CN1296711C - Breast cancer Her-2 immunohisto chemical diagnostic kit - Google Patents
Breast cancer Her-2 immunohisto chemical diagnostic kit Download PDFInfo
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- CN1296711C CN1296711C CNB200410020546XA CN200410020546A CN1296711C CN 1296711 C CN1296711 C CN 1296711C CN B200410020546X A CNB200410020546X A CN B200410020546XA CN 200410020546 A CN200410020546 A CN 200410020546A CN 1296711 C CN1296711 C CN 1296711C
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Abstract
The present invention relates to an Her-2 immunohistochemical diagnostic reagent kit for breast cancers. The content of the kit comprises a nonspecific protein blocker, a mouse anti-human initial antibody, a sheep anti-mouse monoclonal connecting antibody, an enzyme labeled PAMAM reagent, etc., wherein the coloring reagent is prepared from a chromophoric oxidation reactant, a chromophoric agent and a color development buffer solution. An Her-2 immunohistochemical diagnosis for breast cancers is carried out by using the biological nature of a PAMAM dendritic polymer to label the antibodies, not only staining visibility is obviously increased, immunoreactive specificity and sensitivity are improved, but also the detection of Her-2 overexpression is more accurate; patients with Her-2/neu protein overexpression can obtain effective and timely treatment, can reduce unnecessary medical expenses, can improve life quality and can prolong survival time. In vitro diagnostic detection has easy operation, and early diagnoses for tumors are exact, stable and effective.
Description
Technical field
The present invention relates to a kind of clinical tumor diagnosis kit that is used for, particularly a kind of biological nature labelled antibody that utilizes PAMAM dendron shape polymer carries out the kit of immunohistochemical diagnosis to the Her-2 of breast cancer.Also can be used for the detection of the expression of other various cancerous tissue diagnosis of Her-2-2.
Background technology
The breast cancer number of patients sharply rises in recent years, and the incidence of disease occupies first of female tumor.China has ten thousand women of 5-9 that breast cancer takes place every year approximately, and ten thousand women of 3-4 die from breast cancer.In big coastal city such as Shanghai, breast cancer incidence occupies first of female tumor.There are every year ten thousand patient with breast cancers of 12-15 to observe and to treat approximately.In all patient with breast cancers, there is the people of 25-30% that the overexpression of Her-2/neu (EGF-R ELISA) albumen is arranged, the type tumour has more metastatic, danger.U.S. Society of Oncology just recommended " Her-2/neu " as the breast cancer marker thing in 2000, and no matter in diagnosis at that time, the detection when still recurring all has important value.Delivered the hyperplasia of Her-2 and human breast cancer from Si Laiman people such as (Slamon) in 1987, since the prognosis correlative study, the research of Her-2 obtains remarkable progress.U.S. Roche Holding Ag in 1998 releases treatment breast cancer new drug Herceptin (He Saiting) and obtains FDA (food and drug administration) approval listing.And rapidly in Canada, Europe, Japan obtains using.The application of new drug Herceptin has more promoted the development of the clinical examination of Her-2 gene and albumen.To American Society of Clinical Oncology in 2000 " the tumor marker guide " published point out " in the primary breast cancer case, no matter being at that time; still during postoperative recurrence; the overexpression of Her-2 judges that to prognosis the selection of therapeutic scheme etc. all has great importance " again in diagnosis.But the breast cancer diagnosis reagent running program that has had at present is loaded down with trivial details, non-specific height, and specificity is low, can not diagnose pernicious breast cancer rapidly, exactly, observe its prognosis conversion.
