CN101598731B - Immune tissue chemical diagnostic kit used for pathological diagnosis of tumour - Google Patents
Immune tissue chemical diagnostic kit used for pathological diagnosis of tumour Download PDFInfo
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Abstract
The invention discloses an immune tissue chemical diagnostic kit of HAb18G/CD147 cancer landmark used for pathological diagnosis of tumour, including a blocking agent capable of removing endogenous enzyme in a human tissue slice, a sealing agent used for sealing cross protein reaction in the human tissue slice, a first antibody capable of combining with the HAb18G/CD147 cancer landmark in human tumour tissue, a biotin marked secondary antibody capable of combining with the first antibody, and an enzyme marked avidin, chromogenic reagent and a phosphate buffer system used for combining with the secondary antibody. The kit has the characteristics of good specificity, high sensibility, stability and universality, is mainly used for pathologic diagnosis of epithelium-derived malignant tumour (such as liver cancer, lung cancer, esophageal cancer, gastric cancer, colorectal cancer, breast cancer, cervical cancer, ovarian cancer and the like), and is beneficial to warning screen, early diagnosis, etiologic diagnosis, treatment reaction and curative effect monitoring of tumour, recrudescence and transfer and prognosis judgement of tumour.
Description
Technical field
The invention belongs to bioengineering field, relate to a kind of kit for clinical diagnosis, particularly a kind of immunohistochemical diagnosis kit of the HAb18G/CD147 carcinoma marker for the pathologic diagnosis of tumor purposes.
The HAb18G/CD147 carcinoma marker is a kind of novel, wide spectrum and than the carcinoma marker of high specific, with a kind of specific antibody for the HAb18G/CD147 carcinoma marker, in conjunction with general ImmunohistochemistryMethods Methods, make HAb18G/CD147 carcinoma marker immunohistochemical diagnosis kit, be applied to multiple human cancers histopathology sample, carry out pathologic diagnosis of tumor, help early warning screening, early diagnosis, etiologic diagnosis, therapeutic response and the curative effect monitoring of tumour, relapse and metastasis and the prognosis of tumour to judge.
Background technology
In recent years, the number of the infected of various tumours is all in rapid rising, and the medical expense of tumour and the death of tumor patient cause white elephant all can for society and family.People are in the urgent need to the diagnosis that can be used for tumour and technological means or the method for the treatment of.Current, the 3P medical science take prospective medicine, preventive medicine and Personalized medicine as core has become assurance, has improved the important directions of level of human health, and the discovery and application of carcinoma marker is the important key that realizes 3P medical science.
Carcinoma marker (tumor marker) is to be present in the tumour cell or by a kind of material of its secretion, can be distributed in blood, tissue fluid, secretion or the tumour cell.The variation of carcinoma marker and the generation of tumour, development and prognosis have direct relation, as point out the character of tumour, the tissue of understanding tumour occurs, Cell Differentiation, help early warning screening, early diagnosis, etiologic diagnosis, therapeutic response and the curative effect monitoring of tumour, relapse and metastasis and the prognosis of tumour to judge, help diagnosis, molecule parting and the individualized treatment of tumour to instruct.
In recent years, because the fast development of biotechnology, the discovery and application of novel tumor mark has been accelerated in the application of monoclonal antibody technology, protein science technology, bioinformatics greatly.Its technical foundation mostly is by with antibody screening tumour cell chip, cDNA expression library, the methods such as combining yeast double cross, co-immunoprecipitation, mass-spectrometric technique, catch this antibody for antigen molecule.The discovery and application of novel tumor mark has vital role to setting up Prognosis scoveillances such as tumour early warning examination, early diagnosis, etiologic diagnosis, therapeutic response and transfer and relapse etc.
SABC is to utilize the principle of antigen and antibody specific binding, make developer (fluorescein, enzyme, metallic ion, the isotope) colour developing of labelled antibody determine histocyte endoantigen (peptide and protein) by chemical reaction, to its position, qualitative and quantitative research.At present immunohistochemistry widespread use diagnose with clinicopathologia, particularly carry out tumour auxiliary diagnosis and antidiastole with antibody pointedly, become clinicopathologia and diagnose indispensable important means.
