CN112180090A - Kit of monoclonal antibody specifically bound by immune cell p16 - Google Patents
Kit of monoclonal antibody specifically bound by immune cell p16 Download PDFInfo
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- CN112180090A CN112180090A CN202011074981.6A CN202011074981A CN112180090A CN 112180090 A CN112180090 A CN 112180090A CN 202011074981 A CN202011074981 A CN 202011074981A CN 112180090 A CN112180090 A CN 112180090A
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- monoclonal antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57411—Specifically defined cancers of cervix
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
Abstract
The invention provides a kit of a monoclonal antibody specifically bound by an immune cell p16, which relates to the technical field of kits, and comprises the following reagents: 1 ml-1.5 ml of immune cell p16 monoclonal antibody, 0.3 ml-0.5 ml of separation solution, 5 ml-8 ml of diluent, 20 ml-60 ml of PSB working solution, 20 ml-50 ml of 3% H2O2Deionized water, 0.2ml to 0.5ml of neutral resin, 0.2ml to 0.5ml of developing solution A, 0.2ml to 0.5ml of developing solution B, 2ml to 6ml of confining solution and 2ml to 5ml of dimethylbenzene. In the invention, samples are dispersed and filtered by a cell detector, a hybridization capture method is adopted for detection, p16 antibody is used for coating micropores in a glass slide during detection to prepare sample antibody, hematoxylin and bluing solution are used for staining, after thorough washing, a substrate is developed, 5 or more abnormal cells in a smear are stained brown, the reading result is positive, and on the contrary, the number of the stained brown cells is less than 5, even more, the stained cells are judged to be positiveAnd when no coloring cells exist, the interpretation result is negative, the color development effect is good, the marking error is not easy to occur, the operation is convenient, and the method is suitable for screening.
Description
Technical Field
The invention relates to the technical field of kits, in particular to a kit of a monoclonal antibody specifically bound by immune cell p 16.
Background
Cervical cancer is one of the most common female reproductive system malignant tumors in gynecology, the incidence rate is second to breast cancer, and the cervical cancer is the second place of the female malignant tumors, thus seriously threatening the physical health and the life quality of women. China is a high-incidence country of cervical cancer, and the incidence and mortality rate account for 1/3 worldwide. The cervical cancer is the only cancer with definite etiology at present, the high-incidence age of carcinoma in situ is 30-35 years old, the invasive cancer is 45-55 years old, the incidence of the carcinoma tends to be younger in recent years, and the carcinoma can be completely cured through effective early diagnosis and treatment.
Abnormal expression of protein molecules, such as L1 capsid protein, P16 protein and the like, may occur in the process of cervical precancerous lesion or in the body of a cervical cancer patient, these marker proteins can be applied to cervical precancerous screening, P16 protein is a coding product of P16 gene, the degree of cervical precancerous lesion can be predicted, and in immunohistochemistry reaction, an antibody can recognize an antigenic determinant and then is specifically combined with the antigenic determinant. At present, when the kit on the market is used, the color development effect is poor, the labeling error is easy to occur, the operation is inconvenient, the kit is not suitable for screening, and the requirements of medical care personnel cannot be met.
Disclosure of Invention
The invention aims to provide a kit of a monoclonal antibody specifically bound by immune cell p16, which has the advantages of good color development effect, difficulty in occurrence of marking errors, convenience in operation and suitability for screening.
In order to achieve the purpose, the invention is realized by the following technical scheme: a kit for monoclonal antibodies specifically bound by immune cells p16, the kit comprising the following reagents:
1 ml-1.5 ml of immune cell p16 monoclonal antibody;
0.3 ml-0.5 ml of separation liquid;
5ml to 8ml of diluent;
20 ml-60 ml of PSB working solution;
20ml to 50ml of 3% H2O2Deionized water;
0.2ml to 0.5ml of neutral resin;
0.2ml to 0.5ml of color developing solution A;
0.2ml to 0.5ml of color development liquid B;
2 ml-6 ml of confining liquid;
2ml to 5ml of xylene.
As a further scheme of the invention: the color development liquid A is hematoxylin.
As a further scheme of the invention: the color development liquid B is a bluing liquid.
