CN101974088A - Breast globin monoclonal antibody and application thereof in breast cancer diagnosis - Google Patents
Breast globin monoclonal antibody and application thereof in breast cancer diagnosis Download PDFInfo
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- CN101974088A CN101974088A CN2010105299755A CN201010529975A CN101974088A CN 101974088 A CN101974088 A CN 101974088A CN 2010105299755 A CN2010105299755 A CN 2010105299755A CN 201010529975 A CN201010529975 A CN 201010529975A CN 101974088 A CN101974088 A CN 101974088A
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Abstract
The invention discloses a method for screening and preparing two breast globin monoclonal antibodies and application of the monoclonal antibodies in the immunohistochemistry and serum ELISA (Enzyme-Linked Immuno Sorbent Assay) detection of breast cancer. Because the breast globin is the specific expression of a breast tissue, the induction of the method can improve the specificity of the traditional breast cancer clinical diagnosis and can be used for the pathological differential diagnosis of the breast cancer, postoperative blood circulation transfer and invasive early diagnosis. Since used reagents are all immunological detection reagents, the invention is more suitable for clinical application compared with the traditional breast globin nucleic acid detection.
Description
Technical field
The present invention relates to mammary cancer clinical assistant diagnosis technology, be specifically related to adopt the mammary gland globin monoclonal antibody of preparation to carry out the method that mammary cancer immunohistochemical methods and serum ELISA detect.
Background technology
Mammary cancer is one of modal malignant tumour of harm WomanHealth, and sickness rate is increases trend year by year, has become the principal disease of harm WomanHealth.About 1,300,000 examples of the annual de novo mammary cancer in the whole world, about 400,000 people die from this disease, about 200,000 examples of the annual kainogenesis mammary cancer of China, in cities such as Beijing, Shanghai, mammary cancer has occupied first of women's malignant tumour.Think that at present early diagnosis and early treatment are the keys that reduces the mammary cancer mortality ratio.Aspects such as early diagnosis, treatment, the transfer of the monitoring course of disease and judging prognosis in mammary cancer are all significant.Therefore find and identify that the molecular marker with diagnostic significance is significant in the mammary cancer control.
(human mammaglobin is that Watson etc. adopted the difference round pcr to find in breast cancer tissue first in 1996 hMAM) to people's mammary gland globin, only expresses in mammary epithelial cell, and crosses in breast cancer cell and express.O ' Brien etc. find hMAM in 81% patient with breast cancer, in 74% primary breast cancer, find hMAM, but then detect expression less than hMAM in non-breast cancer tissue such as mammary gland fibroadenoma etc., prompting hMAM high specificity in breast cancer detection can be used for the early screening and the diagnosis of mammary cancer.
At present, pathological diagnosis still plays a significant role in the clinical diagnosis of mammary cancer.In the present conventional H E staining analysis negative patients, postoperative 25% above relapse and metastasis.Adopt antibody such as mammary cancer molecular marker such as HER2 to carry out the sensitivity that immunohistochemical methods can suitably improve detection, but because therefore this factor wide expression in kinds of tumors, can't distinguish mammary cancer metastatic carcinoma in late period and organ primary carcinoma.Takeda etc. have compared hMAM expression among 283 routine chests, the metastatic lung cancer patient, found that, the specific degree of hMAM is 98.5% in the lung metastatic breast cancer tissue, can infer thus, adopt hMAM antibody will help to differentiate that for the immunohistochemical methods on basis primary lung cancer, breast tumor and mammary cancer lung shift, thus better guiding clinical diagnosis and treatment.
Studies show that: the transfer of mammary cancer is the major reason that causes death, makes pathology and/or detects the existence that relevant molecular marked compound is judged distant metastasis by peripheral blood, sentinel lymph node, marrow sampling to the patient with breast cancer clinically usually.It is all lower to mammary tissue susceptibility and specificity to be applied to clinical mammary cancer molecular marker at present.Usefulness nido RT-PCR such as Gr ü newald have compared hMAM, EGFR, CK219 and have followed the susceptibility and the specificity of transfer as peripheral blood, and the result shows that hMAM has excellent specificity, and then both can detect in the normal control group, and specificity is relatively poor.Corradini etc. studies show that, the mammary gland globin is the mRNA positive expression in 97% mammary cancer blood sample, and the normal control group is negative, and sensitivity and specificity all are higher than CK19, CK20, EGFR, MUC21 and CEA.The simple and rapid ELISA method of having set up Fanger in 2002 etc. detects breast cancer cell and is secreted into mammary gland globin in the serum, provides new thinking for the blood of judging the mammary cancer postoperative follows to shift and do not have the wound early diagnosis.But, cause detection sensitivity and specificity unsatisfactory because this method adopts the polyclonal antibody of hMAM.