The HAb18G/CD147 molecule is a kind of novel carcinoma marker, and this molecule is the transmembrane glycoprotein of 61kDa, by the molecule that 269 amino acid form, is to screen acquisition with specific monoclonal antibody in human liver cancer cell cDNA expression library.Its monoclonal antibody is the HAb18 (patent No.: ZL02114471.0), be with Freshman liver cancer tissue cell suspension immunity Bab1/C mouse, be prepared from hybridoma technology.
HAb18G/CD147 carcinoma marker immunohistochemical diagnosis kit, be used for pathologic diagnosis of tumor, identify through SABC tissue spectrum, this molecule is high expressed in multiple cancerous tissue, extremely low expression in normal structure and embryonic tissue, be distributed with better specificity, the dyeing phenotype is membranous type or film slurry type.
71.4%), liver cancer (positive rate: 80.0%), cancer of the stomach (positive rate: 68%), lung cancer (positive rate: 54.3%) ImmunohistochemistryResults Results of 2006 routine organization chips shows that the expression rate of HAb18G/CD147 carcinoma marker in following cancer is: breast cancer (positive rate:.And in normal liver tissue, adenoma of liver pathological tissues, express for low; Normal structure, benign tumour and benign disease tissues at mammary gland are negative.Use the responsive Immunohistochemical Staining of multiple height such as ABC-APAAP, the liver cancer tissue section is observed distribution and the location of HAb18G in light microscopic and two levels of Electronic Speculum, the result show the HAb18G/CD147 carcinoma marker optionally high expressed in liver cancer tissue, its positive rate is 75% (39/52), and normal liver tissue is low expresses.Immuno-electron microscope (collaurum) is positioned on the elementary membrane of hepatoma cell membrane, endoplasmic reticulum and envelope shape structure.Result of study is published in Proteomics, and 7 (13): 2151-2161,2007; Histopathology, 2008.
The appearance of monoclonal antibody and the development continuously and healthily of immunohistochemistry detection system make the application of immunohistochemistry technology in modern pathological diagnosis increasingly extensive, and are subjected to the concern and blue or green fan of pathology circle.The earliest with the initial stage seventies only in the laboratory scientific research attempt, went through for 30 years after this technology developed into requisite reliable auxiliary diagnosis means in the routine pathology diagnosis.
In the routine pathology diagnosis, most of cases are to make clear and definite Morphologic Diagnosis in the section of HE dyeing, the case of difficult and problem are arranged at 5-10%.Further diagnosis mainly by specific antibody dyeing, is done to the clinical pathology histotomy by immunohistochemistry technology, analyzes the relation of tumor markers, tumor associated antigen and disease, improves the accuracy of pathological diagnosis with auxiliary diagnosis.
The related neoplasms mark antibody of present clinical use has more than 100 to plant, and the antibody of diagnosing tumor commonly used has 10 kinds approximately, and antibody is used in many genus researchs, mainly is to sell from buying rear packing abroad.Without regular lot number, differ greatly between the antibody of in pathological diagnosis, using and antibody batch, repeatable poor, result not person of modern times anticipates.
Summary of the invention
The object of the invention is to, a kind of immunohistochemical diagnosis kit of the HAb18G/CD147 carcinoma marker for the pathologic diagnosis of tumor purposes is provided.This diagnostic kit is the biological characteristics that utilizes the Ag-Ab specific binding, with the HAb18 specific monoclonal antibody clinical pathology tissue specimen is dyeed, by the SP ImmunohistochemistryMethods Methods, observe the expression of HAb18G/CD147 carcinoma marker in human cancers, clinical practice is in auxiliary diagnosis or the antidiastole of human cancers.This kit has the advantages that specificity is good, susceptibility is high, stable, general.
In order to realize above-mentioned task, the present invention takes following technical solution:
A kind of immunohistochemical diagnosis kit of the HAb18G/CD147 carcinoma marker for the pathologic diagnosis of tumor purposes is characterized in that the content of this kit comprises:
1) be used for to remove the blocking agent of people's histotomy endogenous enzyme, this blocking agent is that 3% superoxol and concentration are that 80% methanol solution forms by concentration;
2) for the nonspecific proteins sealer that seals people's histotomy intersection albumino reaction unspecific staining; This sealer is formulated by 3-5% Normal animal serum and phosphate buffer;
3) first antibody that the HAb18G/CD147 carcinoma marker is combined in human body cancer tissue; This first antibody is the mouse monoclonal antibody of specific binding HAb18G/CD147 carcinoma marker, and its working concentration is 10-100 μ g/mL;
4) second antibody that can be combined with first antibody; This second antibody is the polyclonal antibody of biotin labeled sheep or the anti-mouse IgG of rabbit;
5) be used for enzyme mark Streptavidin in conjunction with biotin on the second antibody; This enzyme mark Streptavidin is the Streptavidin of horseradish peroxidase-labeled, is chromogenic substrate;
6) with the chromogenic reagent of chromogenic substrate effect, this chromogenic reagent is that benzidine (DAB) solution, trishydroxymethylaminomethane (TrisHcl) damping fluid and deionized water are formulated, more than the preparation consumption volume ratio of three kinds of reagent be 1: 1: 50.
7) the used buffer system of kit is phosphate buffer Na
2HPO
4-NaH
2PO
4-NaCl, concentration is 0.01mol/L, pH is 7.2～7.45.
Above-mentioned first antibody is preserved in the liquid at the antibody of 10-100 μ g/mL concentration and is preserved, and this antibody is preserved liquid can keep antibody 2-8 ℃ of lower the preservation 24 months.
The technical characterstic of the HAb18G/CD147 carcinoma marker immunohistochemical diagnosis kit for the pathologic diagnosis of tumor purposes of the present invention is:
1, the first antibody in this kit is the antibody of a high specific, broad spectrum activity.The HAb18G/CD147 tumor markers is to screen acquisition with first antibody in human liver cancer cell cDNA expression library, and HAb18G/CD147 confirms that through to this analysis of the molecular structure molecule is the transmembrane glycoprotein of 61kDa, is comprised of 269 amino acid; Through inquiry Genbank, this molecule is identical with people CD147 gene coded sequence, is positioned No. 19 chromosomes of people, is the member of immunoglobulin superfamily.A large amount of fundamental researchs and applied research working foundation have disclosed biological function and the application value of this tumor markers specific antibody deeply.
2, the immunohistochemical diagnosis kit of this HAb18G/CD147 carcinoma marker, be applicable to people's tissue specimen, fix paraffin-embedded tissue specimen slice with 10% neutral formalin solution, with the cancerous tissue sample of excision, the frozen section of making immediately.
3, in this kit, phosphate buffer system (0.01mol/L, Na that the applicant adopts
2HPO
4-NaH
2PO
4-NaCl, pH 7.2～7.45) consistance and accuracy in whole immunohistochemical experiment procedure, effectively controlled the nonspecific reaction background.
4, in this kit, the first antibody that the applicant adopts is preserved liquid, and its characteristics are preserved liquid for this antibody and contained bovine serum albumin(BSA), collagen, phosphate buffer and NaN
3, this antibody is preserved liquid can keep antibody function, preserves 24 months at 2-8 ℃.Guaranteed the stability of key reagents in the method.
It is good that above technical characterstic has guaranteed that HAb18G/CD147 carcinoma marker SABC kit has specificity, and susceptibility is strong, the characteristic of good stability, simple general-purpose.Use by many hospitals, sheet is read in the dyeing of clinical more than 2000 routine tumor tissues samples, the result shows, HAb18G/CD147 carcinoma marker wide expression (comprises lung cancer in the malignant tumour of epithelium source property, the cancer of the esophagus, cancer of the stomach, colorectal cancer, liver cancer, breast cancer, oophoroma, cervical carcinoma etc.) tissue, the low expression in normal structure and benign disease tissues, also present expression trend in the more other tissue of atypical hyperplasia of cancer and active proliferation sexual cell, it is a kind of novel pointing out this mark, wide spectrum, than the carcinoma marker of high specific, this kit is that diagnosing tumor and prognosis are judged, new approaches and methods has been opened up in assessment.
Description of drawings
Fig. 1 is the colouring method synoptic diagram of HAb18G/CD147 carcinoma marker immunohistochemical diagnosis kit;
Fig. 2 is that the present invention is at different tumor tissues, the microscopically HAb18G/CD147 carcinoma marker SABC kit coloration result collection of illustrative plates of normal structure, wherein (A), the liver cancer tissue of embodiment 1 and the microscopically HAb18G/CD147 carcinoma marker SABC kit coloration result collection of illustrative plates of normal structure, (B) being the cancerous lung tissue of embodiment 2 and the microscopically HAb18G/CD147 carcinoma marker SABC kit coloration result collection of illustrative plates of normal structure, (C) is the human esophageal carcinoma of embodiment 3 and the microscopically HAb18G/CD147 carcinoma marker SABC kit coloration result collection of illustrative plates of normal structure.
The embodiment that provides below in conjunction with accompanying drawing and inventor is described in further detail the present invention.
Embodiment
HAb18G/CD147 carcinoma marker immunohistochemical diagnosis kit for aided Cancer Diagnosis or antidiastole of the present invention, be mainly used in the pathological diagnosis of human cancers (liver cancer, lung cancer, the cancer of the esophagus, cancer of the stomach, colorectal cancer, breast cancer, cervical carcinoma, oophoroma etc.), help early diagnosis, etiologic diagnosis, therapeutic response and the curative effect monitoring of tumour, relapse and metastasis and the prognosis of tumour to judge.Comprise in this kit content:
1, be used for removing the blocking agent of people's histotomy endogenous enzyme, is that 3% superoxol and concentration are that 80% methanol solution preparation in 1: 10 by volume forms by concentration;
2, the nonspecific proteins sealer that is used for sealing people histotomy intersection albumino reaction unspecific staining; This sealer is formulated by 3-5% Normal animal serum and phosphate buffer;
3, first antibody, this first antibody are the mouse monoclonal antibodies of specific binding HAb18G/CD147 carcinoma marker, and its working concentration is 10-100 μ g/mL; This HAb18G/CD147 carcinoma marker high expressed is in the malignant tumour of epithelium source property, and they are liver cancer, lung cancer, the cancer of the esophagus, cancer of the stomach, the carcinoma of the rectum, breast cancer, cervical carcinoma, oophoroma or other known cancer;
4, the second antibody that can be combined with first antibody; This second antibody is the polyclonal antibody of biotin labeled sheep or the anti-mouse IgG of rabbit;
5, be used for enzyme mark Streptavidin in conjunction with biotin on the second antibody; The connection enzyme avidin that this enzyme mark Streptavidin is horseradish peroxidase-labeled is chromogenic substrate;
6, with the chromogenic reagent of chromogenic substrate effect, this chromogenic reagent is that benzidine (DAB) solution, trishydroxymethylaminomethane (TrisHcl) damping fluid and deionized water are formulated, more than the preparation consumption volume ratio of three kinds of reagent be 1: 1: 50.
7, the used buffer system of kit is phosphate buffer Na
2HPO
4-NaH
2PO
4-NaCl, concentration is 0.01mol/L, pH is 7.2～7.45.
First antibody is preserved in the liquid at the antibody of 10-100 μ g/mL concentration and is preserved, and can keep antibody function, preserves 24 months at 2-8 ℃.Antibody is preserved liquid and is contained 1% bovine serum albumin(BSA), 0.1% collagen, 0.01mol/L phosphate buffer, 0.05%NaN
3
The colouring method of HAb18G/CD147 carcinoma marker immunohistochemical diagnosis kit, its principle schematic be as shown in Figure 1:
People's histotomy with aquation after, add an amount of endogenous enzyme blocking agent, reacted 10 minutes, phosphate buffer cleans; Drip 1～2 of nonspecific proteins sealer, reacted 10 minutes; Drip 1～2 of first antibody, 37 ℃ were reacted 2 hours, and phosphate buffer cleans; Drip 1～2 of second antibody, 22-28 ℃ was reacted 20 minutes, and phosphate buffer cleans; Drip 1～2 of enzyme mark streptavidin, 22-28 ℃ was reacted 15 minutes, and phosphate buffer cleans; Drip 1～2 of freshly prepared chromogenic reagent, 22-28 ℃ was reacted 1～10 minute, and phosphate buffer cleans; Bush uniformly dyeing nuclear, gradient alcohol dehydration, dimethylbenzene are transparent, neutral gum mounting, observation by light microscope.Above-mentioned phosphate buffer cleans and is that concussion was cleaned 5 minutes each 3 times.
Phosphate buffer is Na
2HPO
4-KH
2PO
4-NaCl, concentration is 0.01mol/L, pH is 7.2～7.45.But the dyeing background of this phosphate buffer balanced reaction pH of system value, minimizing non-specific responding.
Immunohistochemistry technology is an experimental very strong technology, and its key elements of success mainly contains the antibody that specificity is good, broad spectrum activity is good, reliable reagent and experimental technique, strict quality control requirement.
Below be the specific embodiment that the inventor provides:
HAb18G/CD147 carcinoma marker immunodiagnosis reagent kit experimental technique is as follows, and following examples are called for short " experimental technique " all with reference to the method:
1, clinical tissue case paraffin specimen section, dimethylbenzene dewaxing 20min, 3 times;
2,100% alcohol is sloughed dimethylbenzene, 5min, 2 times; 95%～75% alcohol, 2min/ time;
3,3%H2O2 sealing endogenous peroxydase, 22-28 ℃, 10 minutes;
4, the PBS damping fluid embathed 3 minutes, 3 times;
5, non-specific site in the 5% normal sheep serum sealing tissue, 22-28 ℃, 20 minutes;
6, HAb18 monoclonal antibody (30ug/mL), 37 ℃, 120 minutes;
7, the PBS damping fluid embathed 5 minutes, 3 times;
8, the anti-specificity antiantibody of avidin mark, 22-28 ℃, 15 minutes;
9, the PBS damping fluid embathed 5 minutes, 3 times;
10, horseradish peroxidase-labeled biotin, 22-28 ℃, 20 minutes;
11, the PBS damping fluid embathed 5 minutes, 3 times;
12, DAB colour developing, 22-28 ℃, 2-10 minute;
13, haematine lining transfect cell nuclear, 22-28 ℃, 5 minutes;
14,5%～95% alcohol, 2min/ time; 100% dehydration of alcohol, 5min, 2 times;
15, dimethylbenzene is transparent, 5min, 2 times; Microscopic examination.
The selection of research object: with goldstandard HE decoration method, as the pacing items of determining case group and control group sample, according to the histopathology morphological feature, determine to select research object.
Embodiment 1:
The HAb18G/CD147 carcinoma marker immunohistochemical diagnosis kit that is used for aided Cancer Diagnosis or antidiastole is as follows in the concrete process of the test of hepatic tissue expression:
1. sample is selected: (liver cancer, case is determined in dyeing according to goldstandard HE)
Table 1 liver cancer and hepatic tissue case are selected
2. test operation: see " experimental technique " for details.
3. test findings: (seeing accompanying drawing 2A)
The coloration result of table 2HAb18G/CD147 carcinoma marker diagnostic kit in liver cancer and hepatic tissue
Embodiment 2:
The HAb18G/CD147 carcinoma marker immunohistochemical diagnosis kit that is used for aided Cancer Diagnosis or antidiastole is as follows in the concrete process of the test of lung tissue expression:
1. sample is selected: lung cancer, the result determines case according to goldstandard HE decoration method
Table 3: lung cancer and lung tissue case are selected
2. test operation: see " experimental technique " for details.
