CN113075404A - Immunohistochemical kit for tumor diagnosis - Google Patents
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- G—PHYSICS
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- 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
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- 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
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
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Abstract
The invention provides an immunohistochemical kit for tumor diagnosis, which comprises a DNP-labeled monoclonal antibody or polyclonal antibody group, a PLys-HBr-labeled secondary antibody, an antigen retrieval solution and a phosphate buffer solution. The compound with the Raman spectrum characteristic is marked on the second antibody, so that the SERS quantitative analysis of the tumor tissue antigen is realized, and DNP is marked on the monoclonal antibody or the polyclonal antibody group, so that the DNP and the compound with the Raman spectrum characteristic marked by the second antibody are used as quality control, interference factors are removed, the detection result specificity is higher, and the application prospect is very high.
Description
Technical Field
The invention relates to the field of immunology, in particular to an immunohistochemical kit for tumor diagnosis.
Background
According to the world health organization, over 1200 million people are diagnosed with cancer worldwide each year, and about 760 million people die from cancer. Cancer is one of the major killers of death in both developed and developing countries.
Early screening can significantly improve the survival rate of cancer patients, however, tumors in body cavities often require histopathological examination, i.e., cytological examination or tissue samples taken by scraping, puncturing, biopsy, surgery, and the like. As a destructive procedure, the method is difficult to realize early diagnosis of cancer and may cause complications such as bleeding of tissues, infection, etc. in patients. Therefore, it is of great interest to develop new methods for cancer screening and diagnosis that are non-destructive, rapid and highly sensitive and specific.
Immunohistochemistry is the research of determining the antigens in tissue cells by using the principle of specific combination of antigens and antibodies and developing color developing agents for marking the antibodies through chemical reaction, and carrying out positioning, qualitative and quantitative researches on the antigens. However, the current immunohistochemical diagnostic methods have the problems of complicated operation procedures and high non-specificity, and cannot simply, rapidly and accurately determine the expression of the tumor tissue antigen of a patient.
Therefore, there is an urgent need in medicine to develop a kit for rapid, convenient and accurate in vitro diagnosis of tumor tissue.
Disclosure of Invention
In view of the above, the invention provides an immunohistochemical kit for tumor tissues, which has good specificity, high sensitivity and good stability.
The technical scheme of the invention is realized as follows: the invention provides an immunohistochemical diagnostic kit for tumor diagnosis, which comprises a DNP (deoxyribose nucleic acid) labeled antibody, a PLys-HBr labeled secondary antibody, an antigen retrieval solution and a phosphate buffer solution.
On the basis of the above technical means, preferably, the second antibody has a polyamino acid skeleton or a glucan skeleton coupled to a raman active substance.
In addition to the above technical solutions, it is preferable that the raman active material is a compound containing one or more functional groups of an aromatic ring, an amino group, an aldehyde group, a ketone group, a carboxyl group, an amide group, and a sulfinyl group.
Based on the above technical solution, preferably, the antibody is one of rabbit anti-human CD3 antibody, rabbit anti-human CK20 antibody, rabbit anti-human p53 antibody, MUC-4 antibody, p16 antibody, CK10 antibody, CK14 antibody, CK17 antibody, Bax antibody and MUC-4 antibody.
On the basis of the technical scheme, preferably, the raman active substance is one or a combination of more of p-aminobenzoic acid, tert-butyl peroxybenzoate, 2, 6-di-tert-butyl-4-methylphenol, carboxytetramethylrhodamine and aminoacridine.
On the basis of the above technical scheme, preferably, the antigen retrieval solution is: each 1000ml of the citric acid antigen repairing solution comprises 0.5-1.0g of sodium citrate, 1.5-3g of trehalose, 208-10ml of tween, 0.3-0.5g of EDTA and the balance of water.
Based on the above technical solution, it is preferable that the antibody dilution solution is further included.
On the basis of the above technical solution, preferably, the antibody diluent comprises, by mass: 0.5-2.5% of bovine serum albumin, 0.3-0.5% of animal serum, 0.3-1% of disodium hydrogen phosphate, 0.1-0.3% of potassium dihydrogen phosphate and the balance of water.
On the basis of the technical scheme, the immunohistochemical diagnosis kit for tumor diagnosis is preferably applied to auxiliary diagnosis of breast cancer, skin cancer, bone cancer, prostate cancer, ovarian cancer, cervical cancer, liver cancer, lung cancer, brain cancer, laryngeal cancer, gallbladder cancer, pancreatic cancer, rectal cancer, thyroid cancer, adrenal cancer, colon cancer and gastric precancer.