As a further scheme of the invention: the immune cell p16 monoclonal antibody, the separation solution, the diluent, the PSB working solution, 3% H2O2 deionized water, neutral resin, the developing solution A, the developing solution B, the confining solution and xylene are all stored in an environment with the temperature condition of 2-10 ℃.
A method of using a kit of monoclonal antibodies specifically bound by immune cell p16, comprising the steps of:
s1, placing the immune cell p16 monoclonal antibody in a shaker, and shaking.
S2, adding the separating medium into the numbered test tubes, and pouring the shaken immune cell p16 monoclonal antibody into the numbered test tubes.
S3, placing the prepared test tube into a centrifuge, centrifuging, pouring out the supernatant, and inverting the test tube on absorbent paper.
And S4, adding the mixture into the tube which is centrifuged at the upper part, putting the tube into the centrifuge again for continuous centrifugation, taking out the tube and pouring out the supernatant.
S5, adding a proper amount of diluent, and shaking the test tube.
S6, sucking out the sample by a pipette gun, directly hitting the sample on a 10-hole slide, and standing for 10 min.
And S7, placing the glass slide into an oven at 50 ℃ for 10min, and cooling the glass slide chamber for 2min after baking.
S8, staining the slide.
And S9, dropping a proper amount of neutral resin glue on the glass slide, covering the glass slide with a cover glass and discharging air bubbles.
And S10, dispersing and filtering the sample by using a cell detector, detecting by adopting a hybridization capture method, and comparing the detection result with a clear standard cell to determine the detection result.
As a further scheme of the invention: according to the operation step in S1, the rotation speed of the vibrator is 2000r/min, and the oscillation time is 15 min.
As a further scheme of the invention: the centrifuge is rotated at 800rLmin for 3min according to the operation in S3.
As a further scheme of the invention: excess sample on the slide will be sucked back with a pipette gun after resting for 10min according to the procedure in S6.
As a further scheme of the invention: characterized by comprising the following steps in staining a slide according to the procedure in S8:
s801, adding deionized water for incubation for 13min, washing with PSB working solution for 3 times, and then adding a sealing solution for incubation for 15 min;
s802, adding the color developing solution A, incubating for 3min, washing with distilled water for 2 times, adding the color developing solution B, incubating for 3min, and washing with distilled water for 2 times.
As a further scheme of the invention: according to the operation in S8, when the slide is stained, the method further comprises placing the stained slide in xylene to be substantially transparent.
The invention provides a kit of monoclonal antibodies specifically bound by immune cells p 16. The method has the following beneficial effects:
in the invention, samples are dispersed and filtered by a cell detector, a hybridization capture method is adopted for detection, p16 antibody is used for coating micropores in a glass slide to prepare sample antibody during detection, the antibody can identify antigenic determinant and then is specifically combined with the antigenic determinant, hematoxylin and bluing liquid are used for dyeing, after thorough washing, a substrate is developed, 5 or more abnormal cells in a smear are developed to be brown (no matter cytoplasm or nucleus), the judgment result is positive, on the contrary, the number of the brown cells is less than 5, even no colored cells exist, the judgment result is negative, the development effect is good, the labeling error is not easy to occur, the operation is convenient, and the method is suitable for screening.
Detailed Description
The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.
Example 1:
a kit of monoclonal antibodies specifically bound by immune cell p16, comprising the following reagents:
1 ml-1.5 ml of immune cell p16 monoclonal antibody;
0.3 ml-0.5 ml of separation liquid;
5ml to 8ml of diluent;
20 ml-60 ml of PSB working solution;
20ml to 50ml of 3% H2O2Deionized water;
0.2ml to 0.5ml of neutral resin;
0.2ml to 0.5ml of color developing solution A;
0.2ml to 0.5ml of color development liquid B;
2 ml-6 ml of confining liquid;
2ml to 5ml of xylene.
Specifically, the developing solution A is hematoxylin.
Specifically, the color developing solution B is a bluing solution.
Specifically, the immune cell p16 monoclonal antibody, separation liquid, diluent, PSB working solution, 3% H2O2 deionized water, neutral resin, developing solution A, developing solution B, confining solution and xylene are all stored in an environment with the temperature condition of 2-10 ℃.
A method of using a kit of monoclonal antibodies specifically bound by immune cell p16, comprising the steps of:
(1) and placing the immune cell p16 monoclonal antibody in a shaker for shaking treatment.
(2) And adding the separating medium into the numbered test tubes, and pouring the shaken immune cell p16 monoclonal antibody into the numbered test tubes.
(3) And putting the prepared test tube into a centrifugal machine, carrying out centrifugal treatment, pouring out supernatant, and inverting the test tube on absorbent paper.
(4) Adding the mixture into a test tube with a centrifuged upper part, putting the test tube into the centrifuge again for continuous centrifugation, taking out the test tube and pouring out supernatant.
(5) Add the appropriate amount of diluent and shake the tube.
(6) The sample is sucked out by a pipette gun and directly sprayed on a 10-hole slide, and the slide is kept still for 10 min.
(7) And putting the glass slide into an oven at 50 ℃ for 10min, and cooling the glass slide chamber for 2min after baking.
(8) And staining the glass slide.
(9) And dropping a proper amount of neutral resin glue on the glass slide, covering the glass slide and discharging air bubbles.
(10) Dispersing and filtering the sample by using a cell detector, detecting by adopting a hybridization capture method, and comparing the detection result with a specific standard cell to determine the detection result.
Specifically, according to the operation steps in (1), the rotating speed of the vibrator is 2000r/min, and the oscillation time is 15 min.
Specifically, according to the operation steps in (3), the rotating speed of the centrifuge is 800rLmin, and the centrifugation time is 3 min.
Specifically, according to the procedure in (6), after standing for 10min, the excess sample on the slide is sucked back by a pipette gun.
Specifically, according to the operation procedure in (8), the method comprises the following steps in staining a slide:
(801) adding deionized water for incubation for 13min, then washing with PSB working solution for 3 times, and then adding a sealing solution for incubation for 15 min;
(802) adding color developing solution A, incubating for 3min, washing with distilled water for 2 times, adding color developing solution B, incubating for 3min, and washing with distilled water for 2 times.
Specifically, according to the operation procedure in (8), when the slide glass is stained, the stained slide glass is placed in xylene to be sufficiently transparent.
In order to further illustrate the beneficial effects of the invention, the inventor selects two kits, selects a kit named as a chemical marker color development kit, and briefly describes the first kit below.
Also selected is the so-called fluorescence kit, briefly described below as the second kit.
Meanwhile, the kit prepared by the invention is selected, and the color development effect, the labeling condition and the use time of the monoclonal antibody specifically bound to the immune cell p16 are compared, so that the following data are obtained, and the details are shown in table 1:
TABLE 1 Experimental data sheet
| Color development effect | Marking error conditions | Time of use | |
| First kit | Is not obvious | With errors | 1h |
| Second kit | Is not obvious | With errors | 1h |
| Example 1 | Obvious color development | Without error | 1h |
The experimental data show that the confining liquid for staining the immune cells p16 prepared in the embodiment 1 has the advantages of good color development effect, difficulty in occurrence of marking errors, convenience in operation and suitability for screening and screening.
A kit of monoclonal antibodies specifically bound by immune cell p16, comprising the following reagents: 1 ml-1.5 ml of immune cell p16 monoclonal antibody, 0.3 ml-0.5 ml of separation solution, 5 ml-8 ml of diluent, 20 ml-60 ml of PSB working solution, 20 ml-50 ml of 3% H2O2Deionized water, 0.2ml to 0.5ml of neutral resin, 0.2ml to 0.5ml of developing solution A, 0.2ml to 0.5ml of developing solution, 2ml to 6ml of confining solution and 2ml to 5ml of dimethylbenzene.
Specifically, the developing solution A is hematoxylin, which is a basic dye extracted from folium lignum sappan, and can be used together with mordant to generate hematoxylin after oxidation.
Specifically, the color developing solution B is a bluing solution, and pH is sufficiently controlled to provide a very efficient bluing effect.
Specifically, the immune cell p16 monoclonal antibody, separation liquid, diluent, PSB working solution, 3% H2O2 deionized water, neutral resin, developing liquid A, developing liquid B, confining liquid and xylene are all stored in an environment with the temperature condition of 2-10 ℃, and the effect is achieved by avoiding overhigh or overlow temperature.
A method of using a kit of monoclonal antibodies specifically bound by immune cell p16, comprising the steps of: (1) placing an immune cell p16 monoclonal antibody into a shaker for shaking treatment, wherein the rotation speed of the shaker is 2000r/min, the shaking time is 15min, (2) adding a separation liquid into a numbered test tube, pouring the shaken immune cell p16 monoclonal antibody into the numbered test tube, (3) placing the prepared test tube into a centrifuge, wherein the rotation speed of the centrifuge is 800rLmin, the centrifuging time is 3min, carrying out centrifuging treatment, pouring supernatant, inverting the test tube to absorbent paper, (4) adding the test tube into the test tube which is centrifuged at the upper part, placing the test tube into the centrifuge again for continuing centrifuging, then taking out the test tube, pouring the supernatant, (5) adding a proper amount of diluent, shaking the test tube, (6) sucking out a sample by using an absorbent gun, directly hitting the sample onto a 10-hole slide, standing for 10min, sucking back the redundant sample on the slide by using the absorbent gun after standing for 10min, (7) placing the glass slide into an oven at 50 ℃ for 10min, cooling the glass slide chamber for 2min after baking, (8) dyeing the glass slide, wherein the dyeing process of the glass slide comprises the following steps: (801) adding deionized water for incubation for 13min, then washing with PSB working solution for 3 times, then adding sealing solution for incubation for 15min, (802), adding color development solution A for incubation for 3min, washing with distilled water for 2 times, adding color development solution B for incubation for 3min, washing with distilled water for 2 times, when dyeing the slide, also including putting the dyed slide into dimethylbenzene which is a colorless transparent solvent, (9), dropping a proper amount of neutral resin glue on the slide, covering the slide and discharging air bubbles, which is a bonding agent, the neutral resin glue drops are used for bonding the slide and the cover glass together, sealing the biological tissue slice for long time and keeping, (10), dispersing the sample by using a cell detector and filtering, detecting by adopting a hybridization capture method, comparing the detection result with a clear standard cell, determining the detection result, and the existing 5 or more abnormal cells in the smear are colored brown (no matter of cytoplasm or nucleus), the interpretation result is positive, on the contrary, the number of cells stained brown is less than 5, even no stained cells, the interpretation result is negative, and the abnormal cells have the following characteristics: the cell nucleus occupies more than half of the cell volume, the chromatin is not distributed uniformly, the staining of the cell nucleus is deeper, the shape of the cell nucleus is irregular and has depression or protrusion, and the arrangement shape of the cell is changeable.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various changes and modifications can be made without departing from the inventive concept of the present invention, and these changes and modifications are all within the scope of the present invention.
Claims (10)
1. A kit for monoclonal antibodies specifically bound by immune cells p16, comprising the following reagents:
1 ml-1.5 ml of immune cell p16 monoclonal antibody;
0.3 ml-0.5 ml of separation liquid;
5ml to 8ml of diluent;
20 ml-60 ml of PSB working solution;
20ml to 50ml of 3% H2O2Deionized water;
0.2ml to 0.5ml of neutral resin;
0.2ml to 0.5ml of color developing solution A;
0.2ml to 0.5ml of color development liquid B;
2 ml-6 ml of confining liquid;
2ml to 5ml of xylene.
2. The kit for the monoclonal antibody specifically bound by the immune cell p16, according to claim 1, wherein the developing solution A is hematoxylin.
3. The kit for the monoclonal antibody specifically bound by the immune cell p16, according to claim 1, wherein the developing solution B is a bluing solution.
4. The kit for the monoclonal antibody specifically bound by the immune cell p16, according to claim 3, wherein the immune cell p16 monoclonal antibody, separation solution, dilution solution, PSB working solution, 3% H2O2Deionized water, neutral resin, developing solution A, developing solution B, confining solution and xylene are all stored in an environment with the temperature condition of 2-10 ℃.
