CN109836499A - The monoclonal antibody and its application of anti-human androgen receptor splicing variants 7 - Google Patents
The monoclonal antibody and its application of anti-human androgen receptor splicing variants 7 Download PDFInfo
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Abstract
A kind of antibody specifically binding people AR-V7, the antibody are to use AR-V7 polypeptide fragment as immunogene, the monoclonal antibody with AR-V7 specific binding being prepared by hybridoma；The polypeptide fragment is by choosing segment that sequence is SEQ ID NO.3 and artificial synthesized obtaining in AR-V7 that sequence is SEQ ID NO.2.Anti-human AR-V7 monoclonal antibody of the present invention, can specific recognition and AR-V7, and not with AR and other AR brief introduction variants ining conjunction with, with high degree of specificity.
Description
Technical field
The present invention relates to molecular biology fields, and in particular to a kind of list of anti-human androgen receptor splicing variants 7
Clonal antibody and its application.
Background technique
Prostate cancer is common one of the malignant tumour of urinary system, and it is pernicious swollen to occupy male for its disease incidence in the world
The 2nd of tumor, the death rate occupies the 5th.Androgen receptor (Androgen Receptor, AR), can be with after being activated by androgen
Tenuin A interaction, enhances the motility of cell, this may be related with the invasion transfer of prostate cancer, in forefront
It plays an important role during the occurrence and development of gland cancer.Therefore, AR is the important marker in prostate cancer diagnosis.
Late in the therapeutic process of prostate cancer, it is often used androgen deprivation therapy, but to will lead to cancer thin for this method
Born of the same parents generate drug resistance, thus development be castration-resistant prostate cancer (Castration-Resistant Prostate Cancer,
CRPC).Some researches show that the occurrence and development of prostate gland cancer cell and CRPC are related with the reactivation of AR signal path.Its
In, androgen receptor splicing variants 7 (Androgen Receptor Splice Variant 7, AR-V7) are androgen receptors
One kind of body splicing variants, structure is similar with AR, but it lacks androgen ligands binding domain, can not depend on androgen and hold
Continuous activation.AR-V7 plays a crucial role in the reactivation of AR signal path, and it is right during prostate cancer therapy to further illustrate
The reason of chemotherapeutics develops drug resistance, the expression of AR-V7 have also reacted the chemotherapy of patient for treatment's prostate cancer
Drug resistance sex differernce and chemicals to the therapeutic effect of prostate cancer.Therefore, it is necessary to which seeking one kind can stablize, is convenient
Specific detection AR-V7 method.
Summary of the invention
Based on this, the purpose of the present invention is to provide one kind can be with the monoclonal antibody of specific recognition AR-V7.
To achieve the goals above, it is as follows to provide specific technical solution:
A kind of antibody specifically binding people AR-V7, the antibody are to use AR-V7 polypeptide fragment as immunogene, system
Monoclonal antibody that is standby obtaining and being specifically bound with AR-V7；The amino acid sequence composition of the AR-V7 polypeptide fragment is such as
Shown in SEQ ID NO.3
In wherein some embodiments, the preparation method of above-mentioned antibody the following steps are included:
(1) mouse is immunized in SEQ ID NO.3 polypeptide fragment；
(2) mouse boosting cell after will be immune is merged with SP2/0 myeloma cell, obtains hybridoma；
(3) by the hybridoma secretion after, isolate and purify to get.
In wherein some embodiments, the preparation method of above-mentioned antibody the following steps are included:
(1) mouse is immunized in SEQ ID NO.3 polypeptide fragment；
(2) mouse boosting cell after will be immune is merged with SP2/0 myeloma cell；
(3) positive hybridoma cell for secreting anti-human AR-V7 is obtained using the screening of HAT Selective agar medium；
(4) it is cloned using positive cell strain of the limiting dilution assay to acquisition, obtains the anti-human AR-V7 for generating high-titer
Monoclonal antibody hybridoma cell；
(5) by the high-titer anti-human AR-V7 monoclonal antibody hybridoma cell secretion after, isolate and purify to get.
In wherein some embodiments, above-mentioned preparation method the following steps are included:
(1) it selects and amino acid sequence is prepared as the polypeptide fragment of SEQ ID NO.3；
(2) mouse is immunized in SEQ ID NO.3 polypeptide fragment；
(3) mouse boosting cell after will be immune is merged with SP2/0 myeloma cell, obtains hybridoma；
(4) positive hybridoma cell for secreting anti-human AR-V7 is obtained using the screening of HAT Selective agar medium；
(5) it is cloned using positive cell strain of the limiting dilution assay to acquisition, obtains the anti-human AR-V7 for generating high-titer
Monoclonal antibody hybridoma cell；
(6) it carries out that above-mentioned hybridoma is injected intraperitoneally to Balb/c mouse, takes ascites；
(7) separate from above-mentioned ascites using protein G affinity chromatography, purify and obtain specifically binding people AR-V7
Antibody.
The present invention also provides a kind of high stability antibody reagents, described including the above-mentioned antibody by antibody stabilization dilution agent
Antibody stabilization agent is selected from: BioStab Antibody Stabilizer, Sigma-55514.
