CN111679076A - Detection kit for detecting cyclinD1 and BCL-2 antibodies - Google Patents
Detection kit for detecting cyclinD1 and BCL-2 antibodies Download PDFInfo
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Abstract
The invention discloses a detection kit for detecting cyclinD1 and BCL-2 antibodies, which comprises: a standard curve comparison table of a solid phase carrier, and cyclinD1 and BCL-2 antibodies, a sample diluent, an enzyme-labeled antibody diluent, a developing solution, a stop solution, a washing solution and a strong positive control substance which are coated on the solid phase carrier.
Description
Technical Field
The invention relates to the field of biological detection, in particular to a detection kit for detecting cyclinD1 and BCL-2 antibodies.
Background
There are many methods for cancer diagnosis, but the most clinically used methods are imaging detection methods, including X-ray, ultrasound, CT, nuclear magnetic, etc., which are used more clinically. However, on the one hand, the above-mentioned detection methods require the cancer growth to proliferate to a certain volume, which can be visually identified to obtain a diagnosis. On the other hand, imaging screening involves the accumulation of radiation doses, which if excessive, are themselves suspect for carcinogenesis. In addition, the cost of CT and nuclear magnetism is high, which increases the economic burden of patients and is not suitable for the general investigation of cancer in various parts of human body, so that the imaging method is not convenient for early cancer screening and diagnosis under the prior art.
Molecular biological detection means (including restriction enzyme fragment length polymorphism, single-strand conformation polymorphism and other technologies, denaturing gradient gel electrophoresis and the like), and pathological detection means (such as immunohistochemistry, cell smear and the like). The methods have simple and convenient technology and low price, and are suitable for laboratory detection of a small amount of samples. However, the detection is time-consuming, labor-consuming, slow, and relatively poor in sensitivity, and is not suitable for the screening and monitoring of early cancer. Some special detection kits are available in China, such as external colorectal cancer tumor detection (tumor 8 item detection and the like), which can find the possibility of early tumor latency, susceptibility and the like, but have the problems of high cost, high price (in 2000-3000 yuan), long period (7-10 days), large workload, complex required instruments and the like. Other tumor detection kits (other tumor detection kits) at home and abroad can discover the possibility of early tumor latency, susceptibility and the like, and also have higher cost and high price (different at 400-. Therefore, a new detection method which can be popularized is urgently needed to screen cancers so as to solve the problem that the cancers cannot be widely and generally detected and are discovered early.
As a cancer early screening and diagnosis kit, the kit has been developed, but mainly aims at a single cancer diagnosis method; for example, patent No. 03821077) "cancer diagnostic reagent and diagnostic cancer kit including anti-GPC 3 antibody" invented by english protein science of japan ltd.p., guhaocheng et al, is a diagnostic reagent and diagnostic cancer kit for cancer, which can diagnose cancer by detecting soluble phosphatidylinositolglycan 3(glypican 3, GPC3) in a test sample, GPC3 is closely related to various tumors including liver cancer, malignant melanoma, ovarian cancer, breast cancer, etc. Studies have shown that it plays a different role in different tumors, and may even play an entirely opposite role. Recent studies have shown that GPC3 is highly expressed in liver cancer, but is expressed in small amounts in melanoma, clear cell carcinoma of the ovary, yolk sac tumor, neuroblastoma, hepatoblastoma, Wilm sarcoma cells, etc., and is silent in breast cancer, mesothelioma, epithelial carcinoma of the ovary, and lung cancer; therefore, whether GPC3 can be used as an important diagnostic tool for other tumors besides liver cancer is still under study.
The invention discloses a cancer diagnosis kit containing anti-HLA-G monoclonal antibody and application thereof with the patent number of CN101358964, which is marginally invented by leaves of Sichuan creative biological science and technology Limited company, and relates to an enzyme-linked immunosorbent assay kit prepared from a novel anti-human leukocyte antigen G (human leukocyte antigen-G, HLA-G) monoclonal antibody HGY-2 and an anti-HLA-G monoclonal antibody HGY. HLA-G is a non-classical major histocompatibility I type molecule, has immunosuppressive function of resisting NK cells, T cells and the like, and HLA-G gene polymorphism and molecular expression have important significance in the occurrence and development of maternal and fetal immunity, infection, autoimmune diseases and tumors. HLA-G protein is abnormally or elevated in the expression of various immune system diseases and malignant tumors. Therefore, it can be used as a reference index for the prognosis of various tumors, but it has nonspecific properties, which affect its preventive and diagnostic effects on tumors. In addition, there are some single cancer diagnosis kits, and the existence of these kits can be used for diagnosis of liver cancer, prostate cancer, breast cancer, etc.
Disclosure of Invention
The invention designs and develops a detection kit for detecting cyclinD1 and BCL-2 antibodies, and aims to carry out combined detection on cyclinD1 and BCL-2 antibodies.
The technical scheme provided by the invention is as follows:
a test kit for detecting cyclinD1 and BCL-2 antibodies, comprising: a standard curve comparison table of a solid phase carrier, and cyclinD1 and BCL-2 antibodies, a sample diluent, an enzyme-labeled antibody diluent, a developing solution, a stop solution, a washing solution and a strong positive control substance which are coated on the solid phase carrier.
Preferably, the solid phase carrier is a 96-hole enzyme label plate.
Preferably, the enzyme-labeled antibody is HRP goat anti-human IgG.
Preferably, the sample dilutions contain 1% BSA in PBST buffer.
Preferably, the color developing solution is 0.02% of tetramethylbenzidine.
Preferably, the stop solution is 2M concentrated sulfuric acid.
Preferably, the washing solution is 0.01M PBST buffer containing 0.05% Tween-20, pH 7.4.
Preferably, the preparation process of the standard curve comparison table of the strong positive control substance is as follows:
and 4, selecting 1 part of anti-cyclinD 1 and BCL-2 antibody strong positive in-vitro serum, diluting the antibody coated cyclinD1 and BCL-2 antibody to the optimal concentration, respectively diluting the strong positive serum to make a semilogarithmic curve, taking the diluted serum in the linear segment of the semilogarithmic curve as a standard product of the kit, determining 6 parts of standard product antigen concentration, and drawing a standard curve according to the antigen concentration.
Preferably, in said step 1, cyclinD1 and BCL-2 antibody are diluted to a concentration of 5. mu.g/ml;
in the step 2, the concentration of both the cyclinD1 standard substance and the BCL-2 standard substance is selected to be 0.15 to 10 mu g/ml; and
in said step 4, the antigen concentrations of 6 standard samples were determined to be 1.6. mu.g/ml, 2.4. mu.g/ml, 3.2. mu.g/ml, 4. mu.g/ml, 4.8. mu.g/ml, 5.6. mu.g/ml, respectively.
Preferably, in said step 2,
the standard curve of the cyclinD1 standard substance is that y is-0.0187 x2+0.4497x+0.1812；
The standard curve of the BCL-2 standard substance is that y is-0.0175 x2+0.4118x+0.1702。
Compared with the prior art, the invention has the following beneficial effects:
1. according to the invention, the cyclin D1 and the anti-apoptosis protein BCL-2 are selected and regulated to be combined to carry out ELISA detection screening kit, and the ELISA kit has unique sensitivity and specificity;
2. the application range of the kit is defined by establishing the optimal working concentration of the antigen and the optimal concentration of the monoclonal antibody.
3. The mechanism of malignant tumor is mainly that the cell hyperproliferation or the cell can not die to trigger the overgrowth of the tumor cell, and the cyclin D1 and the anti-die protein BCL-2 play an important role in tumorigenesis. cyclin D1 can promote the cell cycle from G1 to S to promote the proliferation and division of tumor cells, and can promote the function of gene transcription, so that it is known as a protooncogene, and its over-expression can make cell proliferation be out of control and malignant. BCL-2 is closely related to the survival and death of cells and is considered as an important target point for tumor treatment. Therefore, the designed cyclinD1 and BCL-2 combined tumor screening kit can be used for routine screening of outpatient tumor and screening of outpatient rapid tumor blood examination. The two combined detections start from the pathogenesis of the tumor, comprise the detections of proliferation and apoptosis, can mutually make up the deficiency, screen the tumor from the maximum possibility, and are beneficial to the detection of tumor patients.
Drawings
FIG. 1 is a schematic diagram of the standard curves of the cyclinD1 standard and the BCL-2 standard according to the present invention.
FIG. 2 is a schematic diagram of a standard curve according to the antigen concentration according to the present invention.
Detailed Description
The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.
Examples
cyclin D1 antibody (available from Abcam), BCL-2 antibody (available from Santa Cruz Biotechnology), coating solution (pH9.6, 0.05M carbonate buffer), washing buffer (pH7.4, 0.15M PBS), diluent (1% BSA), stop buffer (2M, H, Biotechnology, Inc.), and the like2SO4), substrate buffer (ph5.0, phosphate-citrate buffer), tetramethylbenzidine working solution; a 96-hole enzyme label plate; PLUS 384 full-automatic enzyme marker (MDC, USA).
The experimental process of the preparation method of the high-throughput ELISA kit comprises the following steps:
step one, diluting cyclinD1 and BCL-2 antibody with carbonate buffer solution to the concentration of 5 μ g/ml, adding enzyme labeling plate, each plate 100 μ l, coating overnight at 4 ℃. After sealing, washing the plate for three times, drying in vacuum for 1h, packaging and storing at 4 ℃ for later use;
step two, using PBST + 1% BSA to treat samples to be detected of cyclinD1 and BCL-2 according to the ratio of 1: diluting by 50, respectively adding 100 mu l of diluted sample, cyclinD1 standard substance and BCL-2 standard substance into the enzyme label plate, and standing for 1h at room temperature; adding 1: 10000-diluted HRP sheep anti-human IgG, standing at room temperature for 30min, washing the plate, adding a color development solution, and stopping reaction after color development; detecting an A450 value by a full-automatic enzyme marker, drawing a standard curve by a cyclinD1 standard substance and a BCL-2 standard substance, and calculating the concentrations of a to-be-detected sample cyclinD1 and BCL-2 according to the standard curve; wherein, the concentration of the selected cyclinD1 standard substance and the concentration of the selected BCL-2 standard substance are both 0.15 mu g/ml to 10 mu g/ml; drawing a standard curve as shown in figure 1;
the standard curve of the cyclinD1 standard substance is that y is-0.0187 x2+0.4497x+0.1812；
The standard curve of the BCL-2 standard substance is that y is-0.0175 x2+0.4118x+0.1702；
Step three, diluting the coated samples to be detected by coating buffer solutions in a ratio of 1:500, 1:1000, 1:2000, 1:3000, 1:4000, 1:5000 and 1:6000, adding a row for each dilution, coating each hole with 100 mu l, coating overnight at 4 ℃, and coating the coated cyclinD1 antibody and the coated BCL-2 antibody in a ratio of 1: 500. diluting at a ratio of 1:1000, 1:2000 and 1:3000, adding one vertical row for each dilution, measuring each hole by an ELISA matrix method, determining that the optimal working concentration of a sample to be tested is 1:500 dilution, and the optimal concentration of monoclonal cyclin D1 antibody and monoclonal BCL-2 antibody is 1:2000 dilution;
step four, preparing a standard substance: selecting 1 part of anti-cyclinD 1 and BCL-2 antibody strong positive serum, diluting and coating cyclinD1 and BCL-2 antibody at a ratio of 1:2000, respectively diluting the strong positive serum to make a semilog curve, taking the diluted serum in the linear segment of the semilog curve as a standard product of the kit, and determining the antigen concentrations of 6 parts of the standard product to be 1.6 mu g/ml, 2.4 mu g/ml, 3.2 mu g/ml, 4 mu g/ml, 4.8 mu g/ml and 5.6 mu g/ml by combining the clinical data of the strong positive serum;
standard curves were drawn according to antigen concentration: the absorbance of each of the 6 standard samples is taken as the Y-axis and the natural logarithm of the concentration of each standard sample is taken as the X-axis, and the standard curve is plotted as shown in fig. 2.
The standard curve is y-0.533 x + 0.2605;
step five, determining the composition of the high-flux ELISA kit:
a 96-well ELISA plate coated by cyclinD1 and BCL-2 antibody;
sample diluent: PBST buffer containing 1% (W/V) BSA;
enzyme-labeled antibody: HRP goat anti-human IgG;
color development liquid: 0.02% (W/V) tetramethylbenzidine;
stopping liquid: 2M concentrated sulfuric acid;
washing liquid: 0.01M PBST (phosphate Tween) buffer pH7.4 containing 0.05% Tween-20;
and (5) a standard curve comparison table of a strong positive control substance.
Test examples
1. Quality evaluation of kit
(1) Precision detection
Selecting high, medium and low 3 parts of concentration quality control serum, repeatedly measuring each part for 5 times in the same 1 test, respectively calculating the measurement result, the mean value and the standard deviation thereof according to the standard curve, calculating the variation coefficient CV (%) in the test, measuring for 1 time every 1 day, continuously measuring for 5 times, and calculating the CV (%) value, wherein the measurement result is shown in Table 1.
TABLE 1 results of the precision measurements
(2) Readiness testing
Selecting 2 parts of serum with different antibody concentrations, mixing the serum samples with different concentrations in different proportions to calculate concentration theoretical values according to clinical data, detecting by using a kit to obtain concentration calculated values, and calculating the consistency between the detected values and the theoretical values according to the following formula, namely the recovery rate, wherein the measurement results are shown in table 2;
recovery (%) — calculated concentration/theoretical concentration × 100%;
TABLE 2 results of readiness testing
(3) Stability detection
Selecting 5 parts of anti-cyclinD 1 and BCL-2 antibody positive serum and 3 parts of anti-cyclinD 1 and BCL-2 antibody negative serum, detecting by using the same 1 batch of kit stored at 4 ℃ for 0 and 6 months, comparing the concentration change of cyclinD1 and BCL-2 antibody of each serum, and adopting t test for data statistics, wherein the test result is shown in Table 3;
TABLE 3 stability test results
(4) Utility testing
Selecting 15 serum samples collected clinically, wherein 3 lung cancer patients, 3 stomach cancer patients, 3 colorectal cancer, 3 liver cancer and 3 serum of healthy examiners are diagnosed, and the detection results are shown in table 4;
TABLE 4 results of practical tests
The tests show that the detection by the kit for detecting the cylind 1 and the BCL-2 antibody can well perform stable, efficient and accurate detection on clinically known cancers.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.
Claims (10)
1. A test kit for detecting cyclinD1 and BCL-2 antibodies, comprising: a standard curve comparison table of a solid phase carrier, and cyclinD1 and BCL-2 antibodies, a sample diluent, an enzyme-labeled antibody diluent, a developing solution, a stop solution, a washing solution and a strong positive control substance which are coated on the solid phase carrier.
2. The detection kit for detecting cyclinD1 and BCL-2 antibody according to claim 1, wherein the solid phase carrier is a 96-well enzyme label plate.
3. The detection kit for detecting cyclinD1 and BCL-2 antibody according to claim 2, wherein the enzyme labeled antibody is HRP goat anti-human IgG.
4. The test kit for detecting cyclinD1 and BCL-2 antibodies according to claim 3, wherein the sample diluent contains 1% BSA in PBST buffer.
5. The detection kit for detecting cyclinD1 and BCL-2 antibody according to claim 4, wherein the developing solution is 0.02% tetramethyl benzidine.
6. The detection kit for detecting cyclinD1 and BCL-2 antibody according to claim 5, wherein the stop solution is 2M concentrated sulfuric acid.
7. The test kit for detecting cyclinD1 and BCL-2 antibody according to claim 6, wherein the washing solution is 0.01M PBST buffer solution with 0.05% Tween-20 and pH 7.4.
8. The test kit for detecting cyclinD1 and BCL-2 antibody according to any one of claims 1-7, wherein the standard curve control table of the strong positive control is prepared by the following steps:
step 1, diluting cyclinD1 and BCL-2 antibody with carbonate buffer solution to the concentration of 5 mug/ml, adding an enzyme label plate, each plate is 100 mug, and coating is carried out overnight at 4 ℃;
step 2, diluting samples to be detected of cyclinD1 and BCL-2, respectively adding the diluted samples, cyclinD1 standard substance and 100 mu l of BCL-2 standard substance into the enzyme label plate, and standing for 1h at room temperature; adding diluted HRP goat anti-human IgG after washing the plate, standing at room temperature for 30min, adding a color development solution after washing the plate, and terminating the reaction after color development; detecting an A450 value by a full-automatic enzyme marker, drawing a standard curve by a cyclinD1 standard substance and a BCL-2 standard substance, and calculating the concentrations of a to-be-detected sample cyclinD1 and BCL-2 according to the standard curve;
step 3, diluting the coated samples to be detected in different proportions by using coating buffer solutions, adding a row for each dilution degree, coating 100 mu l of each hole at 4 ℃ overnight, diluting the coated cyclin D1 antibody and the coated BCL-2 antibody in the same proportion, adding a vertical row for each dilution degree and 100 mu l of each hole, performing ELISA matrix method determination, and determining the optimal working concentration of the coated samples to be detected, and the optimal concentrations of the monoclonal cyclin D1 antibody and the monoclonal BCL-2 antibody;
and 4, selecting 1 part of anti-cyclinD 1 and BCL-2 antibody strong positive in-vitro serum, diluting the antibody coated cyclinD1 and BCL-2 antibody to the optimal concentration, respectively diluting the strong positive serum to make a semilogarithmic curve, taking the diluted serum in the linear segment of the semilogarithmic curve as a standard product of the kit, determining 6 parts of standard product antigen concentration, and drawing a standard curve according to the antigen concentration.
9. The detection kit for detecting cyclinD1 and BCL-2 antibody according to claim 8, wherein in step 1, cyclinD1 and BCL-2 antibody are diluted to a concentration of 5 μ g/ml;
in the step 2, the concentration of both the cyclinD1 standard substance and the BCL-2 standard substance is selected to be 0.15 to 10 mu g/ml; and
in said step 4, the antigen concentrations of 6 standard samples were determined to be 1.6. mu.g/ml, 2.4. mu.g/ml, 3.2. mu.g/ml, 4. mu.g/ml, 4.8. mu.g/ml, 5.6. mu.g/ml, respectively.
10. The detection kit for detecting cyclinD1 and BCL-2 antibody according to claim 9, wherein, in step 2,
the standard curve of the cyclinD1 standard substance is that y is-0.0187 x2+0.4497x+0.1812；
The standard curve of the BCL-2 standard substance is that y is-0.0175 x2+0.4118x+0.1702。
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Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060275844A1 (en) * | 2005-04-19 | 2006-12-07 | Linke Steven P | Diagnostic markers of breast cancer treatment and progression and methods of use thereof |
| WO2008077165A1 (en) * | 2006-12-22 | 2008-07-03 | Austrian Research Centers Gmbh - Arc | Set of tumor markers |
| CN101384903A (en) * | 2006-02-09 | 2009-03-11 | 南佛罗里达大学 | Detection of cancer by elevated levels of bcl-2 |
| CN101477127A (en) * | 2008-11-03 | 2009-07-08 | 山东省医药生物技术研究中心 | Human brain natriuretic non-competitive double-antibody sandwich method ELISA detection reagent kit |
| CN106053821A (en) * | 2016-08-04 | 2016-10-26 | 吉林医药学院 | Magnetic bead immunodetection kit for human colorectal cancer tumor stem cell markers CD44 and CD133 |
| CN107223163A (en) * | 2014-12-24 | 2017-09-29 | 豪夫迈·罗氏有限公司 | For the treatment of bladder cancer, diagnosis and method of prognosis |
| CN110198711A (en) * | 2016-11-22 | 2019-09-03 | 第一基因股份有限公司 | Method for detecting cancer |
| CN110716041A (en) * | 2019-10-23 | 2020-01-21 | 郑州大学 | Serum protein marker, kit and detection method for early screening and diagnosis of gastric cancer |
| CN111077312A (en) * | 2020-02-28 | 2020-04-28 | 郑州大学第一附属医院 | Application of group of tumor-associated antigens in preparation of cardiac cancer early screening kit |
-
2020
- 2020-06-15 CN CN202010540409.8A patent/CN111679076A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060275844A1 (en) * | 2005-04-19 | 2006-12-07 | Linke Steven P | Diagnostic markers of breast cancer treatment and progression and methods of use thereof |
| CN101384903A (en) * | 2006-02-09 | 2009-03-11 | 南佛罗里达大学 | Detection of cancer by elevated levels of bcl-2 |
| WO2008077165A1 (en) * | 2006-12-22 | 2008-07-03 | Austrian Research Centers Gmbh - Arc | Set of tumor markers |
| CN101477127A (en) * | 2008-11-03 | 2009-07-08 | 山东省医药生物技术研究中心 | Human brain natriuretic non-competitive double-antibody sandwich method ELISA detection reagent kit |
| CN107223163A (en) * | 2014-12-24 | 2017-09-29 | 豪夫迈·罗氏有限公司 | For the treatment of bladder cancer, diagnosis and method of prognosis |
| CN106053821A (en) * | 2016-08-04 | 2016-10-26 | 吉林医药学院 | Magnetic bead immunodetection kit for human colorectal cancer tumor stem cell markers CD44 and CD133 |
| CN110198711A (en) * | 2016-11-22 | 2019-09-03 | 第一基因股份有限公司 | Method for detecting cancer |
| CN110716041A (en) * | 2019-10-23 | 2020-01-21 | 郑州大学 | Serum protein marker, kit and detection method for early screening and diagnosis of gastric cancer |
| CN111077312A (en) * | 2020-02-28 | 2020-04-28 | 郑州大学第一附属医院 | Application of group of tumor-associated antigens in preparation of cardiac cancer early screening kit |
Non-Patent Citations (2)
| Title |
|---|
| 孙晓杰 等: "肿瘤分子诊断与靶向治疗", 第二军医大学出版社 * |
| 魏国兰等: "抗环瓜氨酸肽抗体定量检测ELISA试剂盒的制备及其诊断类风湿性关节炎的意义", 《中国生物制品学杂志》 * |
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