CN112684175B - Kit for detecting ovarian cancer - Google Patents
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Abstract
The invention relates to the field of tumor immunodetection, in particular to a kit for detecting ovarian cancer, and also discloses application of the antibody in preparation of a tumor treatment and/or diagnosis reagent. The application of the kit provides an effective tool for early screening diagnosis, curative effect detection and prognosis judgment of ovarian cancer.
Description
Technical Field
The invention relates to the field of tumor immunodetection, in particular to a kit for detecting ovarian cancer.
Background
Ovarian malignant tumor is a common malignant tumor of women occurring in reproductive organs, has high incidence rate and is only lower than cervical cancer and endometrial cancer. Because the ovary is at the bottom of the pelvic cavity, the position is special, the early symptoms of the malignant tumor are not obvious, the discovery is difficult, the tumor is transferred at the late stage, the golden stage of the treatment is missed, and the ovarian malignant tumor has the highest death rate in the gynecological malignant tumor, so the ovarian malignant tumor is a main disease which threatens the health and the life of the female. Among ovarian malignancies, epithelial ovarian tumors are the most common, accounting for 85% -90% and 50% -70% of primary ovarian tumors.
Carbohydrate antigen 125(CA125) is the most widely used ovarian epithelial tumor marker in clinical examination at present at home and abroad. It was first discovered by Bast in 1983 that mice were initially immunized with human ovarian serous cystadenocarcinoma cells and then hybridized with myeloma cells to give the monoclonal antibody OC125, which recognizes the glycoprotein CA 125. The amino acid sequence of the carbohydrate antigen is partially characterized by mucin molecules and is therefore designated CA 125. During embryonic development, the epithelial cells of the body cavity secrete CA125 which gradually disappears after birth, but if ovarian tumors reappear, positive detection can be carried out, and the relative molecular weight of the CA125 is more than 200kD, is positioned in the 19P13.2 region of a chromosome, mainly contains N-2-acetylgalactosamine chains, N-2-acetylglucosamine and galactose, is a transmembrane glycoprotein containing 5797 base pairs, and has a circular shape structure. Since CA125 is secreted by epithelial cells in embryonic stage, and is not secreted or rarely secreted in normal condition, when the ovary has malignant lesion, even if the ovary has no clinical expression or is difficult to identify pathologically, the CA125 value is increased, so that the CA125 is a better ovarian cancer diagnosis and screening index and has close relation with the metastasis and prognosis of ovarian cancer.
Disclosure of Invention
Based on the above findings, the primary object of the present invention is to provide a novel CA125 antibody with strong specificity.
The invention provides the following technical scheme:
an anti-CA 125 monoclonal antibody, consisting of a heavy chain and a light chain, wherein the heavy chain and the light chain respectively comprise a variable region and a constant region, wherein the heavy chain variable region is selected from SEQ ID NO. 1, and the light chain variable region is selected from SEQ ID NO. 2.
The heavy and light chain variable regions of the anti-CA 125 monoclonal antibody of the invention have 6 complementarity determining regions. 3 CDR sequences of a heavy chain variable region of the anti-CA 125 monoclonal antibody are respectively SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5; the variable region of light chain has 3 CDR sequences of SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8.
The heavy and light chains of the anti-CA 125 monoclonal antibody of the invention further comprise constant regions, and the antibody light chain constant region further comprises murine kappa and lambda chain sequences. The antibody heavy chain constant region further comprises a murine IgG1, IgG2a, IgG2b, or IgG3, or IgA or IgM sequence.
The invention aims to provide a novel anti-CA 125 monoclonal antibody, which does not cross react with CEA, CA50, CA242, CA199 and the like, and is used for detecting the existence and the content of CA125 protein in a sample.
The invention also aims to provide a CA125 quantitative detection kit, a preparation method and application thereof. The kit comprises the antibody for identifying CA125, and is used for early screening diagnosis, curative effect detection and prognosis judgment of ovarian cancer.
The CA125 quantitative detection kit comprises an anti-CA 125 monoclonal antibody coated enzyme label plate and an anti-CA 125 enzyme labeled antibody.
The anti-CA 125 enzyme-labeled antibody is a CA125 polyclonal antibody marked by horseradish peroxidase. The preparation method of the polyclonal antibody and the method of the enzyme-labeled antibody are all routine operations in the field.
Further, the quantitative detection kit also comprises a CA125 standard substance, a concentrated washing solution, a sample diluent, a substrate solution and a stop solution.