PAMAM (polyamide-amide) is a kind of nanoscale dendron shape polymer, and PAMAM Dendrimer dendrimer is a class new polymers synthetic in recent years and that develop rapidly.This polymkeric substance and traditional polymer phase ratio, can be on nanometer level strict controlling Design molecular size, structure and surface group, thereby have accurate molecular structure, fine and close spherical shapes, monodispersity, fabulous water-soluble.Sa Dun people such as (SATON.) delivers and carries few nuclear acid anhydride acid gene fragment with PAMAM and carry out in the body and experiment in vitro calendar year 2001, proves that it has extraordinary propagation function.Pa Teliannike people such as (PATRIANILK) used PAMAM binding antibody fragment magnetic target therapy prostate cancer in 2002.The patent No. be US2003/0044407A1's " transmission or diagnostic reagent that the immune lipid of labelled antibody or antibody fragment or polymer are used for systematic treating " disclose calendar year 2001 and opened, Ai Site (Chang, Esther H) people such as, use PAMAM mark rare metal and antibody and inject in the body, carry out imaging diagnosis by nuclear magnetic resonance measuring.But because of the toxic action that it decomposes in vivo, metabolism produced it be unclear that, have animal experiment study to show, certain PAMAM dosage can make mouse produce the hemolytic variation.Therefore, experiment is difficult to widespread use in the body.
Studies confirm that in a large number, the Her-2 positive patient is all insensitive to the anti-breast cancer treatment of existing conventional chemotherapy, radiotherapy and hormone, and postoperative easily recurs, and shifts fast, time-to-live is short, and above-mentioned case is all closely related with Her-2/neu (c-erbB-2) protein overexpression.Some development countries have set up some effective Her-2/neu genes and Protein Detection, evaluation criterion, be used to instruct the effect with predicted treatment, but its reagent costliness detect trouble.
Summary of the invention
The Her-2 immunohistochemical diagnosis kit that the purpose of this invention is to provide a kind of breast cancer, it utilizes the biological nature labelled antibody of PAMAM dendron shape polymer, Her-2 immunohistochemical diagnosis to breast cancer, it is visual not only obviously to enlarge dyeing, improve immunoreactive specificity, susceptibility, the detection of HER-2 overexpression is more accurate, and make with Her-2/neu protein overexpression patient and can obtain effectively, treatment timely, reduce the unnecessary health care expenditures of patient, improve patient's life quality and prolong life span, the in-vitro diagnosis detecting operation is easy, accurate to early diagnosis of tumor, stable, effectively.
The object of the present invention is achieved like this: the nonspecific proteins blocking agent that comprises intersection albumino reaction unspecific staining in the blocking-up people histotomy in this kit content, the mouse-anti people's who combines with tissue antigen initial antibodies, the monoclonal of the sheep anti mouse that can be connected with initial antibodies connects antibody, being used for the mark monoclonal connects antibody, has the expansion visual effect, improve the enzyme mark PAMAM reagent of susceptibility, by mutual synergy improve the microscopic examination Color by the color development oxidation agent, colour former, the chromogenic reagent that the colour developing damping fluid is deployed into routinely, the weight ratio of each reagent dosage that splashes into successively during operation is: nonspecific proteins blocking agent: initial antibodies: connect antibody: enzyme mark PAMAM reagent: chromogenic reagent=5～10: 1～10: 5～10: 5～10: 10.
The described nonspecific proteins blocking agent that is used for the albumino reaction of non-specific blocking-up people histotomy intersection is made up of animal blood serum.
Described initial antibodies is the specific antibody at the prepared mouse-anti people of specificity Her-2 antigen in the human body breast cancer tissue.
The described specificity initial antibodies that is used for is made up of the antibody of sheep anti mouse with the connection antibody that is connected bridge between the chromogenic reagent.
The PAMAM that the described enzyme that is used for labelled antibody is marked in the PAMAM reagent is a kind of nanoscale dendron shape polymer, there is highdensity amino group on its surface, positively charged when the pH of physiological environment, combine with electronegative antibody protein, enzyme mark PAMAM reagent is to be mixed with positively charged PAMAM dendron shape polymer by the antibody of horseradish peroxidase-labeled, react at ambient temperature and made in 10～15 minutes, the melting concn of the two is 0.1～10: 1 mol ratio.
Color development oxidation agent in the described developer is made up of 3% aqueous hydrogen peroxide solution, and brown colour former is selected DAB for use, and red colour former is selected AEC for use, and the purple blue colour former is selected BCIP/NBT for use, and the colour developing damping fluid is 0.05Mol Tris-HCl; The modulation of chromogenic reagent, the damping fluid that will develop the color is earlier got 2.5ml and is loaded in another test tube, splashes into two of colour formers, and every 50 μ l splashes into one of color development oxidation agent again, and every 50 μ l spins upside down for several times gently, and is standby after mixing.Can prepare by this ratio according to the quantity of dyeing slice, thin piece.The weight ratio of its consumption is: colour developing damping fluid: colour former: color development oxidation agent=50: 2: 1.
Described human breast carcinoma tissue is the tissue specimen of excision, and its specification of drawing materials is 1 * 1 * 0.5mm, and at 24～72 hours internal fixation in 10% neutral formalin solution.
Because utilization of the present invention contains the biological nature labelled antibody of ammonia functional group macromolecule PAMAM tree ridge portion, so use it for the immunohistochemical in-vitro diagnosis of the Her-2 of breast cancer, has opened up new in-vitro diagnosis method.This is that a kind of special albumen that a part exists in cell or tissue carries out immunological response with corresponding antibody, carries out fractographic diagnostic techniques then.PAMAM is a kind of nanoscale dendron shape polymer that is synthesized, and Dendrimer is made up of the nuclear of centre and the branch that distributes towards periphery.The density of nuclear whole molecule of decision and surface charge, the placement of its size, shape and functional group can accurately be controlled, and presents the monodispersity that can better be controlled on diameter.Simultaneously, it also has better water solubility.Through polyreaction repeatedly, formed highdensity amino group on its surface, positively charged when the pH of physiological environment, can combine with electronegative antibody protein, obviously improve susceptibility and the specificity that detects, this in-vitro diagnosis method is better than other commonsense method of available technology adopting.In addition, the detection of HER-2 expression is the significant observation index to optimum tumor of breast malignant change.The present invention is to utilize PAMAM to come labelled antibody to the test of Her2 albumen, Her-2 immunohistochemical diagnosis to breast cancer, it is visual not only obviously to enlarge dyeing, improve immunoreactive specificity, susceptibility, the detection of Her-2 overexpression is more accurate, and make with Her-2/neu protein overexpression patient and can obtain effectively, treatment timely, reduce the unnecessary health care expenditures of patient, improve patient's life quality and prolong life span, the survival rate that breast cancer women takes place is increased, the in-vitro diagnosis detecting operation of Her-2 overexpression is easy, accurate to early diagnosis of tumor, stable, effectively.It is less that this in-vitro diagnosis method and other method have been compared molecular weight, easier infiltration, the advantage that susceptibility is high.
Kit of the present invention and Her-2 polyclonal antibody kit [Herceptin Test] compare experiment.
Used experiment material: primary breast cancer 106 routine surgical material paraffin organization sections.Use this kit to carry out immunohistochemical staining simultaneously, carry out the detection of polyclonal antibody kit, carry out double blinding respectively and judge, positive and negative case statistical procedures it with serial section.Its result shows; The negative concordance rate of the positive concordance rate of this kit and [Herceptin Test]: 42/50=86.0%, this kit and [Herceptin Test]: 56/56=100%, the diagnosis concordance rate of this kit and [Herceptin Test]: (42+56)/(50+56)=98/106=92.4%.
Conclusion: use this kit to carry out immunohistochemical staining and can obtain good coloration result, its cell membrane positive staining is clear and definite, clear, does not almost have unspecific staining, can obtain judged result accurately easily.Show significant correlativity with the comparative study of [Herceptin Test].Though at positive rate, diagnosis has inconsistent case to exist, the Herciptin Test positive of this and other bibliographical information and genetic test does not see that the false positive results of gene magnification meets.Therefore, this kit specificity is higher, more accurate.
