A monoclonal antibody against CA125 protein and its application

Abstract

The present invention discloses a monoclonal antibody against CA125 protein and its application, and relates to the technical field of monoclonal antibody preparation and synthesis. The complementary determining region sequences of the heavy chain and light chain of the monoclonal antibody are respectively as shown in SEQ ID NO.1-5, or have at least 95% homology with the sequence shown in SEQ ID NO.1-5. The present invention also provides a substance encoding and expressing the above-mentioned monoclonal antibody, and the application of the above-mentioned monoclonal antibody in detecting CA125 protein. The monoclonal antibody provided by the present 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 293F cells after transfection is greater than 1:100000, which can be fully applied to the study of CA125 protein. The chemiluminescent reagent prepared using the monoclonal antibody of the present 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.

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 200kD glycoprotein originating from fetal coelomic epithelial tissue. It was discovered and named by Bast et al. in 1981. CA125 is a membrane protein expressed on the surface of epithelial cells, consisting of an intracellular domain, a transmembrane domain, and an extracellular domain. Its encoding gene is muc16, hence its name MUC16.

[0003] The intracellular region of CA125 is composed of non-tandem repeats, with the C segment containing phosphorylation sites. The extracellular region is composed of multiple tandem repeats, each consisting of a 156-amino acid polypeptide chain, which contains antigenic sites recognized by CA125 monoclonal antibodies, including OC125 and M11. When tumors develop, CA125 expression increases, glycosylation becomes abnormal, and membrane CA125 undergoes hydrolysis, allowing the extracellular segment to enter the blood and the intracellular segment to enter the nucleus, regulating tumor-related effects such as cell proliferation and invasion.

[0004] Since its discovery, CA125 is abnormally elevated in the serum of patients with ovarian cancer. It has been used as a tumor marker for routine ovarian cancer screening, clinical diagnosis, and prognosis. Abnormally elevated serum CA125 occurs primarily in patients with non-mucinous ovarian cancers, such as serous and endometrioid adenocarcinomas. Approximately 82% of patients with ovarian cancer have elevated serum CA125 levels, while only approximately 50% of patients with early-stage ovarian cancer have elevated serum CA125.

[0005] In practice, CA125 is primarily used for preoperative differential diagnosis of suspected ovarian cancer (pelvic masses, ascites, etc.). Although CA125 is not the "gold standard" for ovarian cancer diagnosis, it plays an important role in monitoring the efficacy and prognosis of ovarian cancer. Currently, CA125 quantitative detection kits used in clinical practice in China are primarily imported, resulting in high costs, which limits the further application of CA125 as a routine clinical screening item.

[0006] Therefore, it is very necessary to develop a highly sensitive and 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] An anti-CA125 protein monoclonal antibody, wherein in the heavy chain variable region of the monoclonal antibody, the amino acid sequence of the complementarity 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 complementarity 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 complementarity 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;

[0009] In the light chain variable region of the monoclonal antibody, the amino acid sequence of the complementarity determining region CDR1 is shown in SEQ ID NO.4 or has at least 95% homology to the sequence shown in SEQ ID NO.4, the amino acid sequence of the complementarity determining region CDR2 is shown in SEQ ID NO.5 or has at least 95% homology to the sequence shown in SEQ ID NO.5, and the amino acid sequence of the complementarity determining region CDR3 is shown in SEQ ID NO.6 or has at least 95% homology to 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 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 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 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 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 to 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 to the sequence shown in SEQ ID NO. 16. The light chain constant region of the monoclonal antibody is a kappa chain, and the heavy chain constant region is of the IgG1 type.

[0013] Furthermore, the gene sequence encoding the heavy chain of the monoclonal antibody is shown as SEQ ID NO.16 or has at least 95% homology to 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 to the sequence shown as 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 vector set containing the nucleic acid molecule;

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

[0018] The present invention also provides and claims an anti-CA125 protein compound, comprising any one of the above-mentioned 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 a 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 existing technologies, the present invention is advantageous in that the monoclonal antibody provided by the present invention can effectively recognize natural CA125 tumor cells, with a titer of up to 1:10,000 in the supernatant of B cell culture and greater than 1:100,000 in transfected 293F cells, making it fully applicable to CA125 protein research. The chemiluminescent reagent prepared using the monoclonal antibody of the present invention has the advantages of high specificity, strong anti-interference ability, high detection sensitivity, and good stability, with a detection limit as low as 1 U / ml, and 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 characterization 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 solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort 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 difference in the functions of the 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 this application shall be as defined 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, term " monoclonal antibody " represents the antibody that derives from the colony of substantially homologous antibody, that is, each antibody constituting the colony is identical and / or in conjunction with identical epi-position, except possible variant antibody (for example containing naturally occurring mutation or producing in the production process of monoclonal antibody preparation), such variant is usually present in trace.Different from the polyclonal antibody preparation that generally comprises the different antibodies for different determinants (epi-position), every kind of monoclonal antibody of monoclonal antibody preparation is for the single determinant on antigen.Thus, the modifier " monoclonal " indicates that the antibody derives from the feature of substantially homologous antibody colony, and should not be construed as needing to produce the antibody by any ad hoc method.For example, the monoclonal antibody to be used according to the present invention can be prepared by various techniques, and the technology includes, but is not limited to hybridoma method, recombinant DNA method, phage display method and the method for using all or part of the transgenic animal that comprises human immunoglobulin locus, and such method and other exemplary method for preparing monoclonal antibody 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 (CDRs) and 4 framework regions (FRs). 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 this 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 this 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 (microRNA), 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 by 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, "viral vectors" are vectors transformed from viral genomes, which introduce foreign genes into host cells 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 maintain a constant copy number in daughter cells and express the genetic information it carries.

[0037] In the specification of this application, "enzyme-linked immunosorbent assay (EILSA)" refers to a detection method that utilizes the characteristic that antibody molecules can specifically bind to antigen molecules to bind free foreign proteins and target proteins bound to a solid phase carrier, and uses special markers to conduct qualitative or quantitative analysis thereof. The principle of enzyme-linked immunosorbent assay is: antigens or antibodies can be physically adsorbed on the solid phase 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 conjugate is bound to the corresponding antigen or antibody, 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 different 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, binding will occur, and then the excess antibody is washed away; 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 a colored enzymatic hydrolysis product is 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] Using genetic recombination technology, we synthesized a CA125 tandem repeat polypeptide (shown in SEQ ID NO. 18) and used this recombinant protein as an immunogen to generate antibodies, providing a foundation for the development of a CA125 detection kit and related functional studies. We cloned the CA125 tandem repeat gene into the pGEX-6P-1 prokaryotic expression vector to create a recombinant fusion protein, GST-CA125R. BALB / c mice were immunized with the purified GST-CA125R fusion protein as an immunogen, and hybridoma technology was used to establish a cell line that stably secretes anti-CA125 monoclonal antibodies.

[0041] 2. Animal immunization

[0042] Six 5-8 week old female BALB / c mice were selected for immunization. The recombinant CA125 protein described in Example 1 was diluted to 1 mg / ml with physiological saline and mixed with an equal volume of complete Freund's adjuvant. Emulsification was performed using the double push method. After emulsification, 200 μl was administered subcutaneously to each mouse at multiple sites. Two weeks later, intraperitoneal immunization was performed using the same antigen and the same dose of emulsified incomplete Freund's adjuvant. After an interval of 2 weeks, three more injections were performed using the same dosing route as the second time, with the addition of incomplete Freund's adjuvant. The last immunization was performed one month after the previous one, and the recombinant CA125 protein was administered intraperitoneally (without adjuvant) at 50 μg per mouse. Three days later, tail blood was collected, and serum antibody titers were detected by ELISA.