In wherein some embodiments, in above-mentioned high stability antibody reagent, the concentration of antibody is 1-2 μ g/ml.
It is a further object of the present invention to provide a kind of applications of said monoclonal antibody.Specific technical solution is as follows:
Application of the above-mentioned high stability antibody reagent in the immunohistochemical kit of preparation detection people AR-V7.
Another object of the present invention is to provide a kind of immunohistochemical kits for detecting people AR-V7.
To achieve the above object, specific technical solution is as follows:
A kind of immunohistochemical kit detecting people AR-V7, which includes above-mentioned antibody or above-mentioned
High stability antibody reagent.
In wherein some embodiments, the immunohistochemical kit of above-mentioned detection people AR-V7 further includes HRP- sheep anti-Mouse
IgG working solution and DAB developing solution.
In wherein some embodiments, the immunohistochemical kit of above-mentioned detection people AR-V7 further includes endogenous peroxidating
Object enzyme blocking agent, confining liquid and antibody diluent.
Another object of the present invention is to provide a kind of applications of above-mentioned immunohistochemical kit, and specific technical solution is such as
Under:
Application of the above-mentioned immunohistochemical kit in the product of preparation prediction prostate cancer therapy effect.
Anti-human AR-V7 monoclonal antibody provided by the invention and its application have the following advantages that and effect:
Anti-human AR-V7 monoclonal antibody of the present invention is according to other splicing variants sequences of AR, AR-V7 and AR
Similarities and differences are designed and are prepared for people AR-V7, can specific recognition and AR-V7, and do not become with AR and other AR brief introductions
Allosome combines, and has high degree of specificity.
The present invention also provides AR-V7 monoclonal antibodies in the AR-V7 immunohistochemical kit for preparing detection prostate cancer
Middle application provides the immunohistochemical kit of AR-V7 in detection tissue or cell a kind of, the antibody in the kit by
Sigma antibody-STAB stabilizer is diluted to 1-2 μ g/ml, which is working concentration, without diluting when in use,
It is easy to use；Meanwhile the long period can be stored at room temperature still and have the activity of specific binding AR-V7, there is stability.
Kit of the present invention, on the one hand it includes monoclonal antibody can specific recognition AR-V7, provide one
The detection method of kind AR-V7, required reagent is integrated when on the other hand detecting AR-V7 to immunohistochemistry, makes exempting from for AR-V7
Epidemic disease groupization detects more intuitive, more convenient and quicker.
Detailed description of the invention
Fig. 1 is the result figure detected using anti-human AR-V7 immunohistochemical kit to prostate cancer tissue in embodiment 3；
Fig. 2 is immune group of the embodiment 4 using anti-human AR-V7 immunohistochemical kit to Vcap cell and PC3 cell sheet
Change the result figure of detection；
Fig. 3 is that embodiment 5 is immunized Vcap cell and H1975 cell sheet using anti-human AR-V7 immunohistochemical kit
Groupization detection
Fig. 4 is that the diluted AR-V7 monoclonal antibody of PBS is tied as primary antibody for immunohistochemical experiment dyeing in embodiment 6
Fruit；
Fig. 5 is that the diluted AR-V7 of Sigma BioStab Antibody Stabilizer is used as primary antibody in embodiment 6
In immunohistochemical experiment result coloration result；
Fig. 6 be in embodiment 6 the diluted AR-V7 of Antibody Stabilizer PBS as primary antibody for immunohistochemistry
Experimental result coloration result；
Fig. 7 is in embodiment 6The diluted AR-V7 of HRP-Stab is real for immunohistochemistry as primary antibody
Test result coloration result；
Fig. 8 is the immunohistochemical staining result of Vcap cell in embodiment 7；
Fig. 9 is the immunohistochemical staining result of PC3 cell in embodiment 7.
Specific embodiment
According to following embodiments, the present invention may be better understood.However, it should be readily apparent to one skilled in the art that implementing
Content described in example is merely to illustrate the present invention, is not intended to limit ground of the invention range.
Embodiment 1
According to the mRNA sequence of comparison expression AR, AR-V7 and other AR splicing variants, the specificity of AR-V7 is obtained
The area as a result, to from AR-V7 complete sequence SEQ ID NO.1 mRNA sequence CDS, chooses SEQ ID NO.2, synthetic antigen is determined
Determine cluster polypeptide fragment, the sequence is as follows:
SEQ ID NO.1:
MHHHHHHEVQLGLGRVYPRPPSKTYRGAFQNLFQSVREVIQNPGPRHPEAASAAPPGASLLLQQQQQQQ
QQQQQQQQQQQQQQQQQQQQQQETSPRQQQQQQGEDGSPQAHRRGPTGYLVLDEEQQPSQPQSALECHPERGCVPEP
GAAVAASKGLPQQLPAPPDEDDSAAPSTLSLLGPTFPGLSSCSADLKDILSEASTMQLLQQQQQEAVSEGSSSGRAR
EASGAPTSSKDNYLGGTSTISDNAKELCKAVSVSMGLGVEALEHLSPGEQLRGDCMYAPLLGVPPAVRPTPCAPLAE
CKGSLLDDSAGKSTEDTAEYSPFKGGYTKGLEGESLGCSGSAAAGSSGTLELPSTLSLYKSGALDEAAAYQSRDYYN
FPLALAGPPPPPPPPHPHARIKLENPLDYGSAWAAAAAQCRYGDLASLHGAGAAGPGSGSPSAAASSSWHTLFTAEE
GQLYGPCGGGGGGGGGGGGGGGGGGGEAGAVAPYGYTRPPQGLAGQESDFTAPDVWYPGGMVSRVPYPSPTCVKSEM
GPWMDSYSGPYGDMRLETARDHVLPIDYYFPPQKTCLICGDEASGCHYGALTCGSCKVFFKRAAEGKQKYLCASRND
CTIDKFRRKNCPSCRLRKCYEAGMTLGEKFRVGNCKHLKMTRP
SEQ ID NO.2:EKFRVGNCKHLKMTRP
Using common methods, such as Peptide synthesizer, the polypeptide such as SEQ ID NO.2 sequence is prepared.
Embodiment 2
The antigen determinant polypeptide segment (aa1) and keyhole limpet hemocyanin (KLH carrier protein) that embodiment 1 is prepared
Connection is formed coupling protein aa1-KLH-aa (abbreviation coupling protein).And use the coupling protein as antigen-immunized animal, system
Standby AR-V7 monoclonal antibody specific.The effect of the keyhole limpet hemocyanin is enhancing such as the polypeptide of SEQ ID NO.2 sequence
Immunogenicity.
1, animal immune: by coupling protein and Freund's adjuvant mixed in equal amounts 3 Balb/c mouse of fully emulsified rear injection, often
25 μ g of mouse is immunized 3 times, interval time 15 days altogether.
2, cell fusion, selectivity culture, screening: first 3 days of fusion, mouse peritoneal are injected without 25 μ g of adjuvant coupling protein,
The spleen cell for taking out immune mouse, by 2.0 × 107A SP2/0 myeloma cell and 2.0 × 108It is a through step (1) be immunized
The splenocyte of Balb/c mouse mixes, centrifugation, abandons supernatant, and slight oscillatory mixes, and in 37 DEG C of water-baths, 1ml is added dropwise in 90 seconds
Then 20ml DMEM culture medium is added dropwise in the PEG-1500 aqueous solution that volumetric concentration is 50%, supernatant is abandoned in centrifugation, then washes once,
Centrifugation abandons supernatant, obtains hybridoma.Hybridoma is selected in 96 porocyte culture plates using HAT Selective agar medium,
Total fusion rate > 95% is detected under mirror.The supernatant in monoclonal cell hole is selected to be detected under mirror, picking OD450In the hole of > 0.8
Cell is subcloned, and the final cell clone obtained to AR-V7 reacting positive rate > 99%, as secreting, anti-human AR-V7 is mono-
The hybridoma cell strain of clonal antibody.
3, cell clone: cloning the positive cell strain of acquisition, using limiting dilution assay, clones 3 times, finally obtains
7 plants of anti-human AR-V7 monoclonal antibody hybridoma cell line of high-titer are generated, expands culture, freezes.
4, prepared by ascites: 6-8 week old female Balb/c mouse with after paraffin oil processing 10 days, take hybridoma with 2 ×
106A cell/be only injected intraperitoneally.The ascites for being rich in antibody is obtained after 10 days from mouse peritoneal, measures titer of ascites >
105。
5, Purification: protein G affinity chromatography is used.Protein G affinity column is prepared first, uses PBS
It after balancing pillar, takes ascites to cross column, then cleans pillar with PBS, until OD value is close to zero, with the glycine hydrochloride of 50nmol/L
Salting liquid elution, collects eluent, measures the OD value of each collecting pipe, retain the eluent of peak region, eluent is received after dialysing
Collection.It is the monoclonal antibody of purifying through SDS-PAGE electroresis appraisal, purity is 98% or more.After measured, AR-V7 antibody concentration is
6.02mg/ml。
Embodiment 3: anti-human AR-V7 monoclonal antibody immunity group kit is detected for prostate cancer tissue
The present embodiment provides a kind of immunohistochemical kit and its application, which includes following reagent:
Endogenous peroxydase blocking agent, the H of 3% (v/v)2O2Solution is stored in 2-8 DEG C；
Confining liquid, 5% (w/v) bovine serum albumin(BSA) (BSA) solution that phosphate buffer PBS is prepared as solvent；
Anti- AR-V7 monoclonal antibody is prepared by 2 the method for embodiment, after being diluted to 1mg/ml by PBS, is used
Sigma BioStab Antibody Stabilizer stabilizer is diluted to 1 μ g/ml, is stored in 2-8 DEG C；
HRP- goat anti-mouse igg working solution is bought in neoformation technological development advanced in years Co., Ltd；
20 × DAB developing solution A is bought in neoformation technological development advanced in years Co., Ltd；
20 × DAB developing solution B is bought in neoformation technological development advanced in years Co., Ltd；
DAB buffer is bought in neoformation technological development advanced in years Co., Ltd.
The testing principle of the kit is as follows: being based on the intermolecular complementary structure of the two using immunology antigen and antibody
Property and high affinity can be specifically bound, and so that the enzyme chromogenic reagent of labelled antibody is determined group by redox reaction
Intracellular antigen is knitted, it is positioned, is qualitative.
It is connection AR-V7 monoclonal antibody and structural AR-V7 antigen first；
Second is that HRP- goat anti-mouse igg polymer identifies the AR-V7 monoclonal antibody connected；
Third is that chromogenic substrate is added, and the horseradish peroxidase on polymer can be catalyzed the H in DAB developing solution2O2Point
Solution, makes benzidine be oxidized to biphenyl imines, to make to show yellow or brownish discoloration in histotomy on antigen site；
Finally sample is redyed and mounting.
By microscopic develop the color situation, infer histotomy on AR-V7 there are sites and situation.
The histotomy two for being diagnosed as the fixed paraffin embedding of tissue formaldehyde of prostate cancer is obtained from hospital pathology department
, one is used as negative control, and one using kit described in the present embodiment for detecting AR-V7 protein expression.
1, dewax aquation
1) by 60 DEG C of the paraffin tissue sections of prostate cancer to be detected after roasting piece 30 minutes, it is placed in leaching packet 3 in dimethylbenzene
It is secondary, 10 minutes every time；
3) it is placed in dehydrated alcohol and impregnates 2 times, every time 5 minutes；
4) it is sequentially placed into 95%, 85%, 70% ethyl alcohol and impregnates, each 5 minutes；
5) it is placed in distilled water and impregnates 5 minutes；
6) PBS is rinsed 2 times, every time 3 minutes.
2, antigen retrieval
Slide is placed in the 1mM EDTA antigen retrieval solution boiled (pH=9.0), low fire heating in micro-wave oven
10min, cooled to room temperature.
Notice that the amount for repairing liquid when antigen retrieval must assure that slice is immersed in liquid always, has heated and be cooled to room temperature
Slice must remain in liquid in the process, can not take out slice and cool down in air.
3, endogenous peroxydase is blocked
1) PBS of the histotomy after antigen retrieval is rinsed 3 times, every time 5 minutes；
2) slice is taken out, tissue surrounding liquid, the test serum region on immunohistochemistry pen delineation slice are got rid of and dry；
3) 100~200 μ l endogenous peroxydase blocking agents are added in tissue regions, are incubated at room temperature 10 minutes；
4) PBS solution is rinsed 3 times, every time 5 minutes.
4, it closes:
Confining liquid is added dropwise on organizing region to be measured, until covering region to be measured, after being placed at room temperature for 30min, gets rid of extra liquid
Body.
5, AR-V7 antibody incubation
1) PBS solution is removed, the anti-AR-V7 monoclonal antibody of 100 μ l is added, PBS solution is added in negative control tissue slice,
Incubation at room temperature 60 minutes；
2) PBS solution is rinsed 3 times, every time 5 minutes.
6, enzyme mark polymer is incubated for
1) PBS solution is removed, 100 μ l HRP- goat anti-mouse igg working solutions are added, is incubated at room temperature 20 minutes；
2) PBS solution is rinsed 3 times, every time 5 minutes.
7, it develops the color
The preparation of DAB developing solution: DAB buffer 1ml, 20 × DAB developing solution A 50-100 μ is successively added dropwise in small test tube
L, 20 × DAB developing solution B 50-100 μ l are mixed, are protected from light to prepare DAB developing solution.
PBS solution is removed, the good DAB developing solution of 100~200 μ l Fresh is added, is protected from light is incubated for 3-10 points at room temperature
Clock；After having developed the color, distilled water flushing sample color development stopping.
It is redyed if it is necessary, haematoxylin can be carried out.
Tap water rinses, and 100~200 μ l haematoxylin body cell dyeing liquors is added to be incubated for 10~30 seconds；PBS solution or tap water
Indigo plant is returned in flushing.
Note: the intensity of the haematoxylin dyeing liquid according to effect and the length of incubation time: counterstain result causes carefully
The reaction of pale blue to dark blue color is presented in karyon, and mistake contaminates or insufficient dyeing is likely to jeopardize the judgement of correct result.
8, dehydration, transparent, mounting
1) it sets in 95% ethyl alcohol, impregnates 3 minutes；
2) it sets in dehydrated alcohol, impregnates 3 minutes；
3) dimethylbenzene is transparent；
4) neutral gum and coverslip mounting.
9, biomicroscope diagosis and result judgement
As a result judgment criteria is as follows:
It is positive: to have yellow or brownish discoloration on the cell membrane core of specific cells in histotomy, and without nonspecific
Background stainings；
It is negative: to have no yellow or brownish discoloration in expected cell in tissue.
Coloration result is shown in that Fig. 1, Figure 1A are negative control, and Figure 1B is AR-V7 monoclonal antibody immunity coloration result (AR-V7
It is colored as brown colouration part).It can be seen that without any brown colouration in Fig. 1 negative control, it is only blue-colored, show reality
Testing system, there is no problem, can effectively avoid non-specific background's coloring i.e. false positive.Brown part is positive coloring in Figure 1B,
AR-V7 is mainly expressed in the nucleus of Tumor Tissue Tumors cell, and can see brown colouration largely covering blue
Nucleus distribution.And then coloured without AR-V7 in normal immunocyte, it is normal negative findings, also without appearance
False positive and false negative.Show that AR-V7 in prostate cancer tissue tumour cell karyon, is coloured in distribution of specific in strong positive
Distribution.
Embodiment 4: prostate cancer cell line Vcap cell and PC3 cell sheet are immunized using immuning tissue's kit
Groupization detection
It is detection sample with the prostate cancer cell line Vcap and PC3 of culture, uses immunohistochemistry reagent described in embodiment 3
Box carries out immunohistochemistry detection by the following method:
1, fixed, smear: being added 4% (v/v) paraformaldehyde solution, be placed at room temperature for 15 minutes, distills water washing 3 times；Carefully
Born of the same parents' suspension is coated on anticreep glass slide, and 60 DEG C of roasting piece 1h, PBS are washed 3 times.
2, antigen retrieval
Slide is placed in the 1mM EDTA antigen retrieval solution boiled (pH=9.0), low fire heating in micro-wave oven
10min, cooled to room temperature.
Notice that the amount for repairing liquid when antigen retrieval must assure that slice is immersed in liquid always, has heated and be cooled to room temperature
Slice must remain in liquid in the process, can not take out slice and cool down in air.
3, endogenous peroxydase is blocked
1) PBS of the histotomy after antigen retrieval is rinsed 3 times, every time 5 minutes；
2) slice is taken out, tissue surrounding liquid, the test serum region on immunohistochemistry pen delineation slice are got rid of and dry；
3) 100~200 μ l endogenous peroxydase blocking agents are added in tissue regions, are incubated at room temperature 10 minutes；
4) PBS solution is rinsed 3 times, every time 5 minutes.
4, AR-V7 antibody incubation
1) PBS solution is removed, the 100 μ l AR-V7 antibody reagents of 1 μ g/ml are added, is incubated at room temperature 60 minutes；
2) PBS solution is rinsed 3 times, every time 5 minutes.
5, enzyme mark polymer is incubated for
1) PBS solution is removed, 100 μ l HRP- goat anti-mouse igg working solutions are added, is incubated at room temperature 20 minutes；
2) PBS solution is rinsed 3 times, every time 5 minutes.
6, it develops the color
PBS solution is removed, the good DAB developing solution of 100~200 μ l Fresh is added, is protected from light is incubated for 3-10 points at room temperature
Clock；After having developed the color, distilled water flushing sample color development stopping.
It is redyed if it is necessary, haematoxylin can be carried out.
Tap water rinses, and 100~200 μ l haematoxylin body cell dyeing liquors is added to be incubated for 10~30 seconds；PBS solution or tap water
Indigo plant is returned in flushing.
Note: the intensity of the haematoxylin dyeing liquid according to effect and the length of incubation time: counterstain result causes carefully
The reaction of pale blue to dark blue color is presented in karyon, and mistake contaminates or insufficient dyeing is likely to jeopardize the judgement of correct result.
7, dehydration, transparent, mounting
1) it sets in 95% ethyl alcohol, impregnates 3 minutes；
2) it sets in dehydrated alcohol, impregnates 3 minutes；
3) dimethylbenzene is transparent；
4) neutral gum and coverslip mounting.
8, after resinene solidification, microscopically observation is sliced dyeing effect.
Coloration result is shown in that Fig. 2, Fig. 2A are negative control, and Fig. 2A is to be exempted from using kit described in embodiment 5 to PC3 cell
Epidemic disease group experimental result, wherein coloured part is blue, and no brown colouration, Fig. 2 B is to the immunohistochemistry knot in Vcap cell
Fruit, wherein visible obvious brown colouration part.Because expressing AR in PC3 cell, but without expression AR-V7, and high table in Vcap cell
Up to AR-V7, thus this experimental results showed that, AR-V7 antibody provided by the invention and its immunohistochemical kit have AR-V7
High degree of specificity can effectively avoid non-specific background's coloring, i.e. false positive results.
Embodiment 5 is detected using immunohistochemistry of immuning tissue's kit to Vcap cell and H1975 cell sheet
Using the prostate cancer cell line Vcap of culture and lung adenocarcinoma cell H1975 as sample, using same as Example 4
Step carries out immunohistochemistry detection to Vcap and H1975 cell using kit described in embodiment 3.
As a result as shown in figure 3, Fig. 3 A is the ImmunohistochemistryResults Results figure of H1975 cell sheet, coloring is blue, i.e. AR-V7 is anti-
Body is negative.Fig. 3 B is the immunohistochemical experiment result figure of Vcap cell, and coloring layer is compared with dark-brown, i.e. AR-V7 antibodies positive.
AR-V7 high expression in Prostatic cancer cell lines Vcap, and do not expressed in lung cancer cell line H1975, therefore the visible present invention
The anti-AR-V7 monoclonal antibody and immunohistochemical kit have high degree of specificity, do not occur vacation in other cancer cells
Positive findings.
Embodiment 6: antibody stability influence is studied in antibody stabilization agent
The 6.08mg/ml antibody that embodiment 2 is prepared with phosphate buffer (PBS) after being diluted to 1mg/ml, is used
The Antibody of the BioStab Antibody Stabilizer (55514) of Sigma, CANDOR Bioscience
Stabilizer PBS (131500) and(270500) three kinds of antibody stabilization agent of HRP-Stab, will be above-mentioned
The monoclonal antibody of 1mg/ml is diluted to 1 μ g/ml, and accelerated stability test tests each antibody stabilization agent and stablizes monoclonal antibody effect
The ability of valence.Diluted primary antibody is placed in 37 DEG C of preservations, steps new DAB detection kit pair in 0 day, 4 days, 7 days, utilization in 9 days respectively
Prostate gland cancer cell Vcap and PC3 carry out immunohistochemistry detection, counterstain effect.It is control with the diluted antibody of PBS.It is immune
Groupization test procedure is as follows:
1, fixed, smear: being added 4% (v/v) paraformaldehyde solution, be placed at room temperature for 15 minutes, distills water washing 3 times；Carefully
Born of the same parents' suspension is coated on anticreep glass slide, and 60 DEG C of roasting piece 1h, PBS are washed 3 times.
2. antigen retrieval
Slide is placed in the 1mM EDTA antigen retrieval solution boiled (pH=9.0), low fire heating in micro-wave oven
10min, cooled to room temperature.
Notice that the amount for repairing liquid when antigen retrieval must assure that slice is immersed in liquid always, has heated and be cooled to room temperature
Slice must remain in liquid in the process, can not take out slice and cool down in air.
3. blocking endogenous peroxydase
1) PBS of the histotomy after antigen retrieval is rinsed 3 times, every time 5 minutes；
2) slice is taken out, tissue surrounding liquid, the test serum region on immunohistochemistry pen delineation slice are got rid of and dry；
3) 100~200 μ l endogenous peroxydase blocking agents are added in tissue regions, are incubated at room temperature 10 minutes；
4) PBS solution is rinsed 3 times, every time 5 minutes.
4.AR-V7 antibody incubation
1) PBS solution is removed, the primary antibody solution of various antibody stabilization dilution agents is added, is incubated at room temperature 60 minutes；
2) PBS solution is rinsed 3 times, every time 5 minutes.
5, enzyme mark polymer is incubated for
1) PBS solution is removed, 100 μ l HRP are added and mark HRP- goat anti-mouse igg working solution, are incubated at room temperature 20 minutes；
2) PBS solution is rinsed 3 times, every time 5 minutes.
6, it develops the color
PBS solution is removed, the good DAB developing solution of 100~200 μ l Fresh is added, is protected from light is incubated for 3-10 points at room temperature
Clock；After having developed the color, distilled water flushing sample color development stopping.
It is redyed if it is necessary, haematoxylin can be carried out.
Tap water rinses, and 100~200 μ l haematoxylin body cell dyeing liquors is added to be incubated for 10~30 seconds；PBS solution or tap water
Indigo plant is returned in flushing.
Note: the intensity of the haematoxylin dyeing liquid according to effect and the length of incubation time: counterstain result causes carefully
The reaction of pale blue to dark blue color is presented in karyon, and mistake contaminates or insufficient dyeing is likely to jeopardize the judgement of correct result.
7, dehydration, transparent, mounting
1) it sets in 95% ethyl alcohol, impregnates 3 minutes；
2) it sets in dehydrated alcohol, impregnates 3 minutes；
3) dimethylbenzene is transparent；
4) neutral gum and coverslip mounting.
8. biomicroscope diagosis and result judgement
As a result judgment criteria is as follows:
It is positive: to have yellow or brownish discoloration in histotomy on the nucleus of specific cells, and without nonspecific back
Scape dyeing；
It is negative: to have no yellow or brownish discoloration in expected cell in tissue.
As shown in figs. 4-7, wherein Fig. 4 is used to be immunized coloration result for the diluted AR-V7 monoclonal antibody of PBS as primary antibody
Groupization tests coloration result, and Fig. 5 is Sigma BioStab Antibody Stabilizer, Fig. 6 Antibody
Stabilizer PBS, Fig. 7 areThe diluted AR-V7 of HRP-Stab is used for immunohistochemical experiment as primary antibody
As a result coloration result, wherein A indicates that Vcap cell is corresponding as a result, B indicates that PC3 cell is corresponding as a result, number after A or B
1-4 respectively corresponds the AR-V7 after dilution, when placing the 0th, 4,7,9 day for 37 DEG C, is used for the corresponding result of immunohistochemical assay.
Due to AR-V7 high expression in Vcap cell, and do not expressed in PC3 cell, the equal nothing of PC3 cell in Fig. 4-7
Obvious brown or yellow coloring, and it is royal purple chromatic colorant, effectively exclude false positive results.In the result of Vcap, Sigma is used
BioStab Antibody Stabilizer dilutes to obtain the AR-V7 monoclonal antibody of working concentration, is used for immunohistochemical assay
Afterwards, dyeing effect is apparent dark-brown, effect stability.
And use CANDOR Bioscience Antibody Stabilizer PBS and HRP-
The diluted AR-V7 monoclonal antibody of Stab stabilizer, when placing the 0th, 4,7 day for after immunohistochemical assay, also have compared with
Apparent dark brown chromatic colorant, but when placing the 9th day, it is used for immunohistochemical assay, cell to be without brown or yellow in coloration result
Coloring, and it is royal purple chromatic colorant, antibody failure.Therefore, it is diluted using Sigma BioStab Antibody Stabilizer
The AR-V7 monoclonal antibody arrived has stability.Therefore, Sigma BioStab Antibody Stabilizer is selected to stablize
Agent is good to the stablizing effect of AR-V7 monoclonal antibody.
Embodiment 7: influence of the antibody concentration to immunohistochemistry detection effect
With antibody diluent described in embodiment 3, the monoclonal antibody that embodiment 2 is prepared is diluted to 1mg/ml,
AR-V7 antibody mother liquor is 1mg/ml concentration, dilutes 1000 respectively with BioStab Antibody Stabilizer antibody diluent
Again, 500 times, 250 times, 100 times, the concentration for obtaining AR-V7 antibody are respectively as follows: 1 μ g/ml, 2 μ g/ml, 4 μ g/ml and 10 μ g/ml,
Immunohistochemical experiment is carried out as primary antibody after being placed at room temperature for 7 days.
With each 8 of prostate cancer cell line Vcap and the PC3 slice of culture, it is divided into 4 groups, PC3 paraffin section is made in every group
For negative control, Vcap is sliced for detecting AR-V7 protein expression.Experimental method now, which is provided, according to this kit carries out AR-V7
Detection operation.
1, fixed, smear: being added 4% (v/v) paraformaldehyde solution, be placed at room temperature for 15 minutes, distills water washing 3 times；Carefully
Born of the same parents' suspension is coated on anticreep glass slide, and 60 DEG C of roasting piece 1h, PBS are washed 3 times.
2, antigen retrieval
Slide is placed in the 1mM EDTA antigen retrieval solution boiled (pH=9.0), low fire heating in micro-wave oven
10min, cooled to room temperature.
Notice that the amount for repairing liquid when antigen retrieval must assure that slice is immersed in liquid always, has heated and be cooled to room temperature
Slice must remain in liquid in the process, can not take out slice and cool down in air.
3, endogenous peroxydase is blocked
1) PBS of the histotomy after antigen retrieval is rinsed 3 times, every time 5 minutes；
2) slice is taken out, tissue surrounding liquid, the test serum region on immunohistochemistry pen delineation slice are got rid of and dry；
3) 100~200 μ l endogenous peroxydase blocking agents are added in tissue regions, are incubated at room temperature 10 minutes；
4) PBS solution is rinsed 3 times, every time 5 minutes.
4, AR-V7 antibody incubation
1) PBS solution is removed, is added and presses the diluted AR-V7 antibody reagent of different extension rates, is incubated at room temperature 60 minutes；
2) PBS solution is rinsed 3 times, every time 5 minutes.
5, enzyme mark polymer is incubated for
1) PBS solution is removed, 100 μ l HRP are added and mark HRP- goat anti-mouse igg working solution, are incubated at room temperature 20 minutes；
2) PBS solution is rinsed 3 times, every time 5 minutes.
6, it develops the color
PBS solution is removed, the good DAB developing solution of 100~200 μ l Fresh is added, is protected from light is incubated for 3-10 points at room temperature
Clock；After having developed the color, distilled water flushing sample color development stopping.
It is redyed if it is necessary, haematoxylin can be carried out.
Tap water rinses, and 100~200 μ l haematoxylin body cell dyeing liquors is added to be incubated for 10~30 seconds；PBS solution or tap water
Indigo plant is returned in flushing.
Note: the intensity of the haematoxylin dyeing liquid according to effect and the length of incubation time: counterstain result causes carefully
The reaction of pale blue to dark blue color is presented in karyon, and mistake contaminates or insufficient dyeing is likely to jeopardize the judgement of correct result.
7, dehydration, transparent, mounting
1) it sets in 95% ethyl alcohol, impregnates 3 minutes；
2) it sets in dehydrated alcohol, impregnates 3 minutes；
3) dimethylbenzene is transparent；
4) neutral gum and coverslip mounting.
8, biomicroscope diagosis and result judgement
After resinene solidification, microscopically observation is sliced dyeing effect.
As shown in Fig. 8 and Fig. 9, wherein Fig. 8 is the immunohistochemical staining of Vcap cell as a result, Fig. 9 is the immune of PC3 cell
Histochemical staining is as a result, A is that (antibody working concentration is 1 μ after confrontation AR-V7 monoclonal antibody mother liquor (1mg/ml) dilutes 1000 times
G/ml) detection result schematic diagram；B is 500 times of dilution (antibody working concentration is 2 μ g/ml)；C is 250 times of (antibody works of dilution
Making concentration is 4 μ g/ml)；D is the coloration result figure of (antibody working concentration is 10 μ g/ml) detection after diluting 100 times.