Compared with the prior art, the immunohistochemical kit for tumor diagnosis has the following beneficial effects:
(1) on the basis of IHC detection, the secondary antibody is coupled with a compound with Raman spectrum characteristics, so that the Raman signal of the antibody is obviously enhanced, and the SERS quantitative analysis of the antigen is realized.
(2) According to the invention, DNP is marked on the primary antibody, so that the DNP and a compound with Raman spectrum characteristics marked by the secondary antibody are used as quality control, interference factors are removed, and the specificity of a detection result is higher.
(3) The kit disclosed by the invention marks the Raman reagent to the second antibody, so that the Raman reagent is coupled to the polyamino acid of the second antibody, the expression condition of the tumor tissue antigen of a patient is accurately judged by utilizing the principle of specific combination of the antigen and the antibody, the specificity is high, and the application prospect is very high.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a tissue map of breast cancer according to an embodiment of the present invention;
FIG. 2 is a diagram of a liver cancer tissue detection system according to an embodiment of the present invention;
FIG. 3 is a tissue map of three lung cancers according to an embodiment of the present invention;
FIG. 4 is a tissue detection map of colorectal cancer according to an embodiment of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
The specificity and effectiveness of the kit of the invention are verified by examples 1-4 below.
Example one
An immunohistochemical kit for tumor diagnosis comprises a rabbit anti-human CD3 antibody marked by DNP, a secondary goat anti-mouse IgG marked by PLys-HBr, an antibody diluent, an antigen repairing solution and a phosphate buffer solution.
The antibody diluent comprises the following components in percentage by mass: 0.5% bovine serum albumin, 0.3% animal serum, 0.3% disodium hydrogen phosphate, 0.1% potassium dihydrogen phosphate, and the balance water.
The antigen retrieval liquid is: each 1000ml of the citric acid antigen repairing solution comprises 0.5g of sodium citrate, 1.5g of trehalose, 208ml of tween, 0.3g of EDTA0 and the balance of water.
The phosphate buffer was ph7.4 phosphate buffer: 81g of disodium hydrogen phosphate, 4g of sodium dihydrogen phosphate and 220g of sodium chloride are added into 25L of distilled water to prepare the water-soluble sodium hydrogen phosphate.
The kit comprises the following steps:
1. paraffin section processing
1.1 dewaxing: selecting a paraffin section of breast cancer Her-2 tissue, and baking the section in an electric oven at the temperature of 60-65 ℃ for 60 minutes. Dripping 100 mu l of dimethylbenzene into each slice, and then placing for 20 minutes at the temperature of 60 ℃; finally, incubate for 10 min each in a gradient of aqueous ethanol (100%, 95%, 90%, 80%).
1.2 dewaxing and rehydration: putting the slices into a slice box, and operating according to the following steps: the slices were rinsed with xylene 2 times for 10 minutes each time; soaking and washing the slices with absolute ethyl alcohol for 2 times, 5 minutes each time; soaking and washing the slices with 95% ethanol for 2 times, each time for 5 minutes; the slices were rinsed 2 times with 75% ethanol for 5 minutes each time; the mixture was washed with distilled water 3 times for 3 minutes each.
1.3 antigen retrieval
(1) About 1.0L of the antigen retrieval solution was added to the autoclave and boiled.
(2) And putting the slicing frame into a repair liquid water bath, covering the high-pressure cooker cover, and continuously heating to blow air to the pressure valve of the high-pressure cooker.
(3) Starting timing from the pressure valve blowing of the pressure cooker, 2.5 minutes, shutting off the fire and naturally cooling to 30 ℃.
(4) Cleaning: the microtome racks cooled to room temperature were rinsed 3 times for 3 minutes each in distilled water.
1.4 antibody incubation
(1) First-order labeling: mu.l of rabbit anti-human CD3 antibody was dissolved in phosphate buffer (pH 7.4), 100. mu.l of DNP was added, mixed and stirred well, incubated in a shaker at 37 ℃ for 30 minutes, and placed under UV light for 10 minutes to form DNP-labeled rabbit anti-human CD3 antibody.