[0043] 3. Construction of B cells

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

[0045] The present embodiment adopts SP / 20 myeloma cell strain, and this cell strain growth and fusion efficiency are all good, and doubling time is 10-12h.When fusion, select the good myeloma cell of logarithmic growth phase, cell morphology and activity.Myeloma cell should do adaptive culture before fusion, make cell growth reach 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 flask wall, collect them in a 50ml centrifuge tube or fusion tube, and centrifuge at 1000r / min for 5-10min; discard the supernatant, add 30ml of culture medium to the precipitate, and wash it by centrifugation once for 5-10min; discard the supernatant, and mix the precipitate with 20ml of culture medium for later use.

[0047] (2) Preparation of splenocytes

[0048] BALB / c mice that have completed animal immunization were taken, their eyeballs were removed and blood was collected. The serum was separated by centrifuge and used 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 dish 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 dish were collected in a 50 ml centrifuge tube and centrifuged at 1000 rpm for 10 min. The supernatant was discarded, 10 ml of culture medium was added, and a small amount of 10 dilution was aspirated for 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, fused with PEG, and cultured in HAT selection medium. Ten days later, hybridoma supernatants were screened using ELISA. Positive hybridomas were cloned using limiting dilution. After five rounds of screening, 10 positive hybridoma cell lines were identified.

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

[0053] Recombinant CA125 protein was coated onto a 96-well plate at 10 μg / ml, 50 μl / well, and allowed to adsorb at 37°C for 1 hour. The liquid in the wells was discarded, and the plates were washed three times with washing buffer and patted dry. Each well was blocked with 100 μl of blocking buffer at 37°C for 1 hour, washed twice, and patted dry. Each well was added with 100 μl of culture supernatant from the six hybridoma cell lines to be tested, along with positive, negative, and blank controls. The plates were incubated at 37°C for 0.5 hour, washed four times, and patted dry. A 1:10,000 dilution of horseradish peroxidase-labeled goat anti-mouse polyclonal antibody (IgG) was added to each well of the plate and incubated at 37°C for 30 minutes, washed four times, and patted dry.

[0054] CA125 tumor cell stock solution was fixed to a 96-well plate with 1% formaldehyde solution, adsorbed at 37°C for 2 hours, the liquid in the wells was discarded, and the plates were hand-washed 3 times with detergent and patted dry; 100 μl of blocking solution was added to each well and blocked at 37°C for 1 hour, the liquid in the wells was discarded, and the plates were aspirated dry; 100 μl of culture supernatant of 10 hybridoma cell lines 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 goat anti-mouse polyclonal IgG 1:10000 was taken, 100 μl per well was added to 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 color development 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 values ​​of each well were measured at dual wavelengths of 450nm and 630nm. Blank well values ​​were below 0.02, and negative well values ​​were below 0.1, clearly distinguishing between positive and negative. This indicates that the anti-CA125 hybridoma cells secrete antibodies that specifically recognize both the CA125 protein and CA125 tumor cells. Using the same method, the titer of mouse eye blood reached 1:100,000, suitable for cell fusion.

[0057] 4. Cloning of genes encoding mouse monoclonal antibodies

[0058] Positive clones were identified using antigen-coated ELISA in the cultured hybridoma cell supernatant. Positive clones were harvested, lysed, and RNA extracted. This was then reverse-transcribed into cDNA using a total RNA extraction kit (65°C for 5 minutes). The naturally paired light and heavy chain variable region genes (VH and VL) of the rabbit monoclonal antibody were amplified from the cDNA of the corresponding positive clones using PCR, and the sequences were confirmed by sequencing:

[0059] The amino acid sequences of the complementarity 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 complementarity 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 complementarity 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 and light chain genes of the monoclonal antibody obtained in Step 4 were separately loaded into the pCDN3.4 expression vector and transfected into 293F cells. Recombinant monoclonal antibodies that recognize human CA125 protein were obtained in the culture supernatant 72-96 hours after transfection. Recombinant mouse monoclonal antibodies that recognize human CA125 protein were purified from the transfected culture supernatant using Protein A affinity gel resin. After antibody identification, aliquot and store at -20°C until use.