5. The use method of the kit of monoclonal antibodies specifically bound by the immune cells p16 is characterized by comprising the following steps:
s1, placing the immune cell p16 monoclonal antibody in a shaker, and shaking;
s2, adding a separation solution into the numbered test tubes, and pouring the vibrated immune cell p16 monoclonal antibody into the numbered test tubes;
s3, placing the prepared test tube into a centrifuge, centrifuging, pouring out supernatant, and inverting the test tube on absorbent paper;
s4, adding the mixture into the test tube with the centrifuged upper part, putting the test tube into the centrifuge again for continuous centrifugation, taking out the test tube and pouring out the supernatant;
s5, adding a proper amount of diluent, and shaking the test tube;
s6, sucking out the sample by a liquid suction gun, directly hitting the sample on a 10-hole slide, and standing for 10 min;
s7, placing the glass slide into an oven at 50 ℃ for 10min, and cooling the glass slide chamber for 2min after baking;
s8, staining the slide;
s9, dropping a proper amount of neutral resin glue on the glass slide, covering the glass slide with a cover glass and discharging air bubbles;
and S10, dispersing and filtering the sample by using a cell detector, detecting by adopting a hybridization capture method, and comparing the detection result with a clear standard cell to determine the detection result.
6. The method for preparing a kit of monoclonal antibodies specifically binding to p16 of claim 5, wherein the rotation speed of the vibrator is 2000r/min and the oscillation time is 15min according to the procedure of S1.
7. The method for preparing a monoclonal antibody kit specifically bound by immune cells p16 according to claim 5, wherein the centrifuge is rotated at 800rLmin for 3min according to the procedure of S3.
8. The method for preparing a kit for monoclonal antibodies specifically binding to p16 of claim 5, wherein excess sample on the slide is sucked back by a pipette gun after resting for 10min according to the procedure in S6.
9. The method for preparing a kit of monoclonal antibodies specifically binding to p16 of an immune cell according to claim 5, wherein the method comprises the following steps when staining the slide according to the procedures in S8:
s801, adding deionized water for incubation for 13min, washing with PSB working solution for 3 times, and then adding a sealing solution for incubation for 15 min;
s802, adding the color developing solution A, incubating for 3min, washing with distilled water for 2 times, adding the color developing solution B, incubating for 3min, and washing with distilled water for 2 times.
10. The method for preparing a kit for monoclonal antibodies specifically binding to p16 of claim 5, wherein the method further comprises placing the stained slide in xylene to be substantially transparent when the slide is stained according to the procedure of S8.
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| CN202011074981.6A CN112180090A (en) | 2020-10-09 | 2020-10-09 | Kit of monoclonal antibody specifically bound by immune cell p16 |
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| CN202011074981.6A CN112180090A (en) | 2020-10-09 | 2020-10-09 | Kit of monoclonal antibody specifically bound by immune cell p16 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107202888A (en) * | 2017-07-11 | 2017-09-26 | 广州江元医疗科技有限公司 | A kind of p16 for cervical liquid-based cellsINK4aImmune labeled colour reagent box |
| CN109856383A (en) * | 2019-03-05 | 2019-06-07 | 湖北泰康医疗设备有限公司 | A kind of immunochemistry staining kit for cervical carcinoma auxiliary diagnosis |
| CN110501496A (en) * | 2019-08-28 | 2019-11-26 | 深圳市森盈生物科技有限公司 | A kind of immunocyte p16 detection kit |
| CN110501498A (en) * | 2019-08-28 | 2019-11-26 | 深圳市森盈生物科技有限公司 | A kind of p16 immunologic combined detection reagent kit |
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2020
- 2020-10-09 CN CN202011074981.6A patent/CN112180090A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107202888A (en) * | 2017-07-11 | 2017-09-26 | 广州江元医疗科技有限公司 | A kind of p16 for cervical liquid-based cellsINK4aImmune labeled colour reagent box |
| CN109856383A (en) * | 2019-03-05 | 2019-06-07 | 湖北泰康医疗设备有限公司 | A kind of immunochemistry staining kit for cervical carcinoma auxiliary diagnosis |
| CN110501496A (en) * | 2019-08-28 | 2019-11-26 | 深圳市森盈生物科技有限公司 | A kind of immunocyte p16 detection kit |
| CN110501498A (en) * | 2019-08-28 | 2019-11-26 | 深圳市森盈生物科技有限公司 | A kind of p16 immunologic combined detection reagent kit |
Non-Patent Citations (1)
| Title |
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| 胡艳: "P16/Ki67蛋白检测（cintec plus双染技术）在宮颈上皮内瘤变的诊断及宮颈癌筛查中应用价值探讨", 《中国优秀硕士学位论文全文数据库》 * |
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Application publication date: 20210105 |