(2) Primary antibody incubation: the sections were spun off of excess liquid and spread on a wet box, 100. mu.l of anti-rabbit anti-human CD3 antibody was added dropwise to the tissue sections, incubated at 37 ℃ for 60 minutes, and then washed 3 times with phosphate buffer, 3 minutes each.
Rabbit anti-human CD3 antibody was administered as 1: 10000, i.e. 1. mu.l of the antibody stock solution is diluted with 10ml of the antibody diluent.
(3) Coupling of a secondary antibody: mu.l of the secondary goat anti-mouse IgG was dissolved in phosphate buffer (pH 7.4), 100. mu.l of PLys-HBr was added, mixed and stirred well, incubated in a shaker at 37 ℃ for 30 minutes, and irradiated under an ultraviolet lamp for 10 minutes to form a secondary antibody labeled with PLys-HBr.
(4) And (3) secondary antibody incubation: the sections were spun off of excess liquid and spread on a wet box, 100. mu.l of PLys-HBr-labeled secondary goat anti-mouse IgG was added dropwise, incubated at 37 ℃ for 30 minutes, and then washed 3 times with 3 minutes each with phosphate buffer.
Goat anti-mouse IgG antibodies were administered as 1: 10000, i.e. 1. mu.l of the antibody stock solution is diluted with 10ml of the antibody diluent.
6. Sealing sheet
And (3) dropwise adding a proper amount of neutral gum on the glass slide, covering the glass slide, putting the glass slide into a Raman spectrum analyzer after the glass slide is dried, observing the result, and exporting corresponding detection data at a workstation.
Example two
An immunohistochemical kit for tumor diagnosis comprises a rabbit anti-human CK20 antibody marked by DNP, a secondary antibody marked by aminobenzoic acid, an antibody diluent, an antigen repairing solution and a phosphate buffer solution.
The antibody diluent comprises the following components in percentage by mass: 2.5% bovine serum albumin, 0.5% animal serum, 1% disodium hydrogen phosphate, 0.3% potassium dihydrogen phosphate, and the balance water.
The antigen retrieval liquid is: each 1000ml of the citric acid antigen retrieval solution comprises 1.0g of sodium citrate, 3g of trehalose, 2010ml of tween, 0.5g of EDTA0 and the balance of water.
The phosphate buffer was ph7.4 phosphate buffer: 81g of disodium hydrogen phosphate, 4g of sodium dihydrogen phosphate and 220g of sodium chloride are added into 25L of distilled water to prepare the water-soluble sodium hydrogen phosphate.
The kit comprises the following steps:
1. paraffin section processing
1.1 dewaxing: selecting a liver cancer tissue paraffin section, and baking the section in an electric oven at 62 ℃ for 60 minutes. Dripping 100 mu l of dimethylbenzene into each slice, and then placing for 20 minutes at the temperature of 60 ℃; finally, incubate for 10 min each in a gradient of aqueous ethanol (100%, 95%, 90%, 80%).
1.2 dewaxing and rehydration: putting the slices into a slice box, and operating according to the following steps: the slices were rinsed with xylene 2 times for 10 minutes each time; soaking and washing the slices with absolute ethyl alcohol for 2 times, 5 minutes each time; soaking and washing the slices with 95% ethanol for 2 times, each time for 5 minutes; the slices were rinsed 2 times with 75% ethanol for 5 minutes each time; the mixture was washed with distilled water 3 times for 3 minutes each.
1.3 antigen retrieval
(1) About 1.5L of the antigen retrieval solution was added to the autoclave and boiled.
(2) And putting the slicing frame into a repair liquid water bath, covering the high-pressure cooker cover, and continuously heating to blow air to the pressure valve of the high-pressure cooker.
(3) Starting timing from the pressure valve blowing of the pressure cooker, 2.5 minutes, shutting off the fire and naturally cooling to 30 ℃.
(4) Cleaning: the microtome racks cooled to room temperature were rinsed 3 times for 3 minutes each in distilled water.
1.4 antibody incubation
(1) First-order labeling: mu.l of rabbit anti-human CK20 antibody was dissolved in phosphate buffer (pH 7.4), 100. mu.l of DNP was added, mixed and stirred well, incubated in a shaker at 37 ℃ for 30 minutes, and placed under UV light for 10 minutes to form DNP-labeled rabbit anti-human CK20 antibody.
(2) Primary antibody incubation: the sections were spun off of excess liquid and spread on a wet box, 100. mu.l of anti-rabbit anti-human CK20 antibody was added dropwise to the tissue sections, incubated at 37 ℃ for 60 minutes, and then washed 3 times with phosphate buffer, 3 minutes each.