[0065] 6. Monoclonal Antibody Characterization

[0066] (1) Determination of antibody concentration

[0067] The absorbance values ​​of the monoclonal antibody at 280 nm and 260 nm, A280 and A260, 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 detected 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 detected 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 and activate at room temperature with stirring 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 with ultrasonic waves for 10-20 seconds; quickly add it to 3 ml of 0.4 mg / ml CA125 antibody solution at one time, stir at room temperature for 1.5 hours, centrifuge at 10,000 rpm for 6 minutes after the reaction is complete, and discard the supernatant; add 20 ml of microsphere preservation solution, ultrasonicate on ice for 7 minutes, stir at room temperature for 2 hours, and let it stand at 37°C overnight. The storage concentration of the microspheres is 1.5 mg / ml (0.15%).

[0076] 2. Detection limit test

[0077] Pure CA125 antigen was diluted with physiological saline (0.9% sodium chloride) to concentrations of 1000 U / ml, 100 U / ml, 10 U / ml, 1 U / ml, and 0.1 U / ml. The sample (S), phosphate buffer (R1), and CA125 monoclonal antibody-coupled latex microspheres (R2) were mixed in 20 μl, 180 μl, and 60 μl volumes on a Hitachi 7080 biochemical analyzer and incubated at 37°C for 10 minutes. The absorbance of the incubated solution at a wavelength of 600 nm was measured 10 times for each concentration, and the coefficient of variation (CV) of the 10 tests (CV = standard deviation / mean value * 100%) was calculated. The lowest concentration at which the CV was no 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 with 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 antibodies (R2) were mixed in volumes of 20 μl, 180 μl, and 60 μl, and then incubated at 37°C for 10 min. The absorbance of the incubated solution was measured at a wavelength of 600 nm.

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

[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 of the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection 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 complementarity determining region CDR1 is shown in SEQ ID NO.1, the amino acid sequence of the complementarity determining region CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of the complementarity determining region CDR3 is 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 shown in SEQ ID NO.4, the amino acid sequence of the complementarity determining region CDR2 is KVS, and the amino acid sequence of the complementarity determining region CDR3 is 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 shown in SEQ ID NO.6, the amino acid sequence of the framework region FR2 is shown in SEQ ID NO.7, the amino acid sequence of the framework region FR3 is shown in SEQ ID NO.8, and the amino acid sequence of the framework region FR4 is shown in SEQ ID NO.9; In the light chain variable region, the amino acid sequence of the framework region FR1 is shown in SEQ ID NO.10, the amino acid sequence of the framework region FR2 is shown in SEQ ID NO.11, the amino acid sequence of the framework region FR3 is shown in SEQ ID NO.12, and the amino acid sequence of the framework region FR4 is 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; the amino acid sequence of the light chain of the monoclonal antibody is 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 in SEQ ID NO.16; the gene sequence encoding the light chain of the monoclonal antibody is shown in SEQ ID NO.

17.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the monoclonal antibody according to any one of claims 1 to 4.

6. An expression vector or vector set, characterized in that: The expression vector or vector set comprises the nucleic acid molecule of claim 5.

7. A host cell, characterized in that The host cell comprises the expression vector or vector group according to claim 6.

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

9. The anti-CA125 protein compound according to claim 8, characterized in that The conjugate includes at least one of alkaline phosphatase, horseradish peroxidase, and streptavidin.

10. Use of the monoclonal antibody according to any one of claims 1 to 4, or the anti-CA125 protein compound according to claim 8 or 9, characterized in that: Used to prepare products for detecting CA125 protein.

11. The use according to claim 10, characterized in that The product is a detection kit, a detection test strip or a detection chip.

12. The use according to claim 11, characterized in that The detection kit is an ELISA detection kit, an immunochromatographic 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.

13. The use according to claim 11, characterized in that The detection kit is a colloidal gold detection kit.

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