CN1082187C - Preparation and use of anti-human estrogen acceptor monoclonal antibody and anti-human progestogen acceptor monoclonal antibody - Google Patents

Abstract

The present invention relates to a biologic reagent for detecting an estrogen receptor (ER) and a progesterone receptor (PR) in clinic and scientific research, which comprises a polyclonal antibody and a monoclonal antibody. The biologic reagent has the significance that the biologic reagent can be used for detecting breast cancer and ER and PR level of patients with various hormonal dependent diseases in clinic. After the guide implementation of endocrine therapy in clinic and prognosis estimation, the biologic reagent can also be used for researching the relationship of ER and PR, tumors and various diseases.

Description

A kind of biological reagent and SABC method that detects the estrogen receptor level

The present invention relates to the biological detection reagent that a class is used for clinical detection breast cancer, carcinoma of endometrium and various hormone-dependent neoplasm patient cancerous tissue estrogen receptor (ER) and progesterone receptor (PR) level.

Breast cancer accounts for the first place in the high and rising year by year of the incidence of disease of developed country with the U.S..The seventies, its new number of the infected was 80,000 people/years; The morbidity rate in U.S. every year rises with 3% speed.Reached for 180,000 people/years according to incompletely statistics to 92 years.In China is example with Beijing, and people/100,000 people were ill in 82 years 18.4, and 92 years is that 24.3 people/100,000 people are ill, estimates 2000, and 36 people/100,000 people are with newborn patient's gland cancer, and are also in rising trend.

Treatment means to breast cancer has excision, chemotherapy, radiotherapy and endocrinotherapy etc. at present.When chemotherapy and radiotherapy kill and wound cancer cell, body's immunity to the people also has very big infringement, feel sick, vomiting, alopecia, blood picture change especially white blood cell and reduce to and extremely lowly make the patient be difficult to tolerance so that TD, and that endocrinotherapy has toxicity is atomic, few side effects, continue length and can obviously improve patient's quality of life effective time, is one of important channel of improving the breast cancer curative effect.After Jensen in 1971 etc. had reported that the ER that measures in the breast cancer sample can predict patient's prognosis and helps to select therapeutic scheme, the research that relevant breast cancer and ER, PR concern had had remarkable progress.ER, PR content have following effect clinically in the mensuration breast cancer tissue: (1) provides scientific basis for selecting therapeutic scheme.(2) clinical progress of prediction breast cancer.(3) prediction chemotherapeutic efficacy.(Lv Baozhang etc., acceptor is learned outline P281-283,1991, Science Press, Beijing) biological characteristics to breast cancer is classified before treatment for this reason, and this adopts which kind of methods of treatment and estimation thereof to breast cancer, and more the back is most important.

According to the biological characteristics of breast cancer, it can be divided into two classes clinically.One class breast cancer tissue contains ER and is called hormonal dependent breast cancer, endocrine therapy is effective, ER or content utmost point root are not generally contained in another kind of breast cancer tissue, to such an extent as to detect less than, be called hormonal independent breast cancer, endocrine therapy is invalid, external report (Megvire WL, et al:Estrogen Receptors in Human Breast Cancer, An Overview Estrogen Receptors in Human Breast Cancer, Meguire WL, (eds), pl, New York Raven Press, 1975.) ER positive person, reach about 60% through endocrine therapy is efficient, and the ER negative patient only is about 10%.What now reached common recognition is to check that ER detects the accuracy rate that the PR expression more can improve estimation curative effect and prognosis simultaneously.Efficient 74-90% (the Meguire that reaches of the two positive person's endocrine curative effects of ER (+), PR (+), WL, et al, Progesterone Receptors:Introductionand overview Progesterone Receptors in Normal and NeoPlastic tissue.PI, NewYork Raven Press, 1977; Edwards DP, et al:Estrogen and Progesterone Receptorporteins in breast cancer.Bioc.Biop.Acta.560:457,1979) therefore for avoiding blindly medication, improve the curative effect of breast cancer, measure ER in the breast cancer tissue simultaneously, the PR level is necessary.

The method of present domestic mensuration ER has DCC method (Zhang Huiying etc., the research of mammary gland cancer hormone receptor, the Chinese tumour magazine 5:168 with classics, 1983), also have adopt enzyme or fluorescence labeling estradiol or the anti-estradiol antibody act of mark (permitted very medium, tumour, 4:18,1984; Xu Weiling etc., tumour, 8:160,1988).The latter belongs to indirect reaction, and has fluorescent quenching, needs problems such as fluorescent microscope, be tending towards superseded, with regard to DCC, had following defective: must use fresh specimens, testing process must keep low temperature, required cancer piece is big, needs specific installations such as liquid sudden strain of a muscle, has isotopic contamination, in addition, each sense cycle is three days, and adding DCC liquid can destroy the binding equilibrium between hormone and the acceptor in the mensuration, and the position difference of drawing materials also influences testing result.1986 (Cancer Res.46:4233-4236,1986) such as Guylercq report: European multicenter study seminar totally 8 laboratories respectively to the tissue specimen with a collection of clinical breast cancer patient carried out the enzyme immunization experiment (ER-EIA) of anti-ER monoclonal antibody and simultaneously with biochemical process as contrast.The result shows two kinds of methods there was no significant difference on testing result.But the repeatability of ER one enzyme immunization experiment (variation factor 6% in the laboratory, between the laboratory for 11-19%) to a certain extent than biochemical process (variation factor is 12-32% between the laboratory) for well.Wherein the result of six laboratory ER-enzyme immunoassay and biochemical process correlation analysis shows, can substitute the detection that biochemical process be used for routine clinical ER with the ER-enzyme immunoassay of anti-ER antibody fully.And the ER-enzyme immunoassay can reduce the DCC method because the matrix dilution and the position of the drawing materials error due to improper.When specimen block is not suitable for or measures when not enough the DCC method, this method provides reappearance strong detection means.Thorpe SM et al CancerRes.47＜24pt 1 in 1987＞: 6,572 1987) fresh specimens of 191 routine breast cancer biopsies has also been carried out ER-enzyme immunoassay and DCC method relatively, both height correlations (r=0.97), the data that obtain show, the ER-enzyme immunoassay is more accurate biologically, Graig Allred shows in the report of 1993 (Am J.Clin.Pathol.99:1-3,1993) in addition: the SABC method (Immunohistochemicalmethod) that detects ER is more special, more responsive than DCC method.And point out that, tumour cell little when casting constitutes after a little while, the DCC method easily causes false negative.Be E because of PR again ₂With the product of ER zygotic induction, the 26S Proteasome Structure and Function that ER has been reacted in the existence of PR is all normal, postreceptor defects do not occur, and this explanation detects the significance of PR.In any case reply ER and PR detect simultaneously.

What the detection method classics of estrogen receptor (ER) and progesterone receptor (PR) were commonly used is glucosan activated charcoal parcel absorption method (DCC) method, and its principle is to utilize the combination of aglucon part, with the isotope display result of aglucon mark.(Lippman ME, et al, Cancer Res, 39:1447,1979; China's tumour magazine, 5:168,1983) but there are many problems in the DCC method, as needs special instruments and equipment measurement result; Use isotope must have safeguard procedures; Need the cancerous tissue amount big, there is its volume of tumor operation sample of 20-30% can not satisfy the requirement that the DCC method detects in the real work approximately, and because the position of drawing materials is improper or because tumour histological type difference causes result error, though the DCC method can be quantitatively but can not position observation in addition, so false positive appears in the possible benign cell that contains ER, the PR positive owing to the tumour that is ER, PR feminine gender; Or owing to the heterogeneity and the matrix dilution of tumour causes false negative; This method expense height is time-consuming again; So the DCC method is difficult for applying.Even enzyme immunoassay is because be that to detect acceptor levels with cancer cell solution can not position observation positive reaction composition be cancer cell or normal (or optimum) cell.And immunohistochemistry (IHC) method had both overcome above-mentioned various shortcoming; cheap again, fast, be easy to promote; only need seldom tissue even needle biopsy sample or common pathology paraffin section to get final product; Direct observation ER and PR location and the expression in histocyte under the mirror; can get rid of negative and the normal or optimum composition of tumour cell and be the DCC false positive due to the positive, susceptibility is good, high specificity.This mainly be since in the SABC method the special immunity of antibody and its target antigen-hormone receptor combine determine.Over past ten years.The a large amount of research of external process has relatively confirmed highly to meet (85-90%) between mensuration ER and PR expression SABC method and the DCC method, can the SABC method replace the DCC method.(Graig Allred, AmJ.Clin.Pathol.99:1-3,1993) can satisfy clinical requirement substantially though the SABC method is sxemiquantitative.External SABC method (Marvin T et al Am J.Clin.Pathol.99:8,1993 of adopting more; Heikki J.Helin et al, Cancer 63:1761,1989).Immunohistochemical technique at first must provide anti-ER monoclonal antibody and anti-PR monoclonal antibody (McAb), but because ER, PR antigen especially PR more be difficult to obtain, so domesticly for a long time do not see the report that anti-ER of development and anti-PR antibody are arranged.Domestic have only anti-estradiol (E ₂), the antibody of antiprogestin, this can only indirect detection ER, the PR level, the result is undesirable, has been tending towards superseded.Abroad be with synthetic little peptide be antigen preparation anti-PR monoclonal antibody (172M of DAKO company) be the anti-ER monoclonal antibody of antigen preparation (H08-0149B of ZEMED company) with the aminoterminal AB section of ER, during with the antibody test ER of this antigen preparation or PR, section preparation must pass through micro-wave oven antigen retrieval treatment step, in with high temperature antigen retrieval process, even microslide also is very easy to come off through section preparation after the special silicidation, cause and can not get the result, trivial operations, difficult control of temperature; And, the anti-ER monoclonal antibody and the PR monoclonal antibody fetch long price of external preparation.

One of purpose of the present invention provides special sensitivity, cheap again anti-human estrogen acceptor (ER) easy and simple to handle and the polyclonal antibody and the monoclonal antibody (McAb) of antiprogestin acceptor (PR).Be used to detect other disease patient's of tumor patient and hormonal dependent ER, PR expression.Two of purpose is to set up simple, the special effective method that detects ER, PR.

In order to reach the object of the invention, the inventor thinks and must be started with by antigen.At first the molecular structure of ER is analyzed.The C of ER, D section are the DNA land, and C section high conservative is rich in halfcystine and is folded to form " zinc finger ring " structures of two repetitions.Wherein the D section is positioned at the part of ER and the folding section between the DNA combined function district, has the hinge structure, and promptly the D section is positioned at the surface of ER albumen, make the antibody of antigen preparation with it, the immunity association reaction may more responsive (Kumar V et al, Cell1987,51:941; Acceptor is learned outline P276).The B district is relevant with maintenance ER molecular biological activity.Based on above-mentioned principle, this research is antigen with the expressing protein product of ER-BCD section, and it is clear and definite to have structure, high specificity, and the D district is in the ER protein surface, can improve the susceptibility of antigen-antibody combination.In addition again according to bibliographical information (Glenn S.Et al J.Steroid Biochem.Molec.Biol39 (5A): 687-692,1991) and from PCGENE microcomputer result for retrieval select construction of expression vector between the PR100-1500bp, contain two antigenic determinants of exposing in this section.May be special, responsive with the monoclonal antibody that this expressing protein product is an antigen preparation.Based on above-mentioned analysis foundation, make up PMS-ER, the PMS-PR material of recombinating, the protein product of abduction delivering is an antigen with this, prepares anti-ER, anti-PR monoclonal antibody.

This studies applied ER, PR antigen, its preparation method is at first to connect and compose PMS-ER and PMS-PR recombinant plasmid (seeing Fig. 1 .3) → with the recombinant plasmid transformed Escherichia coli with the PMS-31 expression vector orientation of identical restriction enzyme site again with the selected ER of round pcr amplification and PR gene segment → respectively, make it when 25-35 ℃ of growth is expanded to bacterial turbidity and is 0.6-0.8, again temperature is brought up to 40-45 ℃ and continued to cultivate≤8 hours → collection bacterial sediment → carrying out ultrasonic bacteria breaking → centrifuging and taking supernatant, being prepared property SDS-PAGE electrophoresis, obtain the ER of capacity, PR ,-20 ℃ of preservations are standby.ER, PR expressing protein product confirm that through immunological binding assay we have the immunogenicity identical with natural goods by prepared antigen.

Antibody with its preparation comprises many anti-and monoclonal antibodies.The acquisition of the polyclonal antibody of ER and PR is to be that antigen adds repeatedly immune animal of complete and incomplete freund adjuvant with ER, PR expressing protein product, comprises rabbit, mouse, rat, sheep etc., obtains to have with natural ER, PR the serum of the activity of combining.The MONOCLONAL ANTIBODIES SPECIFIC FOR method, be to adopt hybridoma technology (Kohler G.And Milstein C, Nature 1975,256:495) preparation mouse anti ER monoclonal antibody and mouse anti PR monoclonal antibody, but be not limited to mouse, also available rat (RatHybridomas and Rat Monoclonal Antibodies P75-115,265-270, Bazin H.CRCPress, Inc.Florida 1990).The above-mentioned how anti-and monoclonal antibody that obtains is through three its biologic activity of standard detection.The first usefulness immunizing antigen wrapper sheet, 5 μ g/ml, second usefulness is synthesized little peptide wrapper sheet, Amino acid sequence that should synthetic small peptide is taken from recombinant expression protein product (i.e. a section in (ER, PR antigen) structure, wrapper sheet substrate concentration 5 μ g/ml detect homemade antibody with the ELISA method of routine again and the two all has association reaction; The 3rd usefulness paraffin section that detection confirms the breast carcinoma tissue of the ER or the PR positive through the DCC method carries out immunohistochemical staining with self-control antibody, produces the antigen and antibody specific association reaction.It is special, inexpensive to have sensitivity through experiment confirm antibody of the present invention, does not need antigen retrieval to handle during immunohistochemical staining, and easy to operation, this is one of characteristics that are better than external monoclonal antibody.The hybridoma cell strains of production monoclonal antibody of the present invention is preserved in the CCTCC of Wuhan University, and its preserving number is 96005,96006.

Finish the anti-ER that development has These characteristics, on the basis of anti-PR monoclonal antibody, can reach set up special effectively, sensitive simple again detection ER, the SABC method of PR, at present, utilize the anti-ER of import, during the capable immunohistochemical staining of anti-PR monoclonal antibody, histotomy all must boil the step of antigen retrieval through high temperature, and the anti-ER that utilizes the present invention to develop, anti-PR monoclonal antibody carries out ER, during the PR immunohistochemical staining, do not need antigen retrieval, operation steps is simplified, boil and do not boil indifference through micro-wave oven high temperature through the experiment confirm histotomy again, all can obtain good Color.The positive reaction thing of two kinds of monoclonal antibodies is positioned the karyon of ER, PR positive cell respectively, and background is clear.The advantage of the inventive method is: step is simplified, the shortening time, and easy to operation, good reproducibility, biggest advantage is the shortcoming of frequent flake when having avoided pyroprocessing.Its running program is as follows: the conventional dewaxing of paraffin section entry → 0.3%H ₂O ₂Methyl alcohol soaks, and destroys endogenous peroxydase → conventional PBS and embathes, and seals → add to add two after anti-ER (or anti-PR) monoclonal antibody → routine is embathed and add enzyme labeling streptavidin → embathe afterwards with DAB-H after resist → embathing ₂O ₂Colour developing → haematoxylin redyeing, mounting.Anti-ER monoclonal antibody of the present invention and anti-PR monoclonal antibody are scientific research and clinical all being with a wide range of applications and very high practical value, can not only also provide effective means for clinical formulation therapeutic scheme provides foundation for the relation of further studying hormone receptor and various diseases.

Description of drawings 1.ER-BCD fragment and expression vector PMS-3 1b constitute the recombinant plasmid synoptic diagram.Fig. 2 PMS-ER-BCD expressing protein electroelution result (12.5%SDS-PAGE) that recombinates

1. molecular weight of albumen sign

2.3.4. be reorganization plasmid transformation escherichia coli, abduction delivering 4-6 hour cellular lysate liquid electrophoretic patten;

5.6.7. the purpose band is downcut respectively, concentrates the rear electrophoresis figure through electroelution;

8.9. negative contrast Fig. 3 .PR fragment and expression vector PMS-31b constitute the recombinant plasmid synoptic diagram.Fig. 4. the expression of fusion PMS-PR in Escherichia coli

1. molecular weight of albumen sign;

2.3. be recombinant plasmid PMS-PR transfection Escherichia coli, do not induce electrophoretic patten with abduction delivering;

4.5.6.7. negative contrast is respectively empty carrier and Escherichia coli, does not induce the electrophoretic patten with abduction delivering.Fig. 5. reorganization PMS-P fusion electroelution result (12.5%SDS-PAGE)

1. molecular weight of albumen sign;

2.3. be respectively recombinant plasmid PMS-PR transformed into escherichia coli, do not induce electrophoretic band with abduction delivering;

4.5.6. add the expressing protein amount that the electroelution of 10 μ l, 5 μ l, 2 μ l concentrates respectively;

7. the electrophoretic band that shows 5 μ g bovine serum albumin(BSA)s, for reference protein quantitative.Fig. 6. reorganization PMS-ER expressing protein Western Blot analyzes

1. molecular weight of albumen sign;

2.3.6.7.PMS-ER transformed into escherichia coli;

2.6. abduction delivering;

3.7. do not induce;

4.5. do not induce and induce for the empty carrier transformed bacteria;

2.3. normal mouse serum is one anti-, negative control is not seen painted band;

4.7. mouse-anti ER-BC serum is one anti-, 6 compare with 4.5.7, have the protein band of obvious enhancing, confirm that the ER-BCD protein product has immunogenicity.Fig. 7. the immunoaffinity chromatography purifying is exempted from anti-ER-D antibody (12.5%SDS-PAGE)

M: molecular weight of albumen sign;

1. the anti-ER-D antibody of saturated ammonium sulphate;

2. through the ER-D antibody of ER-BCD immune affinity chromatographic column purifying.Fig. 8. anti-ER-BCD serum Western Blot analyzes

1. turn out to be the breast carcinoma tissue of ER feminine gender through the DCC method;

2.3. be the same breast carcinoma tissue of the ER positive;

1.2. with anti-ER-BCD serum is one anti-, the 3rd, and will resist with ER-BCD expressing protein product and to do one after the absorption of ER-BCD antibody and resist, other staining procedure is identical with 1.2;

2. the visible one special band at the 67KD place;

3.67KD locating band disappears.Fig. 9. anti-ER monoclonal antibody immunohistochemical staining Figure 10 of the present invention. anti-PR monoclonal antibody immunohistochemical staining of the present invention

Describe the present invention below in conjunction with embodiment, be intended to be convenient to understand the present invention better rather than limit the scope of the invention.

Embodiment 1

The inducing, express of the inducing of the abduction delivering of ER, PR recombinant protein and purifying one, ER-BCD recombinant protein, expression and purifying 1.ER-BCD recombinant protein

According to people ER analysis of gene sequences is designed a pair of primer, upstream sequence is 5 '-CGGCGAATTC ATGGGG GGT TTC CCCC-3 ', and downstream sequence is 5 '-CGGCGGATCC TTA GGC AGC TCT CAT GTC TCC-3 '.Carry out PCR, amplification ER-BCD fragment, PCR carries out BamH1, EcoR1 double digestion after finishing, and preparation ER-BCD connects and composes PMS-ER-BCD recombinant plasmid (structure of ER-BCD expression vector is seen Fig. 1) with the PMS-31 expression vector of identical restriction enzyme site again.With PMS-ER-BCD recombinant plasmid transformed Escherichia coli, make it when 25-35 ℃ of growth is expanded to bacterial turbidity and is 0.6-0.8, cultivation temperature is brought up to 40-45 ℃ continue to cultivate≤8 hours.Centrifugal 10 minutes of 10000rpm abandons supernatant, collects thalline, adds 0.1ml with the 1ml thalline and splits the bacterium liquid proportional, makes precipitation multiple outstanding.Sonicated was handled 45 seconds with peak power, placed 10 minutes in the boiling water, in 4 ℃, centrifugal 10 minutes of 10000rpm gets supernatant 20 μ l+20 μ lSDS electrophoretic buffers, the SDS-PAGE electrophoresis of row 12.5%, the expressing protein amount can reach and account for 15% of total protein concentration in the bacterial lysate by analysis.2.ER-BCD the separation of expression product and concentrated

The Escherichia coli that contain recombinant plasmid PMS-ER-BCD are through 25-35 ℃ of amplification, 40-45 ℃ induce after, prepare the ultrasonic bacterium liquid (seeing above-mentioned) that splits, being prepared property SDS-PAGE electrophoresis purifying concentrates PMS-ER albumen.

(1) the thick big PAGE offset plate of preparation 3mm adds electrophoretic buffer in electrophoresis tank.Application of sample,

The constant current electrophoresis is set, and electric current is 25mA.

(2) electrophoresis takes off glue after finishing, and glue is placed in the plate, adds 4 ℃, the dyeing of 0.1M potassium chloride,

Downcut the destination protein band and glue is put into bag filter, in the SDS electrophoretic buffer, constant voltage

200V, electrophoresis 4-5 hour, the adhesive tape in the bag filter is taken out, remaining liquid is at sucrose

In stop when being concentrated into proper volume, to the PBS dialysis, UV spectrophotometer measuring is decided egg

White amount.

(3) SDS-PAGE electrophoresis, Analysis and Identification are carried out in sampling.Purity is better, is the weight of single band

The histone (see figure 2).Electrophoresis result among Fig. 2, row 1 is the molecular weight of albumen sign, 2,3,

4 induce 4-6 hour cellular lysate liquid for Escherichia coli 40-45 ℃ of recombinant plasmid transformed

Electrophoretic patten.Row 5,6,7 carries out wash-out and concentrates rear electrophoresis for the purpose band is downcut respectively,

Row 8,9 negative contrasts are respectively Escherichia coli and transform with the PMS-31 empty expression vector

Escherichia coli, with thermoinducible result, no special protein expression band.Two, the inducing of PR recombinant protein, expression and purifying 1.PR recombinant protein induces, expresses

(1) designs a pair of primer, upstream according to analysis (acceptor outline) to people PR gene complete sequence

Sequence is 5 '-CGGC GAATTC ATG GCT GCG GAG GAG GTT GAG-3 ',

Downstream sequence is this 5 '-CGGC GATCC TTA CTC CGC GCC TTC CTC

CTC-3 ' carries out PCR.

(2) the PCR product is carried out enzyme with restriction endonuclease BamH1, Ecor1 and cut, again with identical restriction enzyme site

The PMS-31 expression vector connect and compose PMS-PR reorganization material (PR expression vector establishment

See Fig. 3) with PMS-PR recombinant plasmid transformed Escherichia coli, it is expanded 25-35 ℃ of growth

When increasing to bacterial turbidity and being 0.6-0.8, cultivation temperature is brought up to 40-45 ℃ continue to cultivate≤8

Hour, centrifugal 10 minutes of 10000rpm abandons supernatant, collects thalline, in the 1ml thalline

0.1ml split the bacterium liquid proportional, make precipitation multiple outstanding.Sonicated was handled 45 seconds with peak power,

Sample was placed in boiling water 10 minutes, and in 4 ℃, centrifugal 10 minutes of 10000rpm gets

Supernatant 10 μ l+10 μ l SDS electrophoretic buffers, the capable 12.5%SDS-PAGE electrophoresis of application of sample,

Remaining sample be stored in-20 ℃ standby.The expressing protein amount, the machine scanning analysis is induced as calculated

The expression product of product accounts for 15% of Tot Prot in the bacterial lysate.Among (see figure 4) Fig. 4

Electrophoresis result the 1st behavior molecular weight of albumen sign, the 2nd, 3 behavior recombinant plasmid PMS-PR

The result that transformed into escherichia coli is not induced and induced.4th, 5,6,7 behavior negative controls,

4th, 5 behavior PMS-31 vector plasmid transformed into escherichia coli are not induced and are induced, the 6th, 7

The behavior Escherichia coli are not induced and induce.2.PR the separation of expression product and concentrated

The Escherichia coli that contain recombinant plasmid PMS-PR are through 25-35 ℃ of amplification, 40-45 ℃ induce after, split bacterium liquid (method is seen above-mentioned) with the ultrasonication preparation.

(1) being prepared property SDS-PAGE electrophoresis, purifying, concentrated PMS-PR albumen.

(2) take off glue after electrophoresis finishes,, downcut the destination protein band and glue with the dyeing of 0.1M potassium chloride

Bar is put into bag filter, in SDS electrophoresis electrophoretic buffer, and electrophoresis 3-4 hour, will be saturating

The adhesive tape of analysing in the bag is taken out, and remaining liquid stops when being concentrated into proper volume in sucrose, PBS

Dialysis, spectrophotometer detects and decides protein content in addition.

(3) SDS-PAGE electrophoretic analysis evaluation is carried out in sampling, is recombinant protein such as Fig. 5 of single band,

Expression of recombinant plasmid fusion protein product electroelution result schematic diagram.The 1st behavior protein molecular

The amount sign, the 2nd, 3 row are respectively recombinant plasmid PMS-PR transfection Escherichia coli and do not induce

With induce, the 3rd row shows the expressing protein band of obvious enhancing; 4th, 5,6 row respectively

Add the concentrated expressing protein amount of electroelution of 10 μ l, 5 μ l, 2 μ l, the 7th row shows 5 μ

L bovine serum albumin(BSA) band, quantitative as reference protein.

Embodiment 2

The evaluation main agents of the evaluation one of ER, PR expressing protein product characteristics, ER expressing protein product:

1. rat anti ER-BC serum (this chamber self-control)

2. mouse anti ER-D serum (this chamber self-control)

3. the oxygen IgG of rabbit Chinese People's Anti-Japanese Military and Political College serum (this chamber self-control)

4. rat monoclonal antibody PAP (this chamber self-control)

5.HRP the anti-mouse IgG of mark rabbit (this chamber self-control)

6. the anti-mouse of biotin labeling horse (Zymde)

7.HPR the streptavidin of mark (Zymde)

8.CNBr-Sepharose?4B (Phamasia)

9.TMB chromogenic substrate (Military Medical Science Institute) method: (one) ELISA method is identified the immunogenicity of ER-BCD protein product

1. the ER-BCD protein product is cushioned liquid with bag and is diluted to 5-10ug/ml, bag is by 96 orifice plates, and 4 ℃ are spent the night.Equally with BSA, HAS with same concentrations bag quilt.

2. with the sealing of 0.1% gelatin, 37 ℃, 30 minutes.

3. be one anti-with mouse anti ER-D serum and rat anti ER-BC serum respectively, 37 ℃, 1 hour.

4. with PBS-0.05%Tween 20 washings, 3 minutes * 3.

5. the rabbit anti-mouse igg (1: 100) that adds the HRP mark, 37 ℃, 40 minutes.

6. washing, method is the same

7. develop the color with tmb substrate: add each 1 of A, B liquid respectively, color development at room temperature 15 minutes.

8. add 12.5% concentrated sulphuric acid cessation reaction.

ER-BCD expression product and mouse anti ER-D serum and rat anti ER-BC section serum all show the specificity association reaction as a result.

(2) Western Blot analyzes

1. with the ER-BCD protein product of separation and purification, carry out the SDS-PAGE electrophoresis after, go electrotransfer again, constant current 150mA, 2 hours, solid support was nitrocellulose filter (a NC film).

2. sealing places plate with the NC film, adds 3% BSA-PBS confining liquid, and 4 ℃ are spent the night.

3. the NC film is dipped in rat anti ER-BC serum, negative control replaces with normal rat serum, and 1: the 50-200 dilution, under the room temperature, slow shaking 1 hour.

4. discard an anti-reactant liquor, 10 minutes/each * 3 of PBS washing are delayed and are shaken rinsing.

5. add the mouse IgG of rabbit Chinese People's Anti-Japanese Military and Political College antiserum (1: 100), under the room temperature, slow shaking 30 minutes.

6.PBS delay and shake rinsing, 10 minutes/time * 3.

7. drip P of Rats AP (1: 100), room temperature is slow shook 30 minutes.

8. colour developing: after the abundant rinsing of NC film (method is the same), immerse freshly prepared 0.5mg/mlDAB, face with before adding H ₂O ₂Lul/ml under the room temperature, when the band colour developing reaches desired depth (1-3 minute), washes film with water at once, immerses among the PBS again.The results are shown in Figure 6 reorganization ER expressing protein Western Blot and analyze synoptic diagram.Article one, be the molecular weight of albumen sign, 2nd, 3,6,7, bar is the PMS-ER transformed into escherichia coli, induce, do not induce, 4,5 is after the PMS-31 transformed into escherichia coli does not induce and induce the sample electricity to change, 2,3 is that normal rat serum is an anti-dyeing, not seeing has painted protein band, and 4-7 is to be one anti-with rat anti ER-BC serum, and the visible the 6th and 4,5,7 compare, have the protein band of obvious enhancing, confirm that the ER-BCD protein product has immunogenicity.(3) the anti-ER-D serum of ER-BCD expressing protein immune affinity chromatographic column purifying

The 1. preparation of affinity column (Zhao Qiang etc., journal of Beijing Medical University 1990,22:307)

(1) after ER-BCD expressing protein electroelution purifying concentrates,, changes liquid repeatedly with PBS and the dialysis of binding buffer liquid.

(2) in the ratio of 10-30mg ER-BCD/g carrier, crosslinked as required amount takes by weighing CNBr-sepharose4B.

(3) operating process

CNBr-sepharose4B

↓ 1mmol/L HCL liquid soaks, and 4 ℃, 15 minutes

↓ 1mmol/L HCL liquid filtering and washing (removing protective agent)

↓

0.1mol/L sodium carbonate, sodium bicarbonate binding buffer liquid are taken out fast and is washed till pH neutrality, 20mg mixes immediately with reorganization ER protein liquid, adds an amount of binding buffer liquid

↓

Shaking table stirs at a slow speed, and room temperature was spent the night in 2 hours or 4 ℃

↓

Suction filtration is measured protein content in the suction filtration liquid

↓

The dress post 0.1mol/L sodium carbonate, sodium bicarbonate binding buffer liquid suspend is washed till no albumen and flows out (0D ₂₈₀＜0.01), mensuration flows down protein content in the liquid

↓

The 1mol/L monoethanolamine soaks the post bed, sealing residue reactive group

↓

0.1mol/L the post secondary is washed in acetate buffer solution and the circulation of binding buffer liquid

↓

0.1mol/L glycocoll-hydrochloride buffer is washed post

↓

0.01mol/L PBS washes post, is dormant state

The calculating of (4) coupling rate:

Coupling rate (%)=(crosslinked total protein concentration-crosslinked back suction filtration washes protein content)/crosslinked total protein concentration * 100%

2. the anti-ER-D serum of affinitive layer purification

Get anti-ER-D serum with 45% saturated ammonium sulphate secondary → 1000rpm PBS dialysis after centrifugal 10 minutes, change liquid 3-4 time, ultraviolet spectrophotometer is surveyed protein content.Stay 1/2 to make contrast experiment, the above-mentioned affinity column purifying of 1/2 usefulness in addition.The flat effect of anti-ER-D antibody and post 30 minutes is washed till 0D with PBS ₂₈₀＜0.01 → 2.5mol/L NaCL liquid is washed till OD280＜0.01 → 2 a times bed volume 0.1mol/L glycocoll-hydrochloride buffer and washes → collect the protein liquid that washes, at any time with 1mol/L NaHCO ₃Extremely neutral in the liquid with pH.Collect the antibody peak that affinity column elutes, the antibody recovery is 20.3% as calculated.The anti-ER-D antibody that elutes carries out SDS-PAGE and detects, and purity improves greatly, the result as shown in Figure 7, the M among Fig. 7 is the molecular weight of albumen sign, 1 is the anti-ER-D antibody through 45% saturated ammonium sulphate, and many protein bands are arranged; The 2nd, the ER-D antibody behind ER-BCD immune affinity chromatographic column purifying, purity obviously improves.Enzyme linked immunosorbent assay (ELISA) is the result show, 25 times of the pure raisings of saltouing of the anti-ER-D serum dilution of the rabbit of affinitive layer purification.SABC result confirms that the antibody of affinitive layer purification is obviously clear than the serum and the pure antibody background of saltouing.

Confirm that through above-mentioned every evaluation the ER-BCD expressing protein has and natural ER same structure, and have the antigenicity identical with natural ER.Two, the evaluation of PR expressing protein product

Main agents:

1. mouse anti PR serum (this chamber self-control): with the synthetic peptide of PR is the mouse anti PR serum of antigen preparation, and this synthetic peptide ammino acid sequence is taken from a section in the recombinant expressed sequence.Concrete preparation method is as follows: with synthetic peptide of PR and keyhole limpet hemocyanin (KLH) coupling (molecular cloning second edition P ₈₅₆Jin Dongyan etc. translate, Science Press, Beijing, 1992), get 20ug/ mouse of this KLH-PR conjugate/time add the complete freund adjuvant of equivalent, fully emulsified, subcutaneous multiple spot foot-pad immunization, the two all immunity of every interval once are total to immunity 4 times, intermittently after one month, strengthen with equivalent antigen, serum is got in bloodletting after 10 days, and survey is tired, packing ,-20 ℃ of preservations are standby.

2. peroxidase (HRP) the mark anti-mouse IgG of rabbit (self-control)

3.TMB chromogenic substrate (available from Military Medical Science Institute)

Method:

Enzyme linked immunosorbent assay (ELISA) method is identified the immunogenicity of PR protein product

1. the PR protein product is cushioned liquid with bag and is diluted to 5ug/ml, bag is by 96 orifice plates, and 4 ℃ are spent the night, and simultaneously with BSA, HSA and irrelevant albumen thereof are with same concentration bag quilt.

2. discard antigen coated liquid, with the sealing of 0.1% gelatin, 37 ℃, 30 minutes.

3. add mouse anti PR serum, 37 ℃, 1 hour.

The 4.PBS-0.05%Tween20 washing, 5 minutes * 3.

5. add the HRP mark anti-mouse IgG of rabbit (1: 100), 37 ℃, 40 minutes.

6.PBS-Tween20 wash, with 4.

7.TMB the substrate colour developing, each 1 of A, B liquid, under the room temperature, 15 minutes.

8.12.5% sulfuric acid cessation reaction.

As a result, PR expression product and mouse anti PR serum show the specificity association reaction, and do not react with the irrelevant albumen that contrasts.

Confirm PR gene expression product biologically active, can be used as antigen preparation antibody.

Embodiment 3

Anti-ER and anti-PR MONOCLONAL ANTIBODIES SPECIFIC FOR one, anti-ER MONOCLONAL ANTIBODIES SPECIFIC FOR:

1. immune programme for children:

Get 5 of female BALB/C mice in age in 6-8 week, get antigen ER-BCD 18-60ug//time, (during preparation PR monoclonal antibody, replacing ER-BCD with the PR expression product) (fully, full freund adjuvant toos many or too much for use later on for the first time to add adjuvant, be not limited to freund adjuvant) subcutaneous multi-point injection, the 7-10 day immunity of every interval once is total to immune 3-5 time, one month at interval, booster immunization once, 3-5 gets spleen and merges after day.

2. prepare the immune mouse spleen cell suspension.

3. prepare common mouse feeder layer cell.

4. myeloma cell's recovery and cultivation.

7-10 day by taking out in the liquid nitrogen, drops into cell in 37 ℃ of water fast before merging, and washes once with serum free medium, adds to contain in the complete culture solution of calf serum, regularly changes liquid, makes the myeloma cell be in vigorous logarithmic growth state before fusion.

5. merge

(1) nutrient solution HAT, HT and PEG (30%w/v) are selected in preparation routinely.

(2) with myeloma cell and immune spleen cell with 1: the 5-10 ratio dropwise adds 0.5-1ml30%PEG in the cell mass after mixing, and adds fine rotation or springing centrifuge tube in 1 minute.

(3) when constantly rotating centrifuge tube, add serum-free medium 1-5ml, add in 1-5 minute.

(4) the 20ml serum-free medium added in 5 minutes in addition.

(5) low-speed centrifugal 500-1000rpm, 5-10 minute, abandon supernatant, add the complete culture solution 50-100ml that contains HAT, mixing gently suspends.

(6) (5) cell suspension kind is placed 96 orifice plates of being with feeder layer, every hole adds 100-200ul.

(7) place 5% carbon dioxide temperature target to cultivate, the hybridoma colony appears in 2-3 week, in time detect, screens, clone.

6. hybridoma detects, clones

(1) with the synthetic peptide of ER-D section (the synthetic peptide bag quilt of PR when detecting the PR monoclonal antibody) wrapper sheet 5ug/ml, liquid is crossed for 4 ℃ in the 50ul/ hole.

(2) PBS washes plate, the sealing of 0.1% gelatin, room temperature 30-60 minute.

(3) discard unnecessary confining liquid, add the supernatant that has in the hybridoma colony growth hole by tag sequence, the 50ul/ hole is established negative control and positive control hole simultaneously, room temperature reaction 1 hour.

(4) PBS that contains 0.05%Tween-20 washes plate 3-4 time.

(5) add the anti-mouse IgG of peroxidase labelling rabbit, 50ul/ hole, room temperature 1 hour.

(6) wash plate, tmb substrate colour developing 15 minutes, 12.5% sulfuric acid cessation reaction.

(7) select the positive reaction hole, get hybridoma, clone with limiting dilution assay.

(8) with said method repeated screening, clone, clone at least continuously 3 times, still the hybridoma liquid nitrogen cryopreservation of stably excreting monoclonal antibody is got its supernatant, further does SABC and detects.

(9) SABC detects:

That A. gets the known ER positive and ER feminine gender (gets known PR when detecting the PR monoclonal antibody ^{+ and}PR ^-) the breast carcinoma tissue paraffin section, conventional dewaxing entry, 0.3%H ₂O ₂-methyl alcohol soaked 30-40 minute, and PBS washes.

B.0.1% gelatin sealing, 30-60 minute.

C. discard unnecessary confining liquid, add supernatant to be checked, 37 ℃, spent the night in 1 hour or 4 ℃.

D.PBS washes 5 minutes * 3,

E. add the anti-mouse IgG of Bioten mark horse, room temperature 30-40 minute.

F.PBS washes 5 minutes * 3, adds HRP mark streptavidin, room temperature 30-40 minute.

G.PBS washes 5 minutes * 3, adds freshly prepared DAB (0.5mg/ml)+H ₂O ₂(1ul/ml), 3-5 minute.

H. conventional dehydration, mounting, microscopy.The positive reaction thing is positioned karyon as a result, is pale brown look dyeing.Two, anti-PR MONOCLONAL ANTIBODIES SPECIFIC FOR

The preparation method of anti-PR monoclonal antibody is with the preparation method of ER monoclonal antibody.The antigen of immunity usefulness changes PR expressing protein product into, when detecting the PR monoclonal antibody, is the antigen wrapper sheet with the synthetic peptide of PR, takes breast cancer tissue's paraffin section of the known PR positive and PR feminine gender when SABC is identified, removes in addition the same ER of other implementation step.

Embodiment 4

The evaluation of the evaluation one of anti-ER monoclonal antibody, anti-PR monoclonal antibody performance, anti-ER monoclonal antibody performance

1. the detection of tiring of anti-ER monoclonal antibody: with the synthetic peptide of ER-D is the antigen wrapper sheet, and enzyme linked immunological is inhaled to have a try and tested (ELISA) method to detect that anti-ER monoclonal antibody tires be 1: 800-1600.And the ER monoclonal antibody nothing to do with albumen reaction that is negative.

2.Western Blot analyzes

That to confirm the ER feminine gender through the DCC value and positive breast cancer sample homogenate in the TED damping fluid, change film again behind the capable again SDS-PAGE electrophoresis, anti-(1) the mouse anti ER-BCD antibody (2) of using respectively is the same with later all the other operation stepss of mouse anti ER-BCD of the pre-absorption of ER expressing protein product.See Fig. 8 through anti-ER-BCD serum coloration result.1 is the breast carcinoma tissue that turns out to be the ER feminine gender through the DCC method among the figure, and 2,3 is the same breast carcinoma tissue of the ER positive.1,2 is all anti-with mouse anti ER-BCD serum work one, and the 3rd, mouse anti ER-BCD serum is done one later on the absorption of ER-BCD expressing protein product resist later staining procedure and 1,2 to carry out synchronously.Last 1 no special band shows; As seen 2 have a special reinforcing band at the 67KD place; And an antiserum is after the pre-absorption of ER-BCD, and this reinforcing band disappears, as among the figure 3.

3. SABC detects the performance of antibody

With anti-hER monoclonal antibody is one anti-, and other SABC operation steps is the same.

(1) anti-ER-BCD serum of self-control and import ER antibody mediated immunity group result are relatively: detected 17 routine fibroadenomas of breast, the proliferation of mammary gland and 8 routine breast cancer paraffin sections, the total coincidence rate 88% (22/25) of yin and yang attribute.The SABC result of antibody of the present invention and import antibody is proportionate.(P＜0.001)

(2) comparison of anti-ER-BCD antibody mediated immunity group result of self-control and DCC value: detect 47 routine breast cancer samples altogether, the yin and yang attribute coincidence rate is 95.5% (43/45), P＜0.001.The positive reaction thing is positioned ER positive cell nuclear, is pale brown look.The breast cancer cell karyon is dyed pale brown look among the (see figure 9) figure, is shown black among this figure.Two, the evaluation of anti-PR monoclonal antibody performance

1. the detection of tiring of anti-PR monoclonal antibody: with the synthetic peptide of PR is the antigen wrapper sheet, and it is 1 that enzyme linked immunosorbent assay (ditto) detects that its culture supernatant of tiring tires: 100-1000, it is 1: 10 that ascites is tired ⁶-10 ⁷, the reaction that is negative of nothing to do with albumen.

2. SABC detects the performance of antibody

Be the anti-30 routine breast cancer tissue paraffin sections that detect with import PR antibody and PR antibody of the present invention respectively, operation steps is as follows respectively:

(1) the anti-PR monoclonal antibody of import operation steps:

The conventional dewaxing of paraffin section entry → 0.3%H ₂O ₂-methanol solution soaks 30 minutes → PBS and embathes in 5 minutes * 3 → citrate buffer in 92-98 ℃ and wash 5 minutes * 3 at room temperature cooling 10 minutes → 0.1% gelatin sealing → PBS after 8-10 minute → add an anti-PR antibody, and 4 ℃ are spent the night, and later step is the same.

(2) anti-PR monoclonal antibody operation steps of the present invention:

Two samples of parallel detection, a sample gives antigen retrieval fully by the running program of import antibody with section, pyroprocessing (hereinafter to be referred as boiling sheet), another sample does not carry out antigen retrieval (hereinafter to be referred as not boiling sheet), and other step is the same.Consequently make the sample Color indifference of anti-PR monoclonal antibody by oneself, and import antibody requires sample to boil, and makes it complicated operation, causes frequent flake to boiling and not boiling.Make by oneself anti-PR antibody mediated immunity group coloration result and DCC value and import antibody as a result coincidence rate reach 90-95%.PR SABC positive reaction thing is positioned PR positive cell karyon, is pale brown look.As shown in figure 10.It promptly is the breast cancer cell karyon that the circular or rotund black in figure center and upper right corner place what dye.

Through above-mentioned every detection, confirm that anti-ER monoclonal antibody of the present invention and anti-PR monoclonal antibody are functional, have the specific bond ability with natural ER, PR.More simple than import like product with its operation steps of carrying out immunohistochemical staining, Color is good, has clinical widely and the research application prospect.Abbreviation is explained and is arranged following ER estrogen receptor PR progesterone receptor ER with the order that occurs in the instructions ⁺(ER ^-) estrogen receptor positive (feminine gender) PR ⁺(PR-) the enzyme immunity test E of the progesterone receptor positive (feminine gender) DCC method glucosan active carbon adsorption ER-ELA estrogen acceptor monoclonal antibody ²Estradiol IHC method immunohistochemical method McAb monoclonal antibody EP-C.D section ER-BCD section estrogen receptor molecular structure divides the recombinant plasmid of A.B.C.D.E.F. section PMS-ER carrier PMS-3lb and ER, can express

PMS-PR is the same for the PMS-ER fusion, and the PR recombinant plasmid that is built into is expressed PMS-

The name code SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS dodecyl sodium sulfate PAGE polyacrylamide gel electrophoresis ELISA EUSA PBS phosphate buffer DAB diaminobenzidine H of PR fusion PCR polymerase chain reaction,PCR PMS-3lb expression vector₂O ₂A kind of Solution H ER human estrogen acceptor that dissolves tissue that the name code NC nitrocellulose filter BSA-PBS bovine serum albumin(BSA) of a sephadex TMB tetramethyl benzidine BSA bovine serum albumin(BSA) HSA human serum albumins HRP horseradish peroxidase Tween-20 detergent of hydrogen peroxide Western Blot immunity marking BamHl restriction endonuclease name code Ecorl restriction endonuclease name code CNBr-Sepharose4B cyanogen bromide activation-phosphate buffered liquor PAP peroxidase anti-peroxidase compound pH represents to talk endlessly the ordinary symbol KLH keyhole limpet hemocyanin HAT hypoxanthine-amino of solution acid alkalinity cool-thymidine HT hypoxanthine-thymidine PEG polyethylene glycol Bioten biotin TED oneself prepares

Claims (5)

1. biological reagent that detects the estrogen receptor level, this reagent is used to detect hormone-dependent neoplasm patient's estrogen receptor level, comprises the monoclonal antibody that is produced by hybridoma MAER-SI CCTCC NO:C96005.

2. biological reagent according to claim 1 is characterized in that described hybridoma is the splenocyte of antigen immune mouse by estrogen receptor-BCD section, forms with myeloma cell's fusion, screening, clone.

3. biological reagent according to claim 2 is characterized in that described antigen is the protein product of the estrogen receptor-BCD section with Protocols in Molecular Biology construction recombination plasmid, abduction delivering.

4. a SABC method that detects the estrogen receptor level is characterized in that usefulness, and the described biological reagent of claim 1 carries out the SABC of estrogen receptor level is detected.

5. method according to claim 4 is characterized in that histopathologic slide is not needed to carry out the step of the antigen retrieval of pyroprocessing.

Images