3. test findings: (seeing accompanying drawing 2B)
The coloration result of table 4:HAb18G/CD147 carcinoma marker diagnostic kit in lung cancer and lung tissue
Embodiment 3:
The HAb18G/CD147 carcinoma marker immunohistochemical diagnosis kit that is used for aided Cancer Diagnosis or antidiastole is as follows in esophageal tissue's concrete process of the test of expression:
1. sample is selected: the cancer of the esophagus, the result determines case according to goldstandard HE decoration method
Table 5 cancer of the esophagus and esophageal tissue's case are selected
2. test operation: see " experimental technique " for details.
3. test findings: (seeing accompanying drawing 2C)
The coloration result of table 6:HAb18G/CD147 carcinoma marker diagnostic kit in the cancer of the esophagus and esophageal tissue
HAb18G/CD147 carcinoma marker immunohistochemical diagnosis kit for aided Cancer Diagnosis or antidiastole of the present invention also can be used for the detection of the expression of other various cancerous tissue HAb18G/CD147 tissues.Method sees " experimental technique " for details.
Below be to adopt the HAb18G/CD147 carcinoma marker immunohistochemical diagnosis kit for aided Cancer Diagnosis or antidiastole of the present invention in the Coloration experiment result of kinds of tumors tissue and benign disease.
Conclusion: the immunohistochemical diagnosis kit that uses the HAb18G/CD147 carcinoma marker for pathologic diagnosis of tumor of the present invention, carry out immunohistochemical staining and can obtain good coloration result, its cell membrane positive staining is clear and definite, clear, almost without unspecific staining, can obtain accurately judged result.
The immunohistochemical diagnosis kit of the HAb18G/CD147 carcinoma marker for the pathologic diagnosis of tumor purposes of the present invention, adopt the ultimate principle of the specific bond of immunology antigen and antibody " one to one ", with specificity HAb18 antibody tissue is dyeed, by distribution and the expression of HAb18G/CD147 carcinoma marker in tissue, the relation of further clear and definite HAb18G/CD147 carcinoma marker and cancer; Use the SP Immunohistochemical Method, make the multistage amplification of antigen-antibody reaction signal, the susceptibility that detects to improve the HAb18G/CD147 carcinoma marker.Therefore, HAb18G/CD147 carcinoma marker immunodiagnosis reagent kit is the specific reaction by PATHOMORPHOLOGICAL OBSERVATION OF PULLORUM and HAb18G/CD147 carcinoma marker, to improve the accuracy of pathological diagnosis, the character of tumour is judged in research, analyze the possibility of tumour origin, and the assessment of prognosis and treatment is all had great importance.
HAb18G/CD147 carcinoma marker immunodiagnosis reagent kit is on probation many clinical disease natural sciences, sheet is read in dyeing by more than 2000 routine clinical tumor tissue specimens, the result shows, HAb18G/CD147 molecule wide expression is in malignant tumour (comprising lung cancer, the cancer of the esophagus, cancer of the stomach, colorectal cancer, liver cancer, breast cancer, oophoroma, the cervical carcinoma etc.) tissue of epithelium source property, in normal structure and the low expression of benign disease tissues, also present expression trend at the other atypical hyperplasias of some cancers, active proliferation cell.As, lung cancer specificity 90%, sensitivity 89.8%; Cancer of the esophagus specificity 93.6%, sensitivity 90.2%; Cancer of the stomach specificity 76%, sensitivity 68%; Breast cancer specificity 83.5%, sensitivity 71.4%; Liver cancer specificity 68%, sensitivity 80.0%; Colorectal cancer specificity 96%, sensitivity 57.9%.The clinical research data show the HAb18G/CD147 molecule be a kind of novel, have broad spectrum activity and than the tumor markers of high specific.HAb18G/CD147 carcinoma marker immunohistochemical diagnosis kit can help types of organization, the tumor-localizing of pathologist diagnosing tumour, the dynamic change of reflection tumour, and judging prognosis, antidiastole tumour/non-Neoplasms by histologic type and characteristics.Make that tumor patient can obtain timely, early stage, correct diagnosis, timely and effectively treatment, and help the patient to the judgement of prognosis curative effect.
HAb18G/CD147 carcinoma marker immunodiagnosis reagent kit compares with Diagnostic Value of Several Serum Tumor Markers associated antibodies and the kit (such as AFP, CEA, CA15-3, CA199, CA125, Her-2, ER, PR etc.) of at present clinical practice, embody tumour and express wide spectrum, sensitivity, specificity height, have high clinical value and wide market application foreground, can be pathologic diagnosis of tumor and prognosis and judge a kind of wide spectrum, new product comparatively accurately are provided.
Claims (2)
1. immunohistochemical diagnosis kit that is used for the HAb18G/CD147 carcinoma marker of pathologic diagnosis of tumor purposes, clinical practice is characterized in that in auxiliary diagnosis or the antidiastole of human cancers the content of this kit comprises:
1) be used for to remove the blocking agent of people's histotomy endogenous enzyme, this blocking agent is that 3% superoxol and concentration are that 80% methanol solution forms by concentration;
2) for the nonspecific proteins sealer that seals people's histotomy intersection albumino reaction unspecific staining; This sealer is formulated by 3-5% Normal animal serum and phosphate buffer;
Described people's histotomy is paraffin section and frozen section;
3) first antibody that the HAb18G/CD147 carcinoma marker is combined in human body cancer tissue; This first antibody is the mouse monoclonal antibody of specific binding HAb18G/CD147 carcinoma marker, and its working concentration is 10-100 μ g/mL;
Described first antibody is preserved in the liquid at the antibody of 10-100 μ g/mL concentration and is preserved, and this antibody is preserved liquid and included 1% bovine serum albumin(BSA), 0.1% collagen, 0.01mol/L phosphate buffer, 0.05% NaN
3, this antibody is preserved liquid can keep first antibody 2-8 ℃ of lower the preservation 24 months;
4) second antibody that can be combined with first antibody; This second antibody is the polyclonal antibody of biotin labeled sheep or the anti-mouse IgG of rabbit;
5) be used for enzyme mark Streptavidin in conjunction with biotin on the second antibody; This enzyme mark Streptavidin is the Streptavidin of horseradish peroxidase-labeled, is chromogenic substrate;
6) with the chromogenic reagent of chromogenic substrate effect, this chromogenic reagent is that benzidine (DAB) solution, trishydroxymethylaminomethane (TrisHcl) damping fluid and deionized water are formulated, more than the preparation consumption volume ratio of three kinds of reagent be 1:1:50;
7) the used buffer system of kit is phosphate buffer Na
2HPO
4-NaH
2PO
4-NaCl, concentration is 0.01mol/L, pH is 7.2 ~ 7.45.
2. the immunohistochemical diagnosis kit of the HAb18G/CD147 carcinoma marker for the pathologic diagnosis of tumor purposes claimed in claim 1 is characterized in that at the colouring method of multiple cancerous tissue, comprising:
People's histotomy with aquation after, add an amount of endogenous enzyme blocking agent, reacted 10 minutes, phosphate buffer cleans; Drip 1 ~ 2 of nonspecific proteins sealer, reacted 10 minutes; Drip 1 ~ 2 of first antibody, 37 ℃ were reacted 2 hours, and phosphate buffer cleans; Drip 1 ~ 2 of second antibody, 22-28 ℃ was reacted 20 minutes, and phosphate buffer cleans; Drip 1 ~ 2 of enzyme mark streptavidin, 22-28 ℃ was reacted 15 minutes, and phosphate buffer cleans; Drip 1 ~ 2 of freshly prepared chromogenic reagent, 22-28 ℃ was reacted 1 ~ 10 minute, and phosphate buffer cleans; Bush uniformly dyeing nuclear, gradient alcohol dehydration, dimethylbenzene are transparent, neutral gum mounting, observation by light microscope; Above-mentioned phosphate buffer cleans and is that concussion was cleaned 5 minutes each 3 times.
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