enzyme conjugates with enzymes through covalent bonds while maintaining their respective immune activity or enzyme activity; after the enzyme conjugates bind to the corresponding antigens or antibodies, the occurrence of the immune reaction can be determined by the color reaction of the added substrate, and the depth of the color reaction is proportional to the amount of the corresponding antigen or antibody in the specimen. Various types of detection methods can be designed according to the substance to be detected and the conditions for detection. The double antibody sandwich method is the most commonly used method for detecting antigens. The principle of the double antibody sandwich method is to adsorb the antiserum containing known antibodies into the small holes of the microtiter plate and wash it once; add the antigen to be tested, if the two are specific, they will bind, and then wash away the excess antibody; add an enzyme-linked antibody that specifically reacts with the antigen to be tested to form a "sandwich" structure; add the substrate of the enzyme, and if colored enzymatic hydrolysis products are seen, it indicates the presence of the corresponding antigen.

[0038] Example 1: Synthesis of recombinant CA125 protein and preparation of anti-CA125 mouse monoclonal antibody

[0039] 1. Synthetic recombinant CA125 protein

[0040] Gene recombination technology was used to synthesize a CA125 tandem repeat sequence polypeptide (as shown in SEQ ID NO.18), and the recombinant protein was used as an immunogen to prepare its antibody, providing a basis for the development of CA125 detection kits and related functional studies. We cloned a CA125 tandem repeat sequence gene into the pGEX-6P-1 prokaryotic expression vector to establish a recombinant fusion protein GST-CA125R. BALB / c mice were immunized with the purified GST-CA125R fusion protein as an immunogen, and a cell line that stably secreted anti-CA125 monoclonal antibodies was established using hybridoma technology.

[0041] 2. Animal immunization

[0042] Six 5-8 week old female BALB / c mice were selected for immunization. The recombinant CA125 protein in Example 1 was diluted to 1 mg / ml with physiological saline and mixed with an equal volume of complete Freund's adjuvant. The mice were emulsified using the double push method. After the emulsification was completed, 200 μl was used for subcutaneous multi-point immunization each time. Two weeks later, the same antigen and the same dose of emulsified incomplete Freund's adjuvant were used for intraperitoneal immunization. After an interval of 2 weeks, 3 injections were continued for immunization. The dosage route was the same as the second time, and incomplete Freund's adjuvant was added. The last immunization was separated from the previous immunization by one month. The recombinant CA125 protein was used for intraperitoneal open spleen immunization at 50 μg / mouse (without adjuvant). After 3 days, tail blood was collected and serum antibody titer was detected by ELISA.

[0043] 3. Construction of B cells

[0044] (1) Cultivation and preparation of myeloma cell lines

[0045] The present embodiment adopts SP / 20 myeloma cell strain, which has good growth and fusion efficiency, and a doubling time of 10-12h. When fusion, select myeloma cells with good logarithmic growth phase, cell morphology and activity. Myeloma cells should be cultured adaptively before fusion to make cell growth reach the optimal state (i.e. logarithmic growth phase).

[0046] On the day of fusion, use an elbow pipette to gently blow the myeloma cells off the bottle wall, collect them in a 50ml centrifuge tube or fusion tube, and centrifuge them at 1000r / min for 5-10min; discard the supernatant, add 30ml culture medium to the precipitate, wash it once by centrifugation, and centrifuge it for 5-10min; discard the supernatant, mix the precipitate with 20ml culture medium for later use.

[0047] (2) Preparation of splenocytes

[0048] Take BALB / c mice that have completed animal immunization, remove the eyeballs to collect blood, and use a centrifuge to separate the serum as the positive control serum for antibody detection.

[0049] Mice were killed by cervical dislocation and immersed in 75% alcohol for 5 min. The spleen was removed aseptically in a clean bench and placed in a plate containing 10 ml of culture medium. The spleen was gently washed and the surrounding connective tissue was removed. The spleen was pierced with a sterile syringe until the spleen turned white. The spleen cells in the plate were collected in a 50 ml centrifuge tube and centrifuged at 1000 r / min for 10 min. The supernatant was discarded, 10 ml of culture medium was added, and a small amount of 10 was taken for dilution counting.

[0050] (3) Cell fusion

[0051] Three days before fusion, positive mice were boosted with immunization, mouse spleen cells were mixed with myeloma cells sp2 / 0 at a ratio of 10:1, PEG fused, and then cultured in HAT selection medium. Ten days later, hybridoma cell supernatants were screened by ELISA, and the screened positive hybridoma cells were cloned by limiting dilution. After five screenings, 10 positive hybridoma cell lines were finally determined.