The anti-CA 125 monoclonal antibody, the CA125 standard substance, the horseradish peroxidase labeled anti-CA 125 polyclonal antibody, the concentrated washing solution, the sample diluent, the substrate solution and the stop solution are respectively put into each reagent bottle, and each reagent bottle is fixed by a sponge bracket and is arranged in the kit body together with an ELISA plate and a sealing plate film which are coated by the CA125 antibody.
Advantageous effects
The invention has the following beneficial effects: the antibody specifically recognizes CA125, and does not cross-react with CEA, CA50, CA242, CA199, and the like; the CA125 detection kit established by the antibody has better sensitivity and specificity, high coincidence rate of the result and a reference reagent, can provide more accurate and reliable detection results, is simple and easy to operate, is quick and sensitive in detection, is simple and popular in an enzyme labeling instrument, is low in price, and provides an effective tool for early screening diagnosis, curative effect detection and prognosis judgment of ovarian cancer.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.
Figure 1 anti-CA 125 antibody specificity: does not react with isocrossing CEA, CA50, CA242, CA 199.
FIG. 2 is a diagram showing the detection range of the CA125 quantitative immunoassay kit.
Detailed Description
The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.
Example 1 anti-CA 125 antibody preparation and purification
5 healthy male BALB/c mice 6-8 weeks old were immunized and emulsified subcutaneously in 15 kU/mouse using Freund's complete adjuvant and CA125 antigen (purchased from Fitzgerald). And emulsifying the mixture with Freund incomplete adjuvant and antigen every 2 weeks, injecting the mixture at multiple points under the skin, 15kU per mouse, taking tail blood serum of the mouse 10 days after 3 immunization, detecting the antibody titer, and selecting the mouse with high serum antibody titer for boosting. Mixing CA125 antigen and incomplete Freund's adjuvant in equal volume, emulsifying, and performing back multi-point injection to enhance immunity once at 15 kU/vaccine.
Taking a BALB/c mouse after boosting immunization, collecting blood through an orbit, breaking a neck, killing the mouse, taking a spleen aseptically, preparing a spleen cell suspension, taking NS-1 cells in a logarithmic phase, and mixing the NS-1 cells according to a ratio of 1: 8-1: 10, cell fusion with PEG4000, 3 days after fusion, complete RPMI1640 medium containing HAT was supplemented, selection culture was performed at 37 ℃ for 2 weeks, HT selection medium was used, and after culture at 37 ℃ for about 2 weeks, culture was performed with complete RPMI1640 medium. And (3) screening positive hybridoma cell strains by adopting an indirect ELISA method. Cloning was performed by limiting dilution. And (5) freezing and storing the hybridoma cells until all the monoclonal cells are positive.
BALB/c mice were injected intraperitoneally with 0.5 ml/mouse, 1 week before hybridoma inoculation. After 1 week, each mouse was inoculated intraperitoneally at about 1X106(ii) individual cells; and after 7-10 days, collecting ascites. Centrifuging ascites at 10000 Xg for 30min, removing precipitate, salting out with 50% ammonium sulfate, coarse extracting, dissolving with PBS, and dialyzing with flowing water for 5 hr; dialyzing and equilibrating with 0.1mol/L phosphate buffer (pH8.0) overnight; and (3) loading, eluting the hybrid protein by using 0.1mol/L phosphate buffer solution (pH8.0), eluting by using citrate eluents with different pH values, collecting elution peaks in sections, concentrating, analyzing and purifying the purity of the monoclonal antibody by 10 percent SDS-PAGE, and determining the titer by an indirect ELISA method. Through indirect ELISA detection, the potency of 6 mAbs can reach 1: 108。
Example 2 identification of antibody specificity
The monoclonal antibody purified in example 1 was cross-reacted with an ELISA plate coated with CEA, CA50, CA242, CA199, and CA125, respectively, by an indirect ELISA method, and the specificity was analyzed. The specific experimental process is to coat CEA, CA50, CA242, CA199 and CA125 (all from Fitzgerald) respectively at a concentration of 0.1mg/ml, add the antibody obtained in example 1 to five different coating antigens respectively, and detect the degree of cross reaction by indirect ELISA. As shown in FIG. 1, the result of the experiment was that CA125-3, which is an antibody having high specificity for CA125, was finally selected.
