CN206270347U - A kind of kit for detecting or treating people's carcinoma of endometrium - Google Patents
A kind of kit for detecting or treating people's carcinoma of endometrium Download PDFInfo
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- CN206270347U CN206270347U CN201620583217.4U CN201620583217U CN206270347U CN 206270347 U CN206270347 U CN 206270347U CN 201620583217 U CN201620583217 U CN 201620583217U CN 206270347 U CN206270347 U CN 206270347U
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Abstract
The utility model passes through will be from people endometrial cancer tumor protein immunization mouse, it is found that a kind of new people's Tumor Markers for Endometrium Carcinoma, it is named as GAB31, it is the CD44 of Aberrant glycosylation, it is characterized in that including sugared structure Galb1-3 (Neu5Aca2-6) GlcNAcb1-4Galb1-4Glcb of epitope on CD44, in addition, by the monoclonal antibody for screening antigen described in specific recognition, obtaining one plant can be with the antibody of antigen described in specific recognition, tested by the immunomagnetic beads for clinical sample, it was found that, cast-off cells microscopy method of the positive rate of the detection method EM2D9-MB of described antibody recognition antigen GAB31 apparently higher than routine.
Description
Technical field
The invention belongs to tumor immunology field.The present invention relates to a kind of novel uterine endometrial carcinomas tumor markers GAB31,
And the monoclonal antibody EM2D9 of anti-GAB31.Cell and histological level are proved：EM2D9 specific recognition people's carcinomas of endometrium
Cell and people's endometrial.The invention further relates to the method that an immunomagnetic beads method detects people's carcinoma of endometrium.The inspection
The antigen that survey method is detected is the carcinoma of endometrium novel markings thing GAB31 of the people in the present invention, and detection antibody is the present invention
In anti-human carcinoma of endometrium GAB31 monoclonal antibody EM2D9.
Background technology
Carcinoma of endometrium is one of most common malignant tumour of female reproductive system, and its incidence of disease is in American-European countries women
Reproductive system malignant tumour is the first.Relative to American-European countries, developing country's onset of endometrial cancer rate is relatively low, but the death rate compared with
It is high.Life and eating habit with China common people tend to westernization, especially developed area, the incidence of disease of carcinoma of endometrium
Just increase year by year, the second of China's female reproductive system malignant tumour is occupied at present, be only second to cervical carcinoma, and age of onset is gradually
Rejuvenation.The cause of disease of carcinoma of endometrium is still not clear so far.Although the method for early diagnosis of carcinoma of endometrium have Transvaginal Ultrasound,
All multi-methods such as endometrial biopsy, diagnostic curettage, endometrial cell, but in asymptomatic general population and danger
The method of examination carcinoma of endometrium in dangerous crowd, does not currently form unified guide or suggestion still in the industry.Thus, explore uterus
The strategy of endometrial carcinomas examination is the hot fields of current medical field carcinoma of endometrium research.The conventional diagnosis of current carcinoma of endometrium
Method is used for screening strategy, all more or less has than larger defect.When Transvaginal Ultrasound cannot determine Endometrial Diseases
The dividing value of inner film thickness；The person that receives endometrial biopsy more or less has a sense of discomfort, and have the person under inspection of significant proportion because
For tissue sampling amount deficiency causes to fail to pinpoint a disease in diagnosis；Diagnostic curettage is invasive operation, includes screening strategy obviously improper and restricted
In the reason for medical level, non-developed regions cannot be popularized；Hysteroscopy high cost, and individually have with diagnostic value
Limit；The characteristics of endometrial cell closest to screening strategy, but, currently lack unified cytology standard.It can be seen that, uterus
The screening strategy of endometrial carcinomas stills need further to probe into.
In recent years, the characteristics of tumor markers is due to its specificity higher and sensitivity, examination, diagnosis to tumour,
Classification, Prognosis scoveillance and treatment provide sizable directive significance.There are some researches show CD44 participates in endometrial carcinoma cell
Invasion and attack and transfer.CD44 belongs to the multi-functional adhesion molecule of cell, is a kind of transmembrane glycoprotein of single-gene coding, and it is widely present
In various kinds of cell and tissue.CD44 has 10 variant exons, thus its can by alternative splicing, be spliced to form it is various different
Structure body, with reference to cell surface respective ligand, and then can adjust various physiology, the pathologic process of cell.Research shows that CD44 divides
Hematopoietic Stem in son expression high in kinds of tumors, and growth with tumour cell, invasion and attack, transfer ability and tumor environment is thin
Going back to the nest for born of the same parents is closely related, and can be used as the mark of tumor stem cell, or can be used as tumor screening, Index for diagnosis and treatment
Target spot.The biological function and expression regulation pattern of CD44 are different because of different condition, such as tumor class, growth conditions.It is various
Factor can influence it to express, such as ERs, transcription factor.The Aberrant glycosylation modification of CD44 can cause tumour cell to be disliked
Property degree change, probe into the abnormal modification of CD44 in carcinoma of endometrium, can be that the examination of carcinoma of endometrium, diagnosis, prognosis be sentenced
Disconnected and treatment provides a kind of new target spot, and the diagnosis and treatment for carcinoma of endometrium will be extremely important.
Utility model content
The present invention extracts total protein immune mouse from fresh people's endometrial, prepares hybridoma cell strain.
One plant of antibody EM2D9 that can be combined with people's endometrial high special is screened with the method for ELISA, the antibody belongs to
IgG1 subclass.Immunohistochemistry confirms that EM2D9 antibody is in strong positive reaction with people's endometrial, and normal with people
Endometrial tissue no cross reaction.
The present invention identifies that the antigen of EM2D9 antibody recognitions is a kind of by immunoprecipitation combination mass spectrum with carbohydrate chip result
The CD44 of Aberrant glycosylation, it is positioned on cell membrane, and its epitope is：Galb1‐3(Neu5Aca2‐6)GlcNAcb1‐
4Galb1-4Glcb belongs to brand-new endometrium carcinoma marker.
EM2D9 antibody coupling nano magnetic particles are prepared into anti-endometrial cancer immunomagnetic beads (EM2D9-MB) by the present invention,
The endometrial carcinoma cell for coming off is captured and be enriched with, through Rui Shi Jim Sa pathology smear stainings, microscopy is carried out under the microscope and is examined
It is disconnected.The kit is applied to early screening, Prognosis scoveillance and the pathology auxiliary diagnosis of tumour patient.
Novelty of the invention is：(1) screen and be prepared for a kind of monoclonal antibody of anti-human carcinoma of endometrium
EM2D9, the antibody and people's endometrial are in strong positive reaction, and with people's normal endometrial tissue no cross reaction；
(2) epitope of EM2D9 antibody recognitions is disclosed for Galb1-3 (Neu5Aca2-6) GlcNAcb1-4Galb1-4Glcb, is
Brand-new endometrium carcinoma marker；(3) develop a kind of highly sensitive for detecting people's intrauterine based on EM2D9 antibody
The immunomagnetic beads method of film cancer.
New detailed description
The invention provides a kind of CD44 of brand-new people's Tumor Markers for Endometrium Carcinoma-Aberrant glycosylation, it is named as
GAB31.And obtain the hybridoma cell strain of the monoclonal antibody for producing anti-GAB31, hybridoma cell strain secretion monoclonal
The epitope of antibody EM2D9, monoclonal antibody EM2D9 identification is Galb1-3 (Neu5Aca2-6) GlcNAcb1-4Galb1-
4Glcb.The monoclonal antibody and people's endometrial are in strong positive reaction, and with people's normal endometrial tissue without intersecting
Reaction.Present invention also offers the immunomagnetic beads (EM2D9- of the external uterine endometrial carcinomas diagnosis based on monoclonal antibody EM2D9
MB) diagnostic method.
Specifically, the invention provides a kind of people Tumor Markers for Endometrium Carcinoma GAB31 of Aberrant glycosylation, it is different
Normal glycosylated CD44, it is characterized in that the sugared structure Galb1-3 (Neu5Aca2-6) comprising epitope on CD44
GlcNAcb1‐4Galb1‐4Glcb。
In a preferred scheme, the amino acid sequence such as SEQ ID No of the CD44:Shown in 1.
It is a further object to provide for the anti-of people's endometrium carcinoma marker GAB31 of the present invention
Body, it being capable of specific recognition institute
State as sugared structure Galb1-3 (Neu5Aca2-6) GlcNAcb1-4Galb1-4Glcb of epitope, it is described anti-
Body is polyclonal antibody or monoclonal antibody, preferably monoclonal antibody.
It is a further object to provide a kind of kit for detecting people's carcinoma of endometrium, it includes the present invention
Above-mentioned antibody EM2D9.In one preferred embodiment, described antibody is that the monoclonal of anti-human carcinoma of endometrium GAB31 resists
Body EM2D9, the monoclonal antibody is by preserving number for the hybridoma cell strain of CGMCC No.11796 is secreted.
It is a further object to provide a kind of conjugate, wherein comprising the anti-human carcinoma of endometrium GAB31 with
Material selected from following components composition is conjugated：Biomarker, antineoplastic, toxin and radioactive activity agent.
It is a further object to provide a kind of for detecting or treating the kit of people's carcinoma of endometrium, wherein wrapping
Containing the above-mentioned antibody of the present invention.
In a preferred embodiment, in described kit, described detection is by monoclonal antibody EM2D9
Coupled to Nano magnetic particle is carried out, and preferably by EM2D9 antibody coupling nano magnetic particles, is prepared into anti-endometrial cancer immunomagnetic beads
(EM2D9‐MB).In a preferred embodiment, the testing sample is the cast-off cells of carcinoma of endometrium containing people uterine cavity scraping
Thing.
It is a further object to provide the miscellaneous of the monoclonal antibody EM2D9 for secreting anti-human carcinoma of endometrium GAB31
Tumor cell strain is handed over, its preserving number is CGMCC No.11796.
Brief description of the drawings
The SABC figure that Fig. 1 .EM2D9 monoclonal antibodies are cut into slices to people's endometrial.
The SABC figure that Fig. 2 .EM2D9 monoclonal antibodies are cut into slices to people's normal endometrial tissue.
Fig. 3 carbohydrate chip testing results.
Fig. 4 kit schematic diagrames, wherein 1 is monoclonal antibody container, 2 is that endometrial carcinoma cell dyes liquid container.
Specific embodiment
The present invention is described in detail hereinafter with reference to embodiment, the embodiment is only intended to illustrate the present invention, without
It is intended to limit the scope of the present invention.The scope of the present invention is specifically limited by appended claims.
Embodiment 1：The preparation and purification of EM2D9 monoclonal antibodies
(1) preparation of hybridoma
1) animal immune and cell culture：It is small using the immune Balb/C of fresh people's endometrial albumen homogenate
Mouse (is bought from Beijing Vital River Experimental Animals Technology Co., Ltd.), and intraperitoneal injection is carried out with every dosage of 20ug total proteins
Mode immune mouse.Mouse is immunized again after two weeks.After mice serum potency reaches requirement, booster immunization once, takes after 3 days
Mouse spleen, prepares lymphoblast suspension, prepares cell fusion.Recovery myeloma cell Sp2/0 (ATCC CRL-1772),
And screened to maintain cell to the sensitiveness of HAT with 8-AG (8 azaguanine).
2) cell fusion：By step 1) prepare lymphocyte suspension merged with myeloma cell, specific method join
According to《Fine works immunological experiment guide》((U.S.) J.E. science root (U.S.) D.H. Margules etc., Science Press, 2009
January publishes).Cell suspension is added and contains culture in feeder cells culture medium after merging.After fusion 24 hours, plus HAT selections
Culture medium (is purchased from Invitrogen companies；HAT is H:Hypoxanthine hypoxanthine, A:Aminopterin methopterins,
T:Thymidine thymidines) carry out selective culture.
3) antibody test：The hybridoma cell strain of secretory antibody is determined by ELISA method.Specific method is：Extract son
Endometrial carcinoma tissue total protein, with 4 DEG C of coatings of 0.05mol/L carbonate buffer solutions (pH9.6) overnight, adds 37 DEG C of 10%BSA
Closing 3 hours.PBST is washed 3 times, then adds 100ul supernatants to be checked, 37 DEG C of incubation 1h.Washing 3 times, adds the peppery of anti-mouse
The secondary antibody IgG-HRP (being purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge) of root peroxidase labelling, 37 DEG C of incubation 1h.Wash
Wash 3 times, add 50ul TMB (being purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge) colour developing, after being stored at room temperature 5min, add
50ul terminate liquids.The OD values that wavelength is 450nm are read with ELIASA.OD values are more than being considered as more than 2 times of negative control OD value
It is positive.
3) cloning of hybridoma and freeze：The positive hybridoma cell that will be filtered out using limiting dilution assay is cloned
Change culture.By the colonized culture of 3 wheels, the hybridoma cell clone Amplification Culture of high-titer monoclonal antibody is filtered out.
A kind of positive hybridoma cell strain obtained in the present invention is the monoclonal hybridoma of anti-human carcinoma of endometrium
Strain, the hybridoma cell strain was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms on December 23rd, 2015
Center (CGMCC, China, Beijing), preserving number is CGMCC No.11796.
(2) preparation and purification of EM2D9 monoclonal antibodies
The hybridoma cell strain (preserving number CGMCC No.11796) of aforementioned stable secrete monoclonal antibody EM2D9 is expanded
Culture, and collect cells and supernatant.Affinitive layer purification is carried out to monoclonal antibody EM2D9 using Protein G, easy steps are：
First Protein G affinity columns (being purchased from " GE " company) are balanced with PBS；Then by the cell culture containing EM2D9 monoclonal antibodies
It is clear to cross Protein G affinity columns；Then wash chromatographic column with PBS, until flow out pillar cleaning solution OD values close to zero；
Pillar is eluted using glycine-HCL solution (PH2.8) of 0.2M, is in charge of collection eluent, determine the OD values of each collecting pipe.Contain
- 20 DEG C freeze after the eluent of EM2D9 monoclonal antibodies is concentrated through PBS.
Embodiment 2：The identification of EM2D9 monoclonal antibodies
People's endometrial and people's normal endometrial tissue are cut using the EM2D9 monoclonal antibodies prepared in embodiment 1
Piece, as a result as shown in Figure 1, 2.Result shows that people's endometrial is presented after being developed the color through EM2D9 monoclonal antibodies immunohistochemical staining
Positive reaction (Fig. 1), and people's normal endometrial tissue is presented negative reaction after being developed the color through EM2D9 monoclonal antibodies immunohistochemical staining
(Fig. 2).
Use the EM2D9 monoclonal antibodies prepared in embodiment 1, SABC detection people endometrial, people's normal uterus
Internal film tissue and other tissues, as a result as shown in table 1.Result shows that EM2D9 antibody is in strong positive with people's endometrial
Reaction, and with people's normal endometrial tissue and other organize no cross reactions.
Table 1
SABC detects immune responses of the anti-human carcinoma of endometrium monoclonal antibody EM2D9 to Various Tissues
Embodiment 3：The preparation of GAB31 antigens
1) total protein extraction：50mg people's endometrial, shreds after grinding to form homogenate, adds three decontaminations of 1ml to split
Solution liquid, 4 DEG C crack 5 minutes, and 12000rpm is centrifuged 20 minutes, takes supernatant, that is, be people's endometrial total protein.
2) immunoprecipitation：Add 100ug EM2D9 monoclonal antibodies, 4 DEG C of incubation 2h.
3) Antigen Identification：Add 50ul Protein G beads, 4 DEG C of incubation 2h.Beads, Ran Houyong are washed with PBS
Glycine-HCL solution (PH 2.8) the wash-out beads of 0.2M, carry out mass spectral analysis.Mass spectral results are as shown in table 2.Mass spectral results
Show, the antigen of EM2D9 is CD44.The sugared lamination data of EM2D9 are (as shown in Figure 3) by analysis finds, the identification of EM2D9 monoclonal antibodies
Epitope behaviour CD44 sugar chain, the epitope is Galb1-3 (Neu5Aca2-6) GlcNAcb1-4Galb1-4Glcb.By
In the only specific CD44 for recognizing people's endometrial, showing that the antigen is Aberrant glycosylation modification of EM2D9 monoclonal antibodies, its
Epitope Galb1-3 (Neu5Aca2-6) GlcNAcb1-4Galb1-4Glcb is only expressed in people's endometrial cell.
Table 2
The Mass Spectrometric Identification of EM2D9 antigens
| Accession | Mass | Score |
| CD44 HUMAN | 176389 | 217 |
| Albumin HUMAN | 42367 | 43 |
| Stratifin HUMAN | 23819 | 32 |
| Actin,gamma 1 HUMAN | 15376 | 32 |
| Desmoglein 1 HUMAN | 38532 | 30 |
| Histone H2B HUMAN | 72513 | 30 |
| Hornerin HUMAN | 31285 | 28 |
| Keratin 13 HUMAN | 63527 | 25 |
| Keratin 15 HUMAN | 19728 | 25 |
| Keratin 6B HUMAN | 24386 | 20 |
Embodiment 4：The preparation of people's carcinoma of endometrium immune diagnostic reagent
Using monoclonal antibody EM2D9 prepared in the present invention, the present invention we by EM2D9 antibody coupling nano magnetic particles, system
It is standby into anti-endometrial cancer immunomagnetic beads (EM2D9-MB).It is mixed with uterine cavity scraping thing, the uterus for coming off is captured and be enriched with
Inner membrance cancer cell, through Rui Shi Jim Sa pathological stainings, is diagnosed under the microscope.The kit is applied to the morning of tumour patient
Phase examination, Prognosis scoveillance and pathology auxiliary diagnosis.Experiment confirms people's carcinoma of endometrium immune diagnostic reagent of the invention and has
Technology is compared and had the positive effect that：(1) sensitivity is high, due to being coupled the enrichment of monoclonal antibody EM2D9 immune magnetic particles, should
Detection method of the sensitivity of method far above conventional exfoliative cytology；(2) high specificity, because the method has been coupled specifically
Property the histiocytic EM2D9 antibody of combination carcinoma of endometrium, it can specific identification endometrial carcinoma cell；(3) it is convenient fast
Victory, saves patient's medical treatment expense, because the method sensitivity is high, high specificity the features such as, the detection to endometrial carcinoma
Rate is high, it is to avoid patient sees a doctor and makes a definite diagnosis repeatedly, saves expense.
Specific experiment method is as follows：
1) uterine cavity scraping thing collection：Endometrial carcinoma comes from The Third Affiliated Hospital of Peking University.
2) enrichment of endometrial cell：The uterine cavity scraping thing temperature of the magnetic particle and patient that will be coupled EM2D9 antibody is incubated
20min, then scrapes magnetic particle thing and separates, so as to be enriched with endometrial carcinoma cell using magnetic frame with the uterine cavity of patient；
3) Rui Shi Jims Sa pathological staining：By step 2) endometrial carcinoma cell that obtains of enrichment carries out Rui Shi Jims Sa disease
Reason dyeing, and dyeing glass slide is prepared for microscopy.
4) microscopy diagnosis：According to the morphological feature of endometrial carcinoma cell, pathological staining result is judged.
EM2D9-MB methods and conventional smear method positive rate comparative result, as shown in table 3.Result shows, uses
EM2D9-MB have detected the uterine cavity scraping thing from the patients with endometrial cancer of The Third Affiliated Hospital of Peking University 32, the sun of EM2D9-MB
Property recall rate be 90.63% (29/32), far above the positive rate 43.75% (14/32) of conventional cast-off cells smear.Cause
This, the GAB31 of endometrial carcinoma cell specifically expressing is a very promising endometrium carcinoma marker, anti-intrauterine
Film cancer immunomagnetic beads (EM2D9-MB) is a kind of simple efficient carcinoma of endometrium diagnostic reagent.
Table 3
Claims (7)
1. a kind of kit for detecting or treating people's carcinoma of endometrium, wherein comprising detection people's endometrial cancer tumor mark
The detection reagent of thing GAB31, the GAB31 is by sugared structure Galb1-3 (Neu5Aca2-6) GlcNAcb1- of epitope
The CD44 of 4Galb1-4Glcb Aberrant glycosylations.
2. it is according to claim 1 detection or treatment people's carcinoma of endometrium kit, it is characterised in that described detection
The reagent of people's Tumor Markers for Endometrium Carcinoma GAB31 includes identification sugar structure Galb1-3 (Neu5Aca2-6) GlcNAcb1-
The monoclