CN101598731A - A kind of immunohistochemical diagnosis kit that is used for the pathologic diagnosis of tumor purposes - Google Patents
A kind of immunohistochemical diagnosis kit that is used for the pathologic diagnosis of tumor purposes Download PDFInfo
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Abstract
The invention discloses a kind of immunohistochemical diagnosis kit that is used for the HAb18G/CD147 carcinoma marker of pathologic diagnosis of tumor purposes, comprising: the blocking agent of removing endogenous enzyme in people's histotomy; Be used for sealing the sealer of people's histotomy intersection albumino reaction; The first antibody that combines with HAb18G/CD147 carcinoma marker in the body tumor tissue; The biotin labeling two that can combine with first antibody is anti-; Be used in conjunction with two anti-enzyme mark Avidins, chromogenic reagent, and phosphatebuffer buffer system etc.This kit has the advantages that specificity is good, susceptibility is high, stable, general, be mainly used in the pathological diagnosis of epithelium source property malignant tumour (liver cancer, lung cancer, the cancer of the esophagus, cancer of the stomach, colorectal cancer, breast cancer, cervical carcinoma, oophoroma etc.), help early warning screening, early diagnosis, etiologic diagnosis, therapeutic response and the curative effect monitoring of tumour, the relapse and metastasis and the prognosis of tumour to judge.
Description
Technical field
The invention belongs to bioengineering field, relate to a kind of kit that is used for clinical diagnosis, particularly a kind of immunohistochemical diagnosis kit that is used for the HAb18G/CD147 carcinoma marker of pathologic diagnosis of tumor purposes.
The HAb18G/CD147 carcinoma marker is a kind of novel, wide spectrum and higher specific carcinoma marker, with a kind of specific antibody at the HAb18G/CD147 carcinoma marker, in conjunction with general SABC method, make HAb18G/CD147 carcinoma marker immunohistochemical diagnosis kit, be applied to multiple epithelium source property malignant tumor tissue Pathologic specimen, carry out pathologic diagnosis of tumor, help early warning screening, early diagnosis, etiologic diagnosis, therapeutic response and the curative effect monitoring of tumour, the 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 press for the diagnosis that can be used for tumour and the technological means or the method for treatment.Current, be that the 3P medical science of core has become assurance, improved the important directions of level of human health with prospective medicine, preventive medicine and individuation medical treatment, the discovery of carcinoma marker is the important key that realizes 3P medical science with using.
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 takes place, cell differentiation, help early warning screening, early diagnosis, etiologic diagnosis, therapeutic response and the curative effect monitoring of tumour, the 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 the application of novel tumor mark have 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, methods such as combining yeast double cross, co-immunoprecipitation, mass-spectrometric technique, catch this antibody at antigen molecule.The discovery of novel tumor mark and application have vital role to setting up prognosis monitorings such as tumour early warning examination, early diagnosis, etiologic diagnosis, therapeutic response and transfer and relapse etc.
SABC is a principle of utilizing antigen to combine with antibody specificity, make developer (fluorescein, enzyme, metallic ion, the isotope) colour developing of labelled antibody determine histocyte endoantigen (polypeptide 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 are formed, 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 Babl/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 the 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%) the SABC result 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 lesion at mammary gland are organized as feminine gender.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 on 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 carcinoma cell film, 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 morphology diagnosis in the section of HE dyeing, and the case that difficult and problem are arranged is 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.
Kind surplus the related neoplasms mark antibody of present clinical use has 100, 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 the back packing abroad.Do not have regular lot number, antibody of using in pathological diagnosis and antibody batch differences is bigger, and is repeatable poor, and the result is person of modern times's meaning not.
Summary of the invention
The objective of the invention is to, a kind of immunohistochemical diagnosis kit that is used for the HAb18G/CD147 carcinoma marker of pathologic diagnosis of tumor purposes is provided.This diagnostic kit is the biological characteristics that utilizes the combination of Ag-Ab specificity, with HAb 18 specific monoclonal antibodies the clinical pathology tissue specimen is dyeed, by SP SABC method, observe the expression of HAb18G/CD147 carcinoma marker in the property malignant tumour of epithelium source, clinical practice is in the auxiliary diagnosis or the antidiastole of epithelium source property malignant tumour.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 that is used for the HAb18G/CD147 carcinoma marker of pathologic diagnosis of tumor purposes is characterized in that the content of this kit comprises:
1) be used for removing the blocking agent of people's histotomy endogenous enzyme, this blocking agent is that 3% superoxol and concentration are that 80% methanol solution is formed by concentration;
2) be used for sealing people's histotomy and intersect the nonspecific proteins sealer of albumino reaction unspecific staining; This sealer is formulated by 3-5% Normal animal serum and phosphate buffer;
3) first antibody that combines with HAb18G/CD147 carcinoma marker in the human body cancer tissue; This first antibody is the mouse monoclonal antibody of specificity in conjunction with the HAb18G/CD147 carcinoma marker, and its working concentration is 10-100 μ g/mL;
4) second antibody that can combine 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 marking Streptavidin in conjunction with the enzyme of 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 preservation liquid can be kept antibody and preserve 24 months down at 2-8 ℃.
The technical characterstic that is used for the HAb18G/CD147 carcinoma marker immunohistochemical diagnosis kit of 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 made up 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 the 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-buffered liquid systems (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 this 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 how tame hospital, sheet is read in the dyeing of routine tumor tissues sample surplus in the of clinical 2000, 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 lesion tissue, also present expression trend in 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, higher specific carcinoma marker, this kit are 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), be 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 describes in further detail the present invention.
Embodiment
The HAb18G/CD147 carcinoma marker immunohistochemical diagnosis kit that is used for cancer auxiliary diagnosis or antidiastole of the present invention, be mainly used in the pathological diagnosis of epithelium source property malignant tumour (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, the relapse and metastasis and the prognosis of tumour to judge.Comprise in this kit content:
1, being 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 is by volume formed by concentration;
2, be used for sealing the nonspecific proteins sealer of people's 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 antibody of specificity in conjunction with the 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 combine 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 marking Streptavidin in conjunction with the enzyme of biotin on the second antibody; This enzyme mark Streptavidin is the connection enzyme avidin 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.
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 successful key element mainly contains the antibody that specificity is good, broad spectrum activity is good, reliable reagent and experimental technique, strict quality 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 this 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:,,, determine to select research object according to the histopathology morphological feature as the pacing items of determining case group and control group sample with goldstandard HE decoration method.
Embodiment 1:
The HAb18G/CD147 carcinoma marker immunohistochemical diagnosis kit that is used for cancer auxiliary 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 cancer auxiliary 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 cancer auxiliary 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
The HAb18G/CD147 carcinoma marker immunohistochemical diagnosis kit that is used for cancer auxiliary 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 dyeing experimental result that is used for the HAb18G/CD147 carcinoma marker immunohistochemical diagnosis kit of cancer auxiliary diagnosis or antidiastole at kinds of tumors tissue and benign disease of the present invention.
Conclusion: use the immunohistochemical diagnosis kit that is used for the HAb18G/CD147 carcinoma marker of 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 do not have unspecific staining, can obtain judged result accurately.
The immunohistochemical diagnosis kit that is used for the HAb18G/CD147 carcinoma marker of 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 SP SABC method, make the multistage amplification of antigen-antibody reaction signal, to improve the susceptibility that the HAb18G/CD147 carcinoma marker detects.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.
How tame HAb18G/CD147 carcinoma marker immunodiagnosis reagent kit is on probation in clinical disease natural sciences, sheet is read in dyeing by routine clinical tumor tissue specimen surplus 2000, 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 lesion tissue, 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 a higher specific tumor markers.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, the types of organization of antidiastole tumour/non-tumour and characteristics.Make that tumor patient can obtain timely, early stage, correct diagnosis, treatment timely and effectively, and help the judgement of patient to the prognosis curative effect.
HAb18G/CD147 carcinoma marker immunodiagnosis reagent kit compares with the kinds of tumors mark associated antibodies and the kit (as AFP, CEA, CA15-3, CA199, CA125, Her-2, ER, PR etc.) of clinical practice at present, embody tumour and express wide spectrum, sensitivity, specificity height, have high clinical value and wide market application prospect, can be pathologic diagnosis of tumor and prognosis and judge a kind of wide spectrum, new product comparatively accurately are provided.
Claims (7)
1, a kind of immunohistochemical diagnosis kit that is used for the HAb18G/CD147 carcinoma marker of pathologic diagnosis of tumor purposes is characterized in that the content of this kit comprises:
1) be used for removing the blocking agent of people's histotomy endogenous enzyme, this blocking agent is that 3% superoxol and concentration are that 80% methanol solution is formed by concentration;
2) be used for sealing people's histotomy and intersect the nonspecific proteins sealer of albumino reaction unspecific staining; This sealer is formulated by 3-5% Normal animal serum and phosphate buffer;
3) first antibody that combines with HAb18G/CD147 carcinoma marker in the human body cancer tissue; This first antibody is the mouse monoclonal antibody of specificity in conjunction with the HAb18G/CD147 carcinoma marker, and its working concentration is 10-100 μ g/mL;
4) second antibody that can combine 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 marking Streptavidin in conjunction with the enzyme of 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 that is used for the HAb18G/CD147 carcinoma marker of pathologic diagnosis of tumor purposes as claimed in claim 1 is characterized in that, described people's histotomy is paraffin section and frozen section.
3, the immunohistochemical diagnosis kit that is used for the HAb18G/CD147 carcinoma marker of pathologic diagnosis of tumor as claimed in claim 1, it is characterized in that, described first antibody is preserved in the liquid at the antibody of 10-100 μ g/mL concentration and is preserved, this antibody is preserved liquid and is included bovine serum albumin(BSA), collagen, phosphate buffer and bacteriostatic agent, and this antibody preservation liquid can be kept antibody and preserve 24 months down at 2-8 ℃.
4, the immunohistochemical diagnosis kit that is used for the HAb18G/CD147 carcinoma marker of pathologic diagnosis of tumor as claimed in claim 1, it is characterized in that described antibody is preserved liquid and contained 1% bovine serum albumin(BSA), 0.1% collagen, 0.01mol/L phosphate buffer and 0.05%NaN
3
5, the immunohistochemical diagnosis kit of the described HAb18G/CD147 carcinoma marker that is used for the pathologic diagnosis of tumor purposes of 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.
6, the described immunohistochemical diagnosis kit that is used for the HAb18G/CD147 carcinoma marker of pathologic diagnosis of tumor purposes of claim 1 is used for early diagnosis, etiologic diagnosis, therapeutic response and the curative effect monitoring of epithelium source property malignant tumour, the relapse and metastasis of tumour and the application that prognosis is judged.
7, application as claimed in claim 6 is characterized in that, described skin source property malignant tumour is meant liver cancer, lung cancer, the cancer of the esophagus, cancer of the stomach, colorectal cancer, breast cancer, cervical carcinoma, oophoroma or other known cancer.
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