Example 3 anti-CA 125 antibody subtype identification
The subtype of the anti-CA 125 monoclonal antibody CA125-3 obtained in example 1 was identified by ELISA (kit from Proteintech). The microplate provided in the kit was already pre-coated with specific antibodies against mouse IgG1, IgG2a, IgG2b, IgG3, IgA, IgM, kappa light chain, lambda light chain, and the anti-CA 125 antibody sample CA125-3 purified in example 1 was added to the sample wells at 50 μ l per well without incubation. Adding 1X goat anti-mouse IgA + IgM + IgG-HRP into sample wells, mixing the sample wells with 50 μ l each, and incubating for 1 h. And (4) deducting liquid in the holes, adding 1XPBST to wash the holes for 3 times, and absorbing the excessive moisture by absorbent paper. Adding color development solution, and developing 100 μ l per well in dark at room temperature for 15 min. The color reaction was stopped by adding 100. mu.l of stop solution. The OD value at 450nm was measured by a microplate reader, and as a result, as shown in Table 1, all the heavy chain subtypes and all the light chain subtypes of the anti-CA 125 monoclonal antibody CA125-3 obtained were IgG 1.
TABLE 1
Example 4 monoclonal antibody sequencing
After the hybridoma cell strain corresponding to CA125-3 is recovered, the hybridoma cell strain is cultured until the total number is 107The cells were centrifuged at 1000rpm for 5min to collect the cells, and RNA was extracted. TRNzol-A + was added to the cell pellet for lysis and allowed to stand at room temperature for 15 min. Mu.l chloroform was added to each ml of TRNzol-A +, vortexed for 15 seconds, and allowed to stand for 3 minutes. The cells were centrifuged at 13000rpm, 4 ℃ for 10 minutes, and the Trizol-A + cell solution was divided into three layers: transferring the water phase dissolved with the RNA into a centrifuge tube, adding isopropanol with the same volume into the water phase, uniformly mixing, and standing at room temperature for 25 minutes. Centrifuging at 13000rpm and 4 deg.C for 10min, discarding the waste liquid to obtain precipitateBottom RNA precipitation. After washing the RNA pellet twice with 75% ethanol, the RNA was dissolved in PEDC water and stored at-80 ℃. Synthesizing first chain cDNA by reverse transcription kit SMARTERRACE, and amplifying antibody variable region DNA sequence corresponding to hybridoma cell by using the first chain cDNA as subsequent template. Designing specific nested PCR primer, the primer sequence used in the amplification reaction is complementary with the first frame region and the constant region of the antibody variable region, and amplifying the target gene by adopting a conventional PCR method. Sequencing the amplified product to obtain the heavy chain variable region sequence SEQ ID NO:1 and light chain variable region sequences SEQ ID NO: 2; the 3 CDR sequences of the heavy chain variable region of the antibody are respectively SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5; the variable region of light chain has 3 CDR sequences of SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8.
Example 5 anti-CA 125 antibody affinity assay
After equilibration of the chip with HBS-EP buffer, the carboxymethylated dextran biosensor chip, i.e. the CM5 chip, was activated with N-ethyl-N' - (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Antigen protein CA125 was diluted to 20. mu.g/ml with 10mM sodium acetate pH4.8, followed by injection at a flow rate of 10. mu.l/min for 3min for antigen coupling, and further ethanolamine at a flow rate of 10. mu.l/min to block unreacted groups. The anti-CA 125 antibody CA125-3 was set up in 6 different concentration gradients of 200. mu.g/mL, 100. mu.g/mL, 50. mu.g/mL, 20. mu.g/mL, 10. mu.g/mL, 2. mu.g/mL, respectively. Starting from low concentrations, the anti-CA 125 antibody CA125-3 was injected at a flow rate of 50. mu.L/min for 3min, followed by HBS-EP buffer for a dissociation time of 10min at a flow rate of 50. mu.L/min. Using 3MMgCl2As a regeneration buffer, the chip was regenerated according to the regeneration procedure. Thereafter, other concentrations of the antibody were measured in the same manner. Calculation of binding Rate (K) by Simultaneous fitting of binding and dissociation sensorgramsa) And dissociation Rate (K)d). Equilibrium dissociation constant (K)d) Using dissociation rate (K)d) Rate of binding (K)a) And (4) calculating. As a result, as shown in Table 2, the affinity of the antibody reached 10-10。
TABLE 2
| Antibodies | Ka(1/Ms) | Kd(1/s) | KD(M) |
| CA125-3 | 1.98x105 | 5.17x10-5 | 2.61x10-10 |
Example 6 anti-CA 125 polyclonal antibody preparation
CA125 antigen (from Fitzgerald) and Freund's complete adjuvant (CFA) are mixed well to form water-like oil bag, 2-2.5kg of healthy rabbits are selected for the first time, and 100 kU/rabbit is injected by intradermal multipoint injection method. Three weeks later, again intradermal multiple immunization, and two weeks later intravenous injections of soluble antigen, 100 kU/mouse, were performed as booster immunizations. Blood was collected from the ear vein 7 days after the booster injection, and the serum was separated and its titer was measured by indirect ELISA. A one-time whole blood sampling method is selected, sterile bleeding is carried out from carotid artery, serum is separated, a proper amount of preservative is added, and the blood is subpackaged into small bottles and stored in a low-temperature refrigerator. Precipitating with 50% ammonium sulfate once and 33% ammonium sulfate twice, and purifying rabbit serum by DEAE chromatography to obtain IgG. The purified IgG titer was determined by indirect ELISA to obtain anti-CA 125 polyclonal antibody.
Example 7 preparation of CA125 quantitative immunoassay kit
Preparing enzyme label plate
The anti-CA 125 monoclonal antibody CA125-3 obtained in example 1 is diluted to 0.5 mu g/ml by using 0.01M phosphate buffer solution, and the pH value is 7.0-7.4 to obtain a coating solution. Adding 3% of skimmed milk powder into 0.01M phosphate buffer solution with the pH value of 7.0-7.4 to prepare confining liquid. Adding the prepared coating solution into the holes of an enzyme-labeled plate, and adding 100 mu L of the coating solution into each hole; the enzyme label plate is placed in an environment of 4 ℃ for coating overnight; adding the prepared confining liquid into the pores of an enzyme-labeled plate, adding 100 mu L of confining liquid into each pore, and placing the pores in a 37 ℃ incubator for 30 minutes; the enzyme label plate is taken out from the incubator, the confining liquid is discarded, and the temperature is kept constant at 37 ℃ for 30 minutes.
(II) preparing enzyme-labeled antibody solution
The polyclonal antibody obtained in example 6 was labeled with horseradish peroxidase to obtain an enzyme-labeled antibody. Firstly, weighing a proper amount of HRP enzyme, dissolving the HRP enzyme in triple distilled water, adding a newly-prepared sodium periodate solution, uniformly mixing, and then placing at 4 ℃ for 30 min; adding ethylene glycol solution, and standing at room temperature for 30 min; adding appropriate amount of purified antibody, mixing, adjusting pH to 9.0, standing at 4 deg.C overnight; adding sodium borohydride, mixing, and standing at 4 deg.C for 2 hr; adding the enzyme-labeled antibody mixed solution into an isovolumetric saturated ammonium sulfate solution, and standing at 4 ℃ for 30 min; after centrifugation, the mixture was dialyzed overnight against a phosphate buffer solution of pH 7.4.
(III) preparing CA125 standard substance
CA125 antigen: commercially available from Fitzgerald corporation.
(IV) preparing concentrated lotion (20X 0.01MPBS)
Mixing 96 parts of sodium chloride, 2.4 parts of potassium chloride, 42.96 parts of disodium hydrogen phosphate dodecahydrate, 2.88 parts of potassium dihydrogen phosphate, 200.05 parts of tween-and 1000 parts of ultrapure water uniformly to obtain the finished product.
(V) sample dilution
Artificial serum commercially available (from Huzhou Yingzhang Biotech Co., Ltd.)
(VI) substrate solution (TMB)
TMB Tetramethylbenzidine (3,3 ', 5, 5' -Tetramethylbenzidine), available from Tiangen, Beijing.
(VII) preparing stop solution (2mol/L sulfuric acid solution)
Diluting concentrated sulfuric acid and ultrapure water by 1: 8 to prepare stop solution.
Example 8 determination of detection Range of CA125 quantitative immunoassay kit
CA125 antigen was diluted with artificial serum in a gradient of 1000, 500, 200, 100, 50, 20, 10, 5, 2, 1U/ml. Detection was performed using the kit of example 7. And drawing a standard curve by taking the CA125 antigen concentration as an abscissa and the corresponding A450 as an ordinate to obtain a fitted regression equation, and selecting the CA125 antigen concentration range with the best linear relation as the optimal detection range of the detection reagent. The finally obtained data are shown in fig. 2, and the detection result shows that when the concentration of CA125 is 5-500U/ml, the linear relation with the corresponding a450 value is good, R2 is 0.993, and the regression equation is as follows: y is 0.0022x +0.321, and the lowest detection line is 5U/ml.
Sequence listing
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Claims (8)
1. A CA125 antigen quantitative detection kit is characterized by comprising an enzyme label plate coated by an anti-CA 125 monoclonal antibody and an anti-CA 125 enzyme label antibody, wherein the anti-CA 125 monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1 shown in SEQ ID NO. 3, CDR2 shown in SEQ ID NO. 4 and CDR3 shown in SEQ ID NO. 5, and the light chain variable region comprises CDR1 shown in SEQ ID NO. 6, CDR2 shown in SEQ ID NO. 7 and CDR3 shown in SEQ ID NO. 8.
2. A CA125 antigen quantitative detection kit is characterized by comprising an enzyme label plate coated by an anti-CA 125 monoclonal antibody and an anti-CA 125 enzyme-labeled antibody, wherein the anti-CA 125 monoclonal antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises an amino acid sequence shown in SEQ ID NO. 1, and the light chain variable region comprises an amino acid sequence shown in SEQ ID NO. 2.
3. The kit for quantitatively detecting the CA125 antigen as claimed in claim 1 or 2, wherein the anti-CA 125 enzyme-labeled antibody is a horseradish peroxidase-labeled anti-CA 125 polyclonal antibody.
4. The kit for quantitatively detecting the CA125 antigen as claimed in claim 3, wherein the kit for quantitatively detecting the CA125 antigen further comprises a CA125 standard, a concentrated washing solution, a sample diluent, a substrate solution and a stop solution.
5. An anti-CA 125 monoclonal antibody, comprising a heavy chain variable region comprising CDR1 of SEQ ID NO. 3, CDR2 of SEQ ID NO. 4 and CDR3 of SEQ ID NO. 5, and a light chain variable region comprising CDR1 of SEQ ID NO. 6, CDR2 of SEQ ID NO. 7 and CDR3 of SEQ ID NO. 8.
6. An anti-CA 125 monoclonal antibody, which is characterized in that the anti-CA 125 monoclonal antibody comprises a heavy chain variable region which comprises an amino acid sequence shown in SEQ ID NO. 1 and a light chain variable region which comprises an amino acid sequence shown in SEQ ID NO. 2.
7. Use of the anti-CA 125 monoclonal antibody of claim 5 or 6 for the preparation of a reagent for detecting CA125 antigen in a cancer patient.
8. Use according to claim 7, characterized in that: the cancer is ovarian cancer.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0288082A2 (en) * | 1987-04-24 | 1988-10-26 | Research Corporation Technologies, Inc. | A tumor specific assay for CA125 ovarian cancer antigen |
| WO1993002358A1 (en) * | 1991-07-25 | 1993-02-04 | Duke University | Method of diagnosing ovarian and endometrial cancer with monoclonal antibodies oxa and oxb |
| CN111537728A (en) * | 2020-07-09 | 2020-08-14 | 广州瑞博奥生物科技有限公司 | Quantitative detection kit for ovarian cancer marker CA125 |
| CN111735949A (en) * | 2020-07-17 | 2020-10-02 | 北京信诺卫康科技有限公司 | Wnt7a and CA125 combined used as early ovarian cancer biomarker and kit |
| CN111735950A (en) * | 2020-07-17 | 2020-10-02 | 北京信诺卫康科技有限公司 | Combination of FGF18 and CA125 as early ovarian cancer biomarker and kit |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5976818A (en) * | 1991-12-16 | 1999-11-02 | The Board Of Trustees Of The University Of Arkansas | Monoclonal antibodies which identify the glycoprotein carrying the CA 125 epitope |
| US9587032B2 (en) * | 2013-06-12 | 2017-03-07 | The Board Of Trustees Of The Leland Stanford Junior University | IgE antibodies for the inhibition of tumor metastasis |
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2021
- 2021-01-23 CN CN202110094481.7A patent/CN112684175B/en active Active
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0288082A2 (en) * | 1987-04-24 | 1988-10-26 | Research Corporation Technologies, Inc. | A tumor specific assay for CA125 ovarian cancer antigen |
| WO1993002358A1 (en) * | 1991-07-25 | 1993-02-04 | Duke University | Method of diagnosing ovarian and endometrial cancer with monoclonal antibodies oxa and oxb |
| CN111537728A (en) * | 2020-07-09 | 2020-08-14 | 广州瑞博奥生物科技有限公司 | Quantitative detection kit for ovarian cancer marker CA125 |
| CN111735949A (en) * | 2020-07-17 | 2020-10-02 | 北京信诺卫康科技有限公司 | Wnt7a and CA125 combined used as early ovarian cancer biomarker and kit |
| CN111735950A (en) * | 2020-07-17 | 2020-10-02 | 北京信诺卫康科技有限公司 | Combination of FGF18 and CA125 as early ovarian cancer biomarker and kit |
Non-Patent Citations (2)
| Title |
|---|
| Development and Characterization of Three Novel Monoclonal Antibodies Against CA-125;Tatiana Michurina et al;《MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY》;20141030;第33卷(第5期);第319-324页 * |
| 卵巢癌相关抗原CA125单克隆抗体的制备;梁勤东等;《中国生物制品学杂志》;20131031;第26卷(第10期);第1458-1462页 * |
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