Coloration result is shown: when AR-V7 monoclonal antibody dilutes 100,250,500 and 1000 times, nothing is appointed in PC3 cell
What brown colouration, showing experimental system, there is no problem, can effectively avoid non-specific background's coloring i.e. false positive, and AR-V7 is mono-
The dyeing of Vcap cellular immunity has obvious brown colouration when clonal antibody dilutes 1000 times and 500 times, when diluting 250 times, 100 times
The Vcap cell section of AR-V7 monoclonal antibody immunity dyeing shows that positive staining effect is poor without apparent brown colouration.Show
When AR-V7 monoclonal antibody dilutes 1000 times and 500 times, i.e., when antibody concentration is 1 μ g/ml and 2 μ g/ml, the immune inspection of AR-V7
It is more appropriate to survey effect.Therefore, 1 μ g/ml is the working concentration of optimal anti-AR-V7 antibody.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (10)
1. a kind of antibody for specifically binding human androgenic receptor splicing variants 7, which is characterized in that the antibody are as follows: use
Amino acid sequence forms the polypeptide fragment as shown in SEQ ID NO.2 as immunogene, be prepared and energy and androgen receptor
The monoclonal antibody that body splicing variants 7 are specifically bound.
2. specifically binding the antibody of human androgenic receptor splicing variants 7 according to claim 1, which is characterized in that institute
State the preparation method of antibody the following steps are included:
(1) mouse is immunized in SEQ ID NO.2 polypeptide fragment；
(2) mouse boosting cell after will be immune is merged with SP2/0 myeloma cell, obtains hybridoma；
(3) by the hybridoma secretion after, isolate and purify to get.
3. specifically binding the antibody of human androgenic receptor splicing variants 7 according to claim 2, which is characterized in that institute
State the preparation method of antibody the following steps are included:
(1) mouse is immunized in SEQ ID NO.2 polypeptide fragment；
(2) mouse boosting cell after will be immune is merged with SP2/0 myeloma cell；
(3) positive for secreting anti-human 7 monoclonal antibody of androgen receptor splicing variants is obtained using the screening of HAT Selective agar medium
Hybridoma；
(4) it is cloned using positive cell strain of the limiting dilution assay to acquisition, obtains the anti-human androgen receptor for generating high-titer
7 monoclonal antibody hybridoma cell of body splicing variants；
(5) it after being secreted by anti-human 7 monoclonal antibody hybridoma cell of androgen receptor splicing variants of the high-titer, separates
Purifying to get.
4. the preparation method of the antibody of specific binding human androgenic receptor splicing variants 7 as described in claim 1, special
Sign is, comprising the following steps:
(1) it selects and amino acid sequence is prepared as the polypeptide fragment of SEQ ID NO.2；
(2) mouse is immunized in SEQ ID NO.2 polypeptide fragment；
(3) mouse boosting cell after will be immune is merged with SP2/0 myeloma cell, obtains hybridoma；
(4) positive hybridoma for obtaining the anti-human androgen receptor splicing variants 7 of secretion using the screening of HAT Selective agar medium is thin
Born of the same parents；
(5) it is cloned using positive cell strain of the limiting dilution assay to acquisition, obtains the anti-human androgen receptor for generating high-titer
7 monoclonal antibody hybridoma cell of body splicing variants；
(6) it carries out that above-mentioned hybridoma is injected intraperitoneally to Balb/c mouse, takes ascites；
(7) separate from above-mentioned ascites using protein G affinity chromatography, purify and obtain specifically binding human androgenic receptor
The antibody of splicing variants 7.
5. a kind of high stability antibody reagent, which is characterized in that including any by antibody stabilization dilution agent such as claim 1-3
The antibody of specific binding human androgenic receptor splicing variants 7 described in, the antibody stabilization agent are selected from: BioStab
Antibody Stabilizer, Sigma-55514.
6. high stability antibody reagent according to claim 5, which is characterized in that the specific binding people AR-V7's
Concentration of the antibody in the antibody stabilization agent is 1-2 μ g/ml.
7. if high stability antibody reagent described in claim 5 or 6 is preparation detects human androgenic receptor splicing variants 7
Application in immunohistochemical kit.
8. a kind of immunohistochemical kit for detecting human androgenic receptor splicing variants 7, which is characterized in that wanted including such as right
Seek antibody described in 1-2 or such as the described in any item high stability antibody reagents of claim 5-7.
9. immunohistochemical kit according to claim 7, which is characterized in that further include the work of HRP- goat anti-mouse igg
Liquid, DAB developing solution, endogenous peroxydase blocking agent, confining liquid and antibody diluent.
10. if the described in any item immunohistochemical kits of claim 7-9 are in the production of preparation prediction prostate cancer therapy effect
Application in product.
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|---|---|---|---|---|
| US20110110926A1 (en) * | 2008-04-16 | 2011-05-12 | Johns Hopkins University | Compositions and methods for treating or preventing prostate cancer and for detecting androgen receptor variants |
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| US20110110926A1 (en) * | 2008-04-16 | 2011-05-12 | Johns Hopkins University | Compositions and methods for treating or preventing prostate cancer and for detecting androgen receptor variants |
| CN105143463A (en) * | 2013-02-25 | 2015-12-09 | 诺华股份有限公司 | Novel androgen receptor mutation |
| WO2015065919A1 (en) * | 2013-10-28 | 2015-05-07 | The Regents Of The University Of California | Treatment of metastatic prostate cancer |
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