[0052] (4) Screening of hybridoma cells (ELISA method)

[0053] Recombinant CA125 protein was coated on a 96-well plate at 10 μg / ml, 50 μl / well, and adsorbed at 37°C for 1 hour; the liquid in the well was discarded, and the plate was washed with washing solution for 3 times, and then patted dry; 100 μl of blocking solution was added to each well, and the plate was blocked at 37°C for 1 hour, washed twice, and then patted dry; 100 μl of the culture supernatant of the 6 hybridoma cells to be tested was added to each well, and positive, negative and blank control standards were set up at the same time; the plate was incubated at 37°C for 0.5 hour, washed 4 times, and then patted dry. Horseradish enzyme-labeled goat anti-mouse polyclonal IgG 1:10000 was taken, and 100 μl was added to each well of the ELISA plate, incubated at 37°C for 30 minutes, washed 4 times, and then patted dry.

[0054] The CA125 tumor cell stock solution was fixed to a 96-well plate with a 1% formaldehyde solution, adsorbed at 37°C for 2 hours, the liquid in the wells was discarded, and the wells were hand-washed 3 times with detergent and patted dry; 100 μl of blocking solution was added to each well, blocked at 37°C for 1 hour, the liquid in the wells was discarded, and the wells were aspirated dry; 100 μl of the culture supernatant of 10 hybridoma cells to be tested was added to each well, and positive, negative and blank control standards were set up at the same time, incubated at 37°C for 0.5 hour, hand-washed 4 times, and aspirated dry; horseradish enzyme-labeled sheep anti-mouse polyclonal IgG 1:10000 was taken, 100 μl was added to each well of the ELISA plate, incubated at 37°C for 30 minutes, hand-washed 4 times, and patted dry.

[0055] Finally, 100 μl / well of TMB substrate colorimetric solution was added, and after 15 min of color development, 50 μl / well of 2 mol / L dilute hydrochloric acid was used to terminate the reaction.

[0056] like Figure 1 As shown, the OD value of each well was measured at 450nm and 630nm. It can be seen that the blank well value is below 0.02, and the negative well value is below 0.1, and the yin and yang are clearly distinguished; it can be judged that the anti-CA125 hybridoma cells secrete antibodies that can specifically recognize both CA125 protein and CA125 tumor cells. The same method was used to measure the titer of mouse eyeball blood, which reached 1:100000 and can be used for cell fusion.

[0057] 4. Cloning of the gene encoding mouse monoclonal antibody

[0058] The supernatant of the cultured hybridoma cells was used to identify positive clones by antigen-coated ELISA. The cells of the positive clones were collected and lysed, and RNA was extracted, which was reverse transcribed into cDNA using a total RNA extraction kit (reverse transcription at 65°C for 5 minutes). The light and heavy chain variable region genes (VH and VL) of the naturally paired rabbit monoclonal antibodies were amplified from the cDNA of the corresponding positive clones by PCR, and the sequences were determined by sequencing:

[0059] The amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the monoclonal antibody are shown in SEQ ID NOs. 1-3; the amino acid sequences of the complementary determining regions CDR1 and CDR3 of the light chain variable region are shown in SEQ ID NOs. 4 and 5; the amino acid sequence of the complementary determining region CDR2 of the light chain variable region is KVS;

[0060] The amino acid sequences of the framework regions FR1, FR2, FR3, and FR4 of the heavy chain variable region of the monoclonal antibody are shown in SEQ ID NOs.6-9; the amino acid sequences of the framework regions FR1, FR2, FR3, and FR4 of the light chain variable region are shown in SEQ ID NOs.10-13.

[0061] The amino acid sequence of the heavy chain of the monoclonal antibody is shown in SEQ ID NO.14, and the amino acid sequence of the light chain is shown in SEQ ID NO.15.

[0062] The gene sequence encoding the heavy chain of the monoclonal antibody is shown in SEQ ID NO.16, and the gene sequence encoding the light chain of the monoclonal antibody is shown in SEQ ID NO.17.

[0063] 5. Production and purification of monoclonal antibodies

[0064] The heavy chain and light chain genes of the monoclonal antibody obtained in step 4 were loaded on the pCDN3.4 expression vector, respectively, and transfected into 293F cells; the culture supernatant containing the recombinant monoclonal antibody recognizing human CA125 protein was obtained 72-96 hours after transfection. The recombinant mouse monoclonal antibody recognizing human CA125 protein was purified from the culture supernatant after transfection using protein A affinity gel resin, and the antibody was subpackaged after identification and stored at -20°C for future use.

[0065] 6. Monoclonal antibody characterization

[0066] (1) Determination of antibody concentration

[0067] The absorbance values ​​A280 and A260 of the monoclonal antibody at 280 nm and 260 nm were determined by UV spectrophotometry. The protein content was calculated according to the following formula:

[0068] Protein content (mg / ml) = (A280 × dilution factor) / 1.35.

[0069] (2) Antibody molecular mass identification

[0070] Monoclonal antibodies were determined by SDS-PAGE. Figure 2 As shown, the heavy chain of the monoclonal antibody is about 46 KD, and the light chain is about 25 KD.

[0071] (3) ELISA titer determination

[0072] The purified ascites monoclonal antibodies were measured by indirect ELISA. Figure 3 As shown, the results showed that the titer after purification was 1:100000.

[0073] Example 2: CA125 monoclonal antibody latex turbidimetric reagent detection limit test

[0074] 1. CA125 monoclonal antibody conjugation

[0075] Take 300 μl of latex microspheres and add them to 3 ml of MES buffer, then add 0.3 ml of 20 mg / ml EDC solution, stir and activate at room temperature for 1 hour; centrifuge at 10,000 rpm for 10 minutes, remove the supernatant, add 3 ml of MES, and disperse in a water bath by ultrasonication for 10-20 seconds; quickly add to 3 ml of 0.4 mg / ml CA125 antibody solution at one time, stir and react at room temperature for 1.5 hours, centrifuge at 10,000 rpm for 6 minutes after the reaction is completed, and discard the supernatant; add 20 ml of microsphere preservation solution, ultrasonicate in an ice bath for 7 minutes, stir at room temperature for 2 hours, and let stand at 37°C overnight. The storage concentration of the microspheres is 1.5 mg / ml (0.15%).

[0076] 2. Detection limit test

[0077] The pure CA125 antigen was diluted with physiological saline (0.9% sodium chloride) to a concentration of 1000U / ml, 100U / ml, 10U / ml, 1U / ml, and 0.1U / ml; the sample (S), phosphate buffer (R1) and latex microspheres (R2) coupled to CA125 monoclonal antibody were mixed in a Hitachi 7080 biochemical analyzer according to the sample addition volume of 20μl, 180μl and 60μl, and then incubated at 37°C for 10min; the absorbance of the incubated solution at a wavelength of 600nm was tested, and each concentration was tested 10 times, and the coefficient of variation CV (CV=standard deviation / average value*100%) of the 10 tests was calculated respectively, and the lowest concentration at which the CV was not higher than 10% was the detection limit of the CA125 monoclonal antibody turbidimetric reagent.

[0078] The results are shown in Table 1 below, which show that the detection limit of the turbidimetric reagent prepared by CA125 monoclonal antibody can reach 1 U / ml.

[0079] Table 1 Detection limit of CA125 monoclonal antibody latex turbidimetric reagent

[0080] Repeat the test 0.1U / ml 1U / ml 10U / ml 100U / ml 1000U / ml 1 7 28 65 528 8563 2 6 27 62 519 8187 3 9 26 60 553 7719 4 8 25 68 561 7897 5 5 26 66 543 7861 6 6 27 65 555 8586 7 9 27 60 513 7579 8 3 22 67 574 7548 9 4 21 68 569 8268 10 7 26 65 594 8202 Standard Deviation 2.01 2.27 2.99 25.57 377.36 average value 6.40 25.50 64.60 550.90 8041.00 CV 31％ 8.9％ 4.6％ 4.6％ 4.0％

[0081] Example 3: Linear range test of CA125 monoclonal antibody latex turbidimetric reagent

[0082] On a Hitachi 7080 biochemical analyzer, the sample (S), phosphate buffer (R1) and latex microspheres coupled to CA125 monoclonal antibody (R2) were mixed in amounts of 20 μl, 180 μl and 60 μl, incubated at 37°C for 10 min, and the absorbance of the incubated solution was tested at a wavelength of 600 nm.

[0083] The results are as follows Figure 4 As shown, it is shown that in the range of [5,2000] U / ml, the linear correlation of the turbidimetric reagent prepared by CA125 monoclonal antibody is as high as above 0.99.

[0084] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A monoclonal antibody against CA125 protein, characterized in that: In the heavy chain variable region of the monoclonal antibody, the amino acid sequence of the complementary determining region CDR1 is as shown in SEQ ID NO.1 or has at least 95% homology to the sequence shown in SEQ ID NO.1, the amino acid sequence of the complementary determining region CDR2 is as shown in SEQ ID NO.2 or has at least 95% homology to the sequence shown in SEQ ID NO.2, and the amino acid sequence of the complementary determining region CDR3 is as shown in SEQ ID NO.3 or has at least 95% homology to the sequence shown in SEQ ID NO.3; In the light chain variable region of the monoclonal antibody, the amino acid sequence of the complementarity determining region CDR1 is as shown in SEQ ID NO.4 or has at least 95% homology with the sequence shown in SEQ ID NO.4, the amino acid sequence of the complementarity determining region CDR2 is KVS or has at least 95% homology with the sequence KVS, and the amino acid sequence of the complementarity determining region CDR3 is as shown in SEQ ID NO.5 or has at least 95% homology with the sequence shown in SEQ ID NO.

5.

2. The monoclonal antibody against CA125 protein according to claim 1, characterized in that: In the heavy chain variable region of the monoclonal antibody, the amino acid sequence of the framework region FR1 is as shown in SEQ ID NO.6 or has at least 95% identity with the sequence shown in SEQ ID NO.6, the amino acid sequence of the framework region FR2 is as shown in SEQ ID NO.7 or has at least 95% identity with the sequence shown in SEQ ID NO.7, the amino acid sequence of the framework region FR3 is as shown in SEQ ID NO.8 or has at least 95% identity with the sequence shown in SEQ ID NO.8, and the amino acid sequence of the framework region FR4 is as shown in SEQ ID NO.9 or has at least 95% identity with the sequence shown in SEQ ID NO.9; In the light chain variable region, the amino acid sequence of the framework region FR1 is as shown in SEQ ID NO.10 or has at least 95% identity with the sequence shown in SEQ ID NO.10, the amino acid sequence of the framework region FR2 is as shown in SEQ ID NO.11 or has at least 95% identity with the sequence shown in SEQ ID NO.11, the amino acid sequence of the framework region FR3 is as shown in SEQ ID NO.12 or has at least 95% identity with the sequence shown in SEQ ID NO.12, and the amino acid sequence of the framework region FR4 is as shown in SEQ ID NO.13 or has at least 95% identity with the sequence shown in SEQ ID NO.

13.

3. The monoclonal antibody against CA125 protein according to claim 2, characterized in that: The amino acid sequence of the heavy chain of the monoclonal antibody is shown in SEQ ID NO.14, or has at least 95% homology to the sequence shown in SEQ ID NO.14; the amino acid sequence of the light chain of the monoclonal antibody is shown in SEQ ID NO.15, or has at least 95% homology to the sequence shown in SEQ ID NO.

15.

4. The monoclonal antibody against CA125 protein according to claim 3, characterized in that: The gene sequence encoding the heavy chain of the monoclonal antibody is shown as SEQ ID NO.16 or has at least 95% homology with the sequence shown as SEQ ID NO.16; the gene sequence encoding the light chain of the monoclonal antibody is shown as SEQ ID NO.17 or has at least 95% homology with the sequence shown as SEQ ID NO.

17.

5. A substance, characterized in that The substance is any one of the following: (1) A nucleic acid molecule encoding the monoclonal antibody according to any one of claims 1 to 3; (2) an expression vector or a vector set, wherein the expression vector or the vector set contains the nucleic acid molecule; (3) A host cell comprising the expression vector or vector set.

6. A compound against CA125 protein, characterized in that: The invention comprises the monoclonal antibody according to any one of claims 1 to 4, and also comprises a conjugate connected to the monoclonal antibody by a covalent bond or a non-covalent bond.

7. The anti-CA125 protein compound according to claim 6, characterized in that The conjugate includes at least one of alkaline phosphatase, horseradish peroxidase and streptavidin.

8. Use of the monoclonal antibody according to any one of claims 1 to 4, or the anti-CA125 protein compound according to claim 6 or 7, characterized in that: Used to prepare products for detecting CA125 protein, or used to detect CA125 protein for purposes other than disease diagnosis and treatment.

9. The use according to claim 8, characterized in that: The product is a detection kit, a detection test strip or a detection chip.

10. The use according to claim 9, characterized in that: The detection kit is a colloidal gold detection kit, an ELISA detection kit, an immunochromatography kit, an immunoturbidimetric detection kit, a magnetic particle detection kit, a chemiluminescence detection kit, an immunofluorescence detection kit or a radioimmunoassay kit; The test strip is a colloidal gold test strip or an ELISA test strip.

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