Description of drawings
Below in conjunction with accompanying drawing the present invention is further described.
Fig. 1 is that a kind of microscopically Her-2 coloration result of the present invention is judged collection of illustrative plates.
Embodiment:
Below in conjunction with embodiment the present invention is further described.Comprise the nonspecific proteins blocking agent in this kit content, mouse-anti people's initial antibodies, the monoclonal of sheep anti mouse connects antibody, enzyme mark PAMAM reagent, the chromogenic reagent that is deployed into by color development oxidation agent, colour former, colour developing damping fluid etc.Will splash into each reagent successively during operation, the weight ratio of its consumption is: nonspecific proteins blocking agent: initial antibodies: connect antibody: enzyme mark PAMAM reagent: chromogenic reagent=5～10: 1～10: 5～10: 5～10: 10.Can be according to actual operating position, packing 20 person-portions, 50 person-portions, isodose each reagent of 100 person-portions in every box according to the above ratio.
The Her-2 immunohistochemical diagnosis kit for preparing breast cancer by every box 50 person-portions
The nonspecific proteins blocking agent is made up of animal blood serum, with animals such as sheep, horse, rabbits, extract its normal blood after, obtain standby behind the separation of serum.Its effect is to intersect albumino reaction in the non-specific inhibition people histotomy.The 6ml/ box
Initial antibodies is the monoclonal antibody specific at the prepared mouse-anti people of specificity Her-2 antigen in the human body breast cancer tissue.The 10ul-100ul/ example, the 2ml/ box.
Connect antibody and form, serve as the bridge that is connected between specificity initial antibodies and the chromogenic reagent by the antibody of sheep anti mouse.The 6ml/ box.
Enzyme mark PAMAM reagent is used for labelled antibody, PAMAM is the tree-shaped polymer of a kind of nanoscale, there is highdensity amino group on its surface, positively charged when the pH of physiological environment, combine with electronegative antibody protein, enzyme mark PAMAM reagent is to be mixed with positively charged PAMAM Dendrimer by the antibody of horseradish peroxidase-labeled, reacts at ambient temperature and makes in 10～15 minutes, and the melting concn of the two is 0.1～10: 1 mol ratio.Can stablize and be dissolved in the damping fluid.The 6ml/ box.
The color development oxidation agent is made up of 3% aqueous hydrogen peroxide solution.The 2ml/ box.
Colour former can be selected a kind of in the following reagent for use according to the needs of colour developing, be that brown colour former is selected DAB (3 for use, 3-diaminobenzidine tetrahydrochloride), red colour former is selected AEC (3-amino-9ethylcarbazole) for use, and the purple blue colour former is selected BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium) for use.The 3ml/ box.
The colour developing damping fluid is selected the Tris-HCl damping fluid of 0.05Mol etc. for use.The 6ml/ box
Embodiment one: the concrete operations step that the present invention is used for the diagnosis of histotomy immunohistochemical staining is as follows:
1, synthetic PAMAM Dendrimer.
PAMAM Dendrimer is by 0 generation in generation to 10, and its molecular diameter belongs to the molecule of nanoscale between 10 to 130 .It is synthetic to be to adopt branch method, and promptly molecule is begun to outgrowth by the core.According to the rule of trees branch ready made monomer is met branch again by the branch of radial order, branch, repeated combination is got up successively.With ethylenediamine (EDA) is initiated core, and the 1st step reaction (a) generates 1 quaternary ester [being called 0.5 generation (G)] with methyl acrylate (MA) by the Michael addition reaction; The 2nd step reaction (b) quaternary ester and excessive ethylenediamine generation ammonolysis reaction generation 1 quaternary amide compound (being called for 1 generation).Repeat the reactions steps that (a) and (b) Michael addition and ammonia are separated, can obtain the PAMAM dendrimer compound of different algebraically.Synthetic product all carries out structural characterization with FT2IR, FAB2MS, 1H NMR and 13C NMR.It below is PAMAM synthetic reaction route.
Different pH value PAMAM are to the difference that influences of solubilising, and test also produces 5.0 generation PAMAM different quality concentration to the PAMAM solution of different quality concentration to solubilising.Prove by experiment in bigger pH value scope and descend stable existence, do not dissociate with different buffer conditions (water, PBS, 0.5mmol/L NaCl etc.).
2, preparation Her-2 antigen.
The fresh tumor tissues of Her-2 strong positive of human breast carcinoma excision extracts total RNA, with the cDNA sequences Design primer of pressing Her-2, carry out pcr amplification and obtain people PCR fragment, open with the same two expression plasmid Pet42b that cut enzyme with double digestion behind the purifying and be connected, obtain the prokaryotic expression plasmid of recombinating, change this plasmid over to Escherichia coli then and increase, after sequence verification, extract the reorganization prokaryotic expression plasmid, and transform expression bacterium BL21 with it.BL21 is inoculated in 37 ℃ of cultivation amplifications in the LB nutrient solution.Collect thalline in damping fluid, behind the broken bacterium of ultrasound wave, centrifugal, wash-out, collection.With the sample behind the WESTERN trace detection purifying.Purity: 96.0%.
Preparation initial antibodies: get anti-Her-2 working cardial cell storehouse hybridoma 2*10
7/ only, lumbar injection extracts ascites through the presensitized BALB/c mouse of paraffin oil after 10-14 days.
Purifying: ammonium sulfate precipitation, the A albumen-anti-Her-2 monoclonal antibody of Sepharose affinity chromatography exchange separation method separation and purification from the hybridoma cell strain nutrient solution.
Tiring of antibody: measure affinity of antibody in the culture supernatant through the ELISA method, reaching the maximum concentration in conjunction with 50% required ascites monoclonal antibody is 7.5 * 10
-6, affinity costant Ka=1.3 * 10
5Mol
-1Utilize the subclass of Roche Holding Ag to measure kit, detect the culture supernatant of 3 strain positive hybridoma cells, tiring with the monoclonal antibody of indirect elisa method detection cell culture supernatant and ascites to reach 10
-5, it is 10 that the purified back of ascites ELISA method detects antibody titer
-6
Antibody specificity: the protein blot analysis measures band and the antibody that molecular weight is 185dp and shows high specificity, reaction sensitivity through immunohistochemical staining.And not with non-Her-2 protein bound.Recombinant soluble antigen Her-2 routine immunization BALB/c mouse, extracting spleen cell and MOPC-21 derive, the BALB/c myeloma cell of IgG secretion molecule (K light chain) is merged preparation antibody, and screening, antibody purification, A albumen-Sepharose affinity chromatography exchange separate.
3, preparation nonspecific proteins blocking agent.
The nonspecific proteins blocking agent is made up of animal blood serum, with animals such as sheep, horse, rabbits, extract its normal blood after, obtain standby behind the separation of serum.
4, preparation connects antibody.
Connecting antibody is that the conventional antibody by sheep anti mouse for preparing is formed.
5, preparation enzyme mark PAMAM reagent.
By the positively charged PAMAM Dendrimer of made in the antibody of horseradish peroxidase-labeled or antibody fragment and the above-mentioned steps 1 (synthetic PAMAMDendrimer) with 0.1～10: the concentration of 1 molar ratio is mixed, room temperature condition reacted 10-15 minute down, made enzyme mark PAMAM reagent.This is fabulous ratio and order that an antibody that makes PAMAM and horseradish peroxidase-labeled mixes, is simple, effectively, stable, associated methods non-chemically.This method than chemistry or additive method effectively or more effective.
6, human breast carcinoma tissue is fixing.
The moment that the tissue of excision exsomatizes just begins various variations take place, as organizes isophagy, corruption etc.Fixing purpose makes this variation stop immediately exactly, preserves its morphology characteristics and antigenicity.The tissue of excision is cut into the tissue specimen of 1 * 1 * 0.5mm at once, is fixed in the conventional 10% neutral formalin solution.Optimal fixation method and time range are 24-72 hour.Be less than 24 hours fixing insufficient, surpass 72 hours antigenicities and begin to weaken.
7, the pathological tissue wax stone is suitable for.
Liquid-solid fixed through neutral buffered formalin, be used for processed conventionally and paraffin-embedded tissue, be suitable for this test.Do not recommend alcohol and other formalin substitute.
8, antigenicity is recovered to handle.
Paraffin-embedded histotomy, after dewaxing, put into 0.1M, pH 6.2 aqueous solution of citric acid, after the microwave device heat treated reaches 100 ℃ of boilings rapidly, reduce the irradiation dynamics and keep 90-98 ℃, 15-30 minute, take out container then and place room temperature dyeing again about 20 minutes, its antigenicity is recovered, increased staining power.
9, the modulation of chromogenic reagent.
Before using chromogenic reagent, the damping fluid that will develop the color is got 2.5ml and is loaded in another test tube, splashes into two of colour formers (every about 50 μ l, as follows), splashes into one of color development oxidation agent again, spins upside down gently for several times, and is standby after mixing.Can prepare by this ratio according to the quantity of dyeing slice, thin piece.The weight ratio of its consumption is: colour developing damping fluid: colour former: color development oxidation agent=50: 2: 1:.
10, dying operation step.
After above-mentioned histotomy is cleaned with distilled water, an amount of 3% aqueous hydrogen peroxide solution processing adds nonspecific proteins blocking agent 1-2 after 10 minutes and drips, reacted 10 minutes, do not wash after sopping up reactant liquor, adding the initial antibodies amount according to the size of organizing adjusts from 10 μ l to 100 μ l, after 30 minutes, adding connection antibody 1-2 dripped 30 minutes, enzyme-added mark PAMAM reagent 1-2 dripped 30 minutes, add modulated chromogenic reagent 100 μ l, 3-5 minute, more than between each step all PBS wash 3 times each 3 minutes, with the PBS around the clean tissue of clean filter paper sassafras, each step all can not make dry don't fail to keep organizing moistening of tissue, nuclear staining, and the bush uniformly dyeing is examined 1 minute, dehydration, transparent, mounting, microscopic examination, above-mentioned reaction is all at room temperature carried out.
11, this test is applicable to artificial and automatic staining.This test can operation automatically on supporting automatic staining instrument.
12, coloration result is judged.
0+: cell membrane cancer cell negative or the cell membrane stained positive is less than 10% (being judged as feminine gender during simple cell slurry dyeability)
1+: almost seldom have the intact cell film positive or have the cancer cell of intact cell film stained positive to be less than or equal 10% of whole cancer cells
2+: the intact cell film is weak-and the cancer cell of moderate stained positive is more than or equal to 10% of whole cancer cells
3+: the cancer cell of the strong stained positive of intact cell film is more than or equal to 10% of whole cancer cells
The result judges: 0+, 1+ are negative, 2+, 3+ positive (seeing Fig. 1 for details).
Embodiment two:
This antibody also is applicable to tumour puncture examination of castoff cells.Cellular incubation on cancerous tissue tumour puncture cell smear dyeing or the microslide; The puncture cell is evenly coated in and was fixed in immediately after to be dried on the microslide in the 10% neutral formalin solution 10 minutes.Cellular incubation is attached on the slide because of cancer cell on the microslide, can directly put into immobile liquid 10 minutes.Distilled water directly enters the 8th step among the embodiment one after cleaning, and uses 0.3% hydrogen peroxide methanol solution (H instead
2O
21ml: methyl alcohol; 99ml) blocking-up endogenous enzymes, 30 minutes reaction backs of room temperature PBS cleans 3 times.Other later steps are with embodiment one.
Embodiment three:
This kit also can be used for the detection of the expression of other various cancerous tissue diagnosis of Her-2-2.Method together
Embodiment one.
Claims (7)
1, a kind of Her-2 immunohistochemical diagnosis kit of breast cancer, it is characterized in that: the nonspecific proteins blocking agent that comprises intersection albumino reaction unspecific staining in the blocking-up people histotomy in the box content, the mouse-anti people's who combines with tissue antigen initial antibodies, the monoclonal of the sheep anti mouse that can be connected with initial antibodies connects antibody, being used for the mark monoclonal connects antibody, has the expansion visual effect, improve the enzyme mark PAMAM reagent of susceptibility, by mutual synergy improve the microscopic examination Color by the color development oxidation agent, colour former, the chromogenic reagent that the colour developing damping fluid is deployed into, the weight ratio of each reagent dosage that splashes into successively during operation is: nonspecific proteins blocking agent: initial antibodies: connect antibody: enzyme mark PAMAM reagent: chromogenic reagent=5～10: 1～10: 5～10: 5～10: 10.
2, the Her-2 immunohistochemical diagnosis kit of breast cancer according to claim 1 is characterized in that: the described nonspecific proteins blocking agent that is used for the albumino reaction of non-specific blocking-up people histotomy intersection, form by animal blood serum.
3, the Her-2 immunohistochemical diagnosis kit of breast cancer according to claim 1 is characterized in that: described initial antibodies is the monoclonal antibody specific at the prepared mouse-anti people of specificity Her-2 antigen in the human body breast cancer tissue.
4, the Her-2 immunohistochemical diagnosis kit of breast cancer according to claim 1 is characterized in that: the described specificity initial antibodies that is used for is made up of the antibody of sheep anti mouse with the connection antibody that is connected bridge between the chromogenic reagent.
5, the Her-2 immunohistochemical diagnosis kit of breast cancer according to claim 1, it is characterized in that: the PAMAM that the described enzyme that is used for labelled antibody is marked in the PAMAM reagent is the tree-shaped polymer of a kind of nanoscale, there is highdensity amino group on its surface, positively charged when the pH of physiological environment, combine with electronegative antibody protein, enzyme mark PAMAM reagent is to be mixed with positively charged PAMAM dendron shape polymer by the antibody of horseradish peroxidase-labeled, react at ambient temperature and made in 10～15 minutes, the melting concn of the two is 0.1～10: 1 mol ratio.
6, the Her-2 immunohistochemical diagnosis kit of breast cancer according to claim 1, it is characterized in that: the color development oxidation agent in the described chromogenic reagent is made up of 3% aqueous hydrogen peroxide solution, brown colour former is selected DAB for use, red colour former is selected AEC for use, the purple blue colour former is selected BCIP/NBT for use, and the colour developing damping fluid is the Tris-HCl of 0.05Mol; The modulation of chromogenic reagent, the damping fluid that will develop the color is earlier got 2.5ml and is loaded in another test tube, splash into two of colour formers, every 50 μ l splashes into one of color development oxidation agent again, every 50 μ l, spin upside down gently for several times, it is standby to mix the back, can prepare by this ratio according to the quantity of dyeing slice, thin piece, and the weight ratio of its consumption is: colour developing damping fluid: colour former: color development oxidation agent=50: 2: 1.
7, the Her-2 immunohistochemical diagnosis kit of breast cancer according to claim 1, it is characterized in that: described human breast carcinoma tissue is the tissue specimen of excision, its specification of drawing materials is 1 * 1 * 0.5mm, and at 24～72 hours internal fixation in 10% neutral formalin solution.
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| CN101256190B (en) * | 2008-03-27 | 2012-09-19 | 北京美康生物技术研究中心 | CA15-3, CEA, CA19-9, CA12-5, SF mammary cancer colloidal gold five joint inspection diagnostic reagent kit |
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