Rabbit anti-human CK20 antibody was administered as 1: 10000, i.e. 1. mu.l of the antibody stock solution is diluted with 10ml of the antibody diluent.
(3) Coupling of a secondary antibody: mu.l of secondary goat anti-mouse IgG is dissolved in phosphate buffer (pH 7.4), 100. mu.l of aminobenzoic acid is added, the mixture is mixed and stirred uniformly, the mixture is placed in a shaking table at 37 ℃ for incubation for 30 minutes, and the shaking table is placed under an ultraviolet lamp for illumination for 10 minutes, so that an aminobenzoic acid labeled IgG polymer is formed.
(4) And (3) secondary antibody incubation: the sections were spun off of excess liquid and spread on a wet box, 100. mu.l of aminobenzoic acid-labeled secondary goat anti-mouse IgG was added dropwise, incubated at 37 ℃ for 30 minutes, and then washed 3 times with phosphate buffer, 3 minutes each.
Goat anti-mouse IgG antibodies were administered as 1: 10000, i.e. 1. mu.l of the antibody stock solution is diluted with 10ml of the antibody diluent.
6. Sealing sheet
And (3) dropwise adding a proper amount of neutral gum on the glass slide, covering the glass slide, putting the glass slide into a Raman spectrum analyzer after the glass slide is dried, observing the result, and exporting corresponding detection data at a workstation.
EXAMPLE III
An immunohistochemical kit for tumor diagnosis comprises a rabbit anti-human p53 DNP antibody, a tert-butyl peroxybenzoate labeled secondary antibody, an antibody diluent, an antigen retrieval solution and a phosphate buffer solution.
The antigen retrieval liquid is: each 1000ml of the citric acid antigen repairing solution comprises 0.8g of sodium citrate, 2.5g of trehalose, 209ml of tween, 0.4g of EDTA0 and the balance of water.
The antibody diluent comprises the following components in percentage by mass: 1.5% bovine serum albumin, 0.4% animal serum, 0.8% disodium hydrogen phosphate, 0.2% potassium dihydrogen phosphate, and the balance water.
The phosphate buffer was ph7.4 phosphate buffer: 81g of disodium hydrogen phosphate, 4g of sodium dihydrogen phosphate and 220g of sodium chloride are added into 25L of distilled water to prepare the water-soluble sodium hydrogen phosphate.
The kit comprises the following steps:
1. paraffin section processing
1.1 dewaxing: selecting a lung cancer tissue paraffin section, and baking the section in an electric oven at 62 ℃ for 60 minutes. Dripping 100 mu l of dimethylbenzene into each slice, and then placing for 20 minutes at the temperature of 60 ℃; finally, incubate for 10 min each in a gradient of aqueous ethanol (100%, 95%, 90%, 80%).
1.2 dewaxing and rehydration: putting the slices into a slice box, and operating according to the following steps: the slices were rinsed with xylene 2 times for 10 minutes each time; soaking and washing the slices with absolute ethyl alcohol for 2 times, 5 minutes each time; soaking and washing the slices with 95% ethanol for 2 times, each time for 5 minutes; the slices were rinsed 2 times with 75% ethanol for 5 minutes each time; the mixture was washed with distilled water 3 times for 3 minutes each.
1.3 antigen retrieval
(1) About 1.5L of the antigen retrieval solution was added to the autoclave and boiled.
(2) And putting the slicing frame into a repair liquid water bath, covering the high-pressure cooker cover, and continuously heating to blow air to the pressure valve of the high-pressure cooker.
(3) Starting timing from the pressure valve blowing of the pressure cooker, 2.5 minutes, shutting off the fire and naturally cooling to 30 ℃.
(4) Cleaning: the microtome racks cooled to room temperature were rinsed 3 times for 3 minutes each in distilled water.
1.4 antibody incubation
(1) First-order labeling: mu.l of rabbit anti-human p53 antibody was dissolved in phosphate buffer (pH 7.4), 100. mu.l of DNP was added, mixed and stirred well, incubated in a shaker at 37 ℃ for 30 minutes, and placed under UV light for 10 minutes to form DNP-labeled rabbit anti-human p53 antibody.
(2) Primary antibody incubation: the sections were spun off of excess liquid and spread on a wet box, 100. mu.l of anti-rabbit anti-human p53 antibody was added dropwise to the tissue sections, incubated at 37 ℃ for 60 minutes, and then washed 3 times with phosphate buffer, 3 minutes each.
Rabbit anti-human p53 antibody was administered as 1: 10000, i.e. 1. mu.l of the antibody stock solution is diluted with 10ml of the antibody diluent.
(3) Coupling of a secondary antibody: mu.l of the secondary goat anti-mouse IgG was dissolved in phosphate buffer (pH 7.4), 100. mu.l of tert-butyl peroxybenzoate was added, mixed and stirred well, incubated in a shaker at 37 ℃ for 30 minutes, and placed under an ultraviolet lamp for 10 minutes to form a tert-butyl peroxybenzoate-labeled IgG polymer.
(4) And (3) secondary antibody incubation: the sections were spun off of excess liquid and spread on a wet box, 100. mu.l of dimethyl sulfoxide-labeled secondary goat anti-mouse IgG was added dropwise, incubated at 37 ℃ for 30 minutes, and then washed 3 times with phosphate buffer, 3 minutes each.
Goat anti-mouse IgG antibodies were administered as 1: 10000, i.e. 1. mu.l of the antibody stock solution is diluted with 10ml of the antibody diluent.
6. Sealing sheet
And (3) dropwise adding a proper amount of neutral gum on the glass slide, covering the glass slide, putting the glass slide into a Raman spectrum analyzer after the glass slide is dried, observing the result, and exporting corresponding detection data at a workstation.
Example four
An immunohistochemical kit for tumor diagnosis comprises a DNP-labeled p16 antibody, aminoacridine and a 2, 6-di-tert-butyl-4-methylphenol-labeled secondary antibody, an antibody diluent, an antigen retrieval solution and a phosphate buffer solution.
The antigen retrieval liquid is: each 1000ml of the citric acid antigen retrieval solution comprises 1.0g of sodium citrate, 3g of trehalose, 2010ml of tween, 0.5g of EDTA0 and the balance of water.
The antibody diluent comprises the following components in percentage by mass: 2.5% bovine serum albumin, 0.5% animal serum, 1% disodium hydrogen phosphate, 0.3% potassium dihydrogen phosphate, and the balance water.
The phosphate buffer was ph7.4 phosphate buffer: 81g of disodium hydrogen phosphate, 4g of sodium dihydrogen phosphate and 220g of sodium chloride are added into 25L of distilled water to prepare the water-soluble sodium hydrogen phosphate.
The kit comprises the following steps:
1. paraffin section processing
1.1 dewaxing: selecting a rectal cancer tissue paraffin section, and baking the section in an electric oven at 62 ℃ for 60 minutes. Dripping 100 mu l of dimethylbenzene into each slice, and then placing for 20 minutes at the temperature of 60 ℃; finally, incubate for 10 min each in a gradient of aqueous ethanol (100%, 95%, 90%, 80%).
1.2 dewaxing and rehydration: putting the slices into a slice box, and operating according to the following steps: the slices were rinsed with xylene 2 times for 10 minutes each time; soaking and washing the slices with absolute ethyl alcohol for 2 times, 5 minutes each time; soaking and washing the slices with 95% ethanol for 2 times, each time for 5 minutes; the slices were rinsed 2 times with 75% ethanol for 5 minutes each time; the mixture was washed with distilled water 3 times for 3 minutes each.
1.3 antigen retrieval
(1) About 1.5L of the antigen retrieval solution was added to the autoclave and boiled.
(2) And putting the slicing frame into a repair liquid water bath, covering the high-pressure cooker cover, and continuously heating to blow air to the pressure valve of the high-pressure cooker.
(3) Starting timing from the pressure valve blowing of the pressure cooker, 2.5 minutes, shutting off the fire and naturally cooling to 30 ℃.
(4) Cleaning: the microtome racks cooled to room temperature were rinsed 3 times for 3 minutes each in distilled water.
1.4 antibody incubation
(1) First-order labeling: mu. l p16 antibody was dissolved in phosphate buffer (pH 7.4), 100. mu.l of DNP was added, mixed and stirred well, incubated in a shaker at 37 ℃ for 30 minutes and placed under an ultraviolet lamp for 10 minutes to form DNP-labeled p16 antibody.
(2) Primary antibody incubation: the sections were spun off of excess liquid and spread on a wet box, 100. mu.l of p16 antibody was added dropwise to the tissue sections, incubated at 37 ℃ for 60 minutes, and then washed 3 times with 3 minutes each with phosphate buffer.
The p16 antibody was administered as 1: 10000, i.e. 1. mu.l of the antibody stock solution is diluted with 10ml of the antibody diluent.
(3) Coupling of a secondary antibody: mu.l of a secondary goat anti-mouse IgG was dissolved in a phosphate buffer (pH 7.4), 50. mu.l of aminoacridine and 50. mu.l of 2, 6-di-t-butyl-4-methylphenol were added, mixed and stirred uniformly, incubated in a shaker at 37 ℃ for 30 minutes, and irradiated under an ultraviolet lamp for 10 minutes to form an aminoacridine-labeled IgG polymer.
(4) And (3) secondary antibody incubation: the sections were spun off of excess liquid and spread on a wet box, 100. mu.l of aminoacridine-labeled secondary goat anti-mouse IgG was added dropwise, incubated at 37 ℃ for 30min, and then washed 3 times with phosphate buffer, 3min each time.
Goat anti-mouse IgG antibodies were administered as 1: 10000, i.e. 1. mu.l of the antibody stock solution is diluted with 10ml of the antibody diluent.
6. Sealing sheet
And (3) dropwise adding a proper amount of neutral gum on the glass slide, covering the glass slide, putting the glass slide into a Raman spectrum analyzer after the glass slide is dried, observing the result, and exporting corresponding detection data at a workstation.
The Raman spectra of examples 1-4 were measured by confocal Raman spectrometer with a measurement range of 600-1800cm-1The excitation wavelength was 785nm, the sampling time was 2s, the excitation light power was 1mw, and the resulting spectra are shown in fig. 1-4.
As shown in fig. 1 to 4, raman signals at characteristic peak positions can be obtained from the raman spectrogram, and tumor tissues can be judged according to the peak values corresponding to the raman spectrogram.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (9)
1. An immunohistochemical kit for tumor diagnosis, comprising a DNP-labeled antibody, a PLys-HBr-labeled secondary antibody, an antigen retrieval solution and a phosphate buffer solution.
2. The immunohistochemical kit for tumor diagnosis according to claim 1, wherein the second antibody has a polyamino acid skeleton or a dextran skeleton coupled to a raman active substance.
3. The immunohistochemical kit for tumor diagnosis according to claim 2, wherein said raman active substance is a compound containing one or more functional groups of aromatic ring, amino group, aldehyde group, ketone group, carboxyl group, amide group and sulfinyl group.
4. The immunohistochemical kit for tumor diagnosis according to claim 1, wherein said antibody is one of a rabbit anti-human CD3 antibody, a rabbit anti-human CK20 antibody, a rabbit anti-human p53 antibody, a MUC-4 antibody, a p16 antibody, a CK10 antibody, a CK14 antibody, a CK17 antibody, and a Bax antibody.
5. The immunohistochemical kit for tumor diagnosis according to claim 3, wherein said Raman-active substance is one or more combinations of p-aminobenzoic acid, tert-butyl peroxybenzoate, 2, 6-di-tert-butyl-4-methylphenol, carboxytetramethylrhodamine, and aminoacridine.
6. The immunohistochemical kit for tumor diagnosis according to claim 1, wherein said antigen retrieval solution is: each 1000ml of the citric acid antigen repairing solution comprises 0.5-1.0g of sodium citrate, 1.5-3g of trehalose, 208-10ml of tween, 0.3-0.5g of EDTA and the balance of water.
7. The immunohistochemical kit for tumor diagnosis according to claim 1, further comprising an antibody diluent.
8. The immunohistochemical kit for tumor diagnosis according to claim 7, wherein said antibody diluent comprises, in mass percent: 0.5-2.5% of bovine serum albumin, 0.3-0.5% of animal serum, 0.3-1% of disodium hydrogen phosphate, 0.1-0.3% of potassium dihydrogen phosphate and the balance of water.
9. The immunohistochemical kit for tumor diagnosis according to claims 1 to 8, wherein the kit is used for auxiliary diagnosis of breast cancer, skin cancer, bone cancer, prostate cancer, ovarian cancer, cervical cancer, liver cancer, lung cancer, brain cancer, larynx cancer, gallbladder cancer, pancreatic cancer, rectal cancer, thyroid cancer, adrenal cancer, colon cancer and precancerous stomach cancer.
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