For this reason, this research adopts bioinformatics technique to analyze the epitope of hMAM, exploitation is with synthesizing above-mentioned epitope gene, clonal expression and purifying protein, immune mouse prepares monoclonal antibody, set up corresponding immunohistochemical methods and ELISA detection technique, the blood that satisfies the differential diagnosis of mammary cancer pathology, postoperative follows the demand that shifts and do not have the wound early diagnosis.
Summary of the invention:
In order to solve the problem that the clinical early diagnosis of mammary cancer lacks the mammary gland-specific molecular marked compound, the invention provides the monoclonal antibody of the specific expressed hMAM molecule of a kind of mammary tissue.In order effectively to distinguish mammary cancer metastatic carcinoma in late period and organ primary carcinoma, the invention discloses a kind of immunohistochemistry technique based on above-mentioned antibody, overcome the Metastasis in Breast Cancer pathological diagnosis specificity problem on the low side that original technology uses other antibody to cause.In addition, the present invention also on the basis of above-mentioned monoclonal antibody, has set up the double antibodies sandwich ELISA detection technique of hMAM molecule, by the detection to peripheral blood hMAM content, satisfies the clinical demand that the blood circulation of mammary cancer postoperative is shifted and do not had the wound early diagnosis.
The object of the invention provides two strain hMAM specific monoclonal antibody.
The specific monoclonal antibody of two strain hMAM disclosed by the invention institute to deserved epitope respectively as shown in sequence table:
(1) sequence in the sequence table 01
(2) sequence in the sequence table 02
The invention also discloses the purposes of above-mentioned two strain monoclonal antibodies in preparation immunohistochemical methods and double-antibody sandwich elisa detection technique.
The present invention compared with prior art has following characteristics:
1. invention is based on mammary gland globin specific detection
At present, the equal wide expression of the molecular marked compound of breast cancer detection is in multiple tissue and cancer cells, and mammary gland globin hMAM only expresses in mammary tissue and breast cancer cell, therefore, compare with other tumour molecular marked compounds and to have higher specificity, can satisfy clinical blood and follow the demand that shifts and do not have the wound early diagnosis mammary cancer immunohistochemical methods, postoperative.
2. invention is to be based upon on the basis of the immunodetection of mammary gland globin
In view of the critical role of hMAM in the mammary cancer clinical detection, the detection of the mammary gland globin that extensively adopts in the world at present mostly is gene test, and complicated operation is to personnel's equipment requirements height.The present invention sets up hMAM immunohistochemical methods and double-antibody sandwich elisa technology respectively on the basis of preparation hMAM monoclonal antibody, need not expensive equipment, clinical expansion simple to operate, suitable.
For achieving the above object, the contriver has at first synthesized the gene of hMAM, inserts in the pBVIL1 carrier.Sequencing is identified positive colony, and preserves bacterial classification.After 42 ℃ of thermal inductions, adopt ion exchange chromatography and the corresponding hMAM antigen of dextrane gel method purifying.
Utilize the epitope of information biology software analysis hMAM, its aminoacid sequence adopts the synthetic respectively above-mentioned epitope of synthetic peptide technology shown in sequence 1-sequence 4 in the sequence table, be used for follow-up hybridoma screening.
The hMAM antigen immune mouse of employing dna recombinant expression is got mouse spleen and the SP2/0 cell merges, the screening positive hybridoma cell, adopt above-mentioned synthetic peptide screening positive colony, acquisition can be secreted the cell strain of hMAM specific antibody, preparation mouse odd contradictive hydroperitoneum, purifying hMAM monoclonal antibody.
Adopt breast cancer tissue's section to carry out the specificity of the immunohistochemical methods checking hMAM monoclonal antibody that obtains, adopt the double-antibody sandwich elisa technology to set up the detection technique of hMAM molecule in the serum simultaneously.
The present invention is achieved by the following technical programs:
The present invention obtains to express efficiently in E.coli in order to help the hMAM antigen gene, selects colibacillary bias codon to design and synthesize the chimeric antigen gene; To expression vector, introduce XhoI and two restriction enzyme sites of XbaI for the ease of gene clone, to be fit to expression vector pBVIL1 (referring to Chinese patent ZL00100695.9: expression vector pBVIL1 and construction process thereof and purposes) at the two ends of connecting arm.Insert among the carrier pBVIL1 behind the double digestion, make up corresponding expression plasmid.Sequencing proves that each gene fragment has obtained correct insertion.
HMAM chimeric antigen expression plasmid behind the Transformed E .coli HB101, is cultivated by thermal induction, gets full bacterium liquid and carries out the SDS-PAGE evaluation, proves that plasmid has all obtained to efficiently express.Adopt affinity chromatography and gel-filtration purifying, obtain the pure product of hMAM antigen.
With 3 immune mouses of hMAM antigen of purifying, the separating spleen cell carries out cytogamy with myeloma cell SP2/0, and screening positive clone adopts limiting dilution assay to carry out subclone.
Adopt bioinformatics technique to analyze the epitope of hMAM, carry out the hybridoma antibody activity with the synthetic peptide of hMAM epi-position and identify.Acquisition can be secreted the cell strain of hMAM specific antibody.The abdominal injection hybridoma, preparation mouse odd contradictive hydroperitoneum adopts ammonium sulphate precipitation in conjunction with ProteinG affinitive layer purification hMAM monoclonal antibody.Adopt the SDS-PAGE method to identify antigen purity, identify the monoclonal antibody hypotype.
Set up the immunohistochemical methods test kit of patient with breast cancer's pathological tissue sample; Development double-antibody sandwich enzyme connection serum hMAM quantitative measurement technology, and carry out clinical evaluation.
Experimental result proves that the present invention screens two strain monoclonal antibodies altogether, difference called after hMAM-1E12 and hMAM-4B11, and special positive reaction can take place with breast cancer cell in the mammary cancer immunohistochemical methods of foundation, mainly is positioned in the mammary cancer endochylema; To positive rate 102 examples in the 127 routine mammary cancer serum, sensitivity is 80.3%, and the positive only has 5 examples in 133 routine normal control serum, and specificity is 96.2%, and is approaching with external detection level, can be applicable to early diagnosing mammary cancer.
Description of drawings
The mammary gland globin of accompanying drawing 1 purifying
Accompanying drawing 2 epitope bioinformatic analysis mammary gland globin sequences
The immunohistochemical reaction of accompanying drawing 3hMAM-1E12 monoclonal antibody and mammary cancer paraffin section
Accompanying drawing 4 mammary cancer and normal control serum mammary gland globin expression level are relatively
Embodiment
The antigenic cloning and expression of embodiment 1 chimeric hMAM
1. the structure of chimeric hMAM antigen presentation plasmid
1.1 chimeric hMAM antigen gene is synthetic
According to the antigenic aminoacid sequence of above-mentioned hMAM, adopt the intestinal bacteria optimal codon to entrust the gene of the synthetic above-mentioned sequence of Shanghai English fine horse biotechnology Services Co., Ltd.
1.2 the structure of chimeric epitope antigen expression plasmid
1.2.1PCR product and expression vector pBVIL1 double digestion: get each 30 μ l of above synthetic gene product and expression vector and be put in respectively in the Eeppendorf centrifuge tube, each adds 10 * buffer (H), 4 μ l, Xba I (10u/ μ l) and each 1 μ l of XhoI (12u/ μ l), add sterile purified water to 40 μ l, put 37 ℃ of water-bath enzymes and cut and spend the night.
Enzyme is cut the agarose gel electrophoresis purifying and the recovery of product, and PCR product and carrier pBVIL1 carry out purifying with 1.2% sepharose behind double digestion, and concrete grammar is undertaken by the method for " molecular cloning " (Science Press, second edition).The a small amount of glue that purified genes adopts Shanghai China Shun biotechnology company limited to produce reclaims test kit and reclaims, and carries out purifying by the test kit specification sheets.
1.2.2 connect: the carrier after the above-mentioned enzyme of adding is cut in sterilization Eeppendorf centrifuge tube and goal gene each 1 μ l, 10 * T4 DNA Ligasebuffer, 1 μ l, T4 DNA Ligase (12u/ul) 1 μ l, add sterile purified water to 10 μ l, put 16 ℃ and spend the night.
1.2.3 transform: in Bechtop, get 100 μ l competent cells (competent cell is undertaken by the method for " molecular cloning " (Science Press, second edition)) suspension with aseptic suction nozzle and in Eppendorf, add above-mentioned connector 5 μ l, rotate mixing gently, ice bath 30 minutes.Transfer to immediately in 42 ℃ of water-baths and placed 2 minutes, every pipe adds 0.5ml LB substratum (not added with antibiotic), and 30 ℃ of shaking baths were cultivated after 60 minutes, respectively gets 0.2ml and is applied to respectively on the LB agar culture plate and (contains microbiotic), after room temperature is dried, put 37 ℃ of thermostat containers and be inverted overnight incubation.Select several bacterium colonies, be inoculated in respectively (5ml/ pipe) among the LB, overnight incubation is respectively got 0.1ml next day and is transferred to (2ml/ pipe) among the LB, cultivated 3 hours for 32 ℃, inducing culture 4h in 42 ℃ of shaking baths receives bacterium, identify with SDS-PAGE, select goal gene to obtain the high expression level bacterial strain, and order-checking is identified.
2. antigenic expression and purification
2.1 the cultivation of expression strain: the expression strain 20 μ l that get-70 ℃ of preservations are inoculated in (100ml LB/500ml triangular flask) in the LB substratum, 30 ℃ of air shaking table overnight incubation, next day in 5% ratio transferred species in LB substratum (the same), 30 ℃ of air shaking tables were cultivated about 3 hours, when the OD600 value reaches 0.7, immediately culturing bottle is forwarded in 42 ℃ of shaking baths to inducing culture 4 hours.Bacterium liquid is merged, and centrifugal 20 minutes of 6000rpm abandons supernatant, the collecting precipitation part.
2.2 extraction inclusion body: will precipitate and claim weight in wet base, will precipitate with the 20mmol/L pH8.0 TE damping fluid of 10 times of volumes and hang, adding N,O-Diacetylmuramidase (1mg/ml suspension), at room temperature magnetic agitation is 10 minutes.Ultrasonic disruption bacterium in ice bath surpassed for 30 seconds at every turn, surpassed altogether 10 times.8 ℃, 1,2000rpm, centrifugal 20 minutes, abandon supernatant, precipitation is washed once with 1mol/L NaCl (preparing with TE), washes 2 times collecting precipitation again with TE.With 8M urea (preparing) dissolving with PH8.0TE, add 1% beta-mercaptoethanol, again in 20 ℃, 1,2000rpm centrifugal 10 minutes, goes precipitation to get supernatant.
2.3 purifying: above-mentioned dissolved inclusion body solution is crossed Q-Sepharose FF anion-exchange column, with balance liquid (pH8.0,20mmol/L TF contains the 6mol/L urea, 0.1% beta-mercaptoethanol) after the cleaning, NaCl (preparing) wash-out with different concns with balance liquid, collect each elution peak, identify, collect 0.05mol/L NaCl elution peak through SDS-PAGE.After Sephardex G-50 gel-filtration column, collect first elution peak.Identify result such as accompanying drawing 1 with SDS-PAGE.
The selection of embodiment 2hMAM epitope
The epitope of hMAM sequence is predicted that the result contains 8-27aa, 28-47aa and 48-66aa, 66-93aa totally 4 main antigen sections as shown in Figure 1 by information biology software, its sequence is respectively shown in sequence 1-sequence 4 in the sequence table.
The screening and the preparation of embodiment 3hMAM monoclonal antibody
1. animal immune: chimeric antigen 100 a μ g/ complete Freund's adjuvant, adopt subcutaneous and abdominal injection method 3 mouse of immunity respectively, it is immune to carry out second time at interval 4 weeks, and every injected in mice antigen dose is 50 a μ g/ incomplete Freund's adjuvant; Merge preceding 3 days through the abdominal cavity booster immunization, every injected in mice antigen dose is that 50 μ g/ only do not add adjuvant.The survey mouse antibodies is tired, and chooses the highest mouse of antibody titer and carries out cytogamy.
2. cytogamy: fusogen is under the effect of 50%PEG, the ratio of SP2/0 and splenocyte is the cell suspension after merging at 1: 10, adds the RPMI-1640 that contains HAT, joins respectively in 6 96 orifice plates, place 37 ℃ of 5%CO2, cultivate in the constant incubator.
3. the screening of positive hybridoma cell: wrap respectively by elisa plate with above-mentioned 4 synthetic peptides, filter out two strain positive colonies altogether with indirect elisa method, name hMAM-1E12 and hMAM-4B11 respectively, corresponding epitope is respectively sequence 1 and sequence 2.
4. clone and subclone: adopt limiting dilution assay, select the hybridoma of the high monoclonal antibody of positive value to clone and subclone, reach 100% until positive rate.
5. the male BALB/c mouse abdominal injection 0.5ml whiteruss in age in ascites MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying: 10-12 week, every mouse peritoneal injection treats that through the hybridoma suspension 0.5ml of physiological saline washing mouse ascites gathers the back and collects after 10 days.Carry out the ascites purifying with sad-saturated ammonium sulphate salting-out process, dialysis treatment, frozen after the packing at-20 ℃.
6. antibody subtype is identified: get cells and supernatant, carry out according to the mouse resource monoclonal antibody subgroup identification test kit specification sheets of Hbt company.The SDS-PAGE electrophoresis is identified the gel peak, and the uv-spectrophotometric instrument is measured protein content.
Embodiment 4hMAM immunohistochemical methods method
1. paraffin section dewaxes: dimethylbenzene 3 * 10min, 100% ethanol 5min, 95% ethanol 5min, 80% ethanol 5min, 70% ethanol 5min, PBS 3 * 5min.
2. protease K digesting: under 37 ℃ of the freshly prepared 25 μ l 20mg/ml Proteinase Ks, slide is hatched 5min.With PBS washing 3 times, each 5min. is to repair antigen, PBS washing three times, each 5min.
3. remove endogenous peroxydase: slide is soaked 15min sealing endogenous peroxydase in the 3%H2O2-methanol solution, then with PBS washing three times, each 5min.
4. sealing: with 3%BSA-PBS confining liquid sealing 2h.
5. adding specific antibody: with the anti-hMAM monoclonal antibody that the dilution in 1: 200 of 3%BSA-PBS confining liquid obtains, 37 ℃ of wet boxes are hatched 1h.PBS washing three times, each 5min.
6. it is anti-to add good horseradish peroxidase (HRP) mark two of dilution, and wet box is hatched 30min under the room temperature.Wash slide 5 times with PBS, each 5min.
7. colour developing: (50mg DAB is dissolved in the TB of 0.05mol/L (pH7.6), is settled to 100ml, adds H2O230～40 μ l of 30% after filtering before the colour developing) lucifuge colour developing 30s-5min.React with the distilled water color development stopping.
8. redye: phenodin is redyed 3min, uses distilled water flushing.
9. dehydration and transparent: 70% ethanol, 1 * 5min, 80% ethanol, 1 * 5min, 90% ethanol, 1 * 5min, 100% ethanol, 1 * 5min, dimethylbenzene 2 * 10min, neutral gum mounting.
Embodiment 5hMAM double-antibodies sandwich ELISA
With two strain antibodies respectively with the different concns bag by elisa plate, again with two strain antibodies mark vitamin H respectively, combined crosswise, the square formation volumetry filters out the best of breed scheme.
2. coated antibody is in 96 orifice plates (5 μ g/ml), 100 μ l/ holes, 4 ℃ spend the night (16 hours);
The 3.3%BSA-PBS sealing, 120 μ l/ holes, room temperature 6 hours is got rid of confining liquid, and drying at room temperature is standby;
4. every hole adds 50 μ l test serums, adds 50 μ l biotin labeling antibody again, and (1000 times of dilutions of working concentration) hatched 1 hour for 37 ℃ behind the abundant mixing; Wash plate 5 times;
5. the horseradish peroxidase 100 μ l (dilution in 1: 1000) that add the avidin mark are hatched 30min for 37 ℃; Wash plate 5 times; 6.TMB colour developing: A.B liquid respectively adds 50 μ l, hatches 10min for 37 ℃; Stop buffer 50 μ l stop; Survey the OD450nm value.
Adopt double-antibodies sandwich ELISA to detect the situation that exists of patient with breast cancer's serum hMAM, serum specimen all derives from a section mammary gland sample storehouse outside 307 hospitals of PLA.Untreated women with breast cancer 127 examples (2004.5-2007.1 is adenocarcinoma infiltrating), 51.75 years old mean age, median age 51 years old; Physical Examination women 133 examples (2006.1-2007.1), 45.57 years old mean age, median age 46 years old.The result is as shown in Figure 4: positive rate 102 examples in the 127 routine mammary cancer serum, and sensitivity is 80.3%, and the positive only has 5 examples in 133 routine normal control serum, and specificity is 96.2%.
Claims (3)
1. two strain mammary gland globin monoclonal antibodies, the aminoacid sequence of its identification is respectively as shown in sequence table:
(1) sequence 1 in the sequence table
(2) sequence 2 in the sequence table.
2. according to the purposes of the described monoclonal antibody of claim 2 in mammary cancer immunohistochemical methods reagent.
3. according to the purposes of monoclonal antibody described in the claim 1 in mammary gland globin immunoenzyme connection serum detection reagent.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108864272A (en) * | 2018-07-03 | 2018-11-23 | 中国人民解放军军事科学院军事医学研究院 | A kind of mammaglobin Staphylococal Protein A epitope and its application |
| CN109580952A (en) * | 2018-12-28 | 2019-04-05 | 河南省生物工程技术研究中心有限公司 | A kind of markers for breast cancer hMAM test strip and preparation method thereof |
| CN112300283A (en) * | 2019-07-29 | 2021-02-02 | 深圳华大生命科学研究院 | Monoclonal antibody for resisting human pancreatic cancer and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO1996038463A1 (en) * | 1995-05-31 | 1996-12-05 | Washington University | Dna sequence and encoded mammary-specific breast cancer protein |
| CN1277614A (en) * | 1997-09-18 | 2000-12-20 | 华盛顿大学 | Mammaglobin a secreted mammary-specific breast cancer protein |
| CN1646563A (en) * | 2000-04-17 | 2005-07-27 | 科里克萨有限公司 | Compositions and methods for the therapy and diagnosis of breast cancer |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996038463A1 (en) * | 1995-05-31 | 1996-12-05 | Washington University | Dna sequence and encoded mammary-specific breast cancer protein |
| CN1277614A (en) * | 1997-09-18 | 2000-12-20 | 华盛顿大学 | Mammaglobin a secreted mammary-specific breast cancer protein |
| CN1646563A (en) * | 2000-04-17 | 2005-07-27 | 科里克萨有限公司 | Compositions and methods for the therapy and diagnosis of breast cancer |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108864272A (en) * | 2018-07-03 | 2018-11-23 | 中国人民解放军军事科学院军事医学研究院 | A kind of mammaglobin Staphylococal Protein A epitope and its application |
| CN108864272B (en) * | 2018-07-03 | 2021-04-27 | 中国人民解放军军事科学院军事医学研究院 | Mammaglobin A epitope and application thereof |
| CN109580952A (en) * | 2018-12-28 | 2019-04-05 | 河南省生物工程技术研究中心有限公司 | A kind of markers for breast cancer hMAM test strip and preparation method thereof |
| CN112300283A (en) * | 2019-07-29 | 2021-02-02 | 深圳华大生命科学研究院 | Monoclonal antibody for resisting human pancreatic cancer and preparation method and application thereof |
| CN112300283B (en) * | 2019-07-29 | 2024-01-02 | 深圳华大生命科学研究院 | Anti-human pancreatic cancer monoclonal antibody and preparation method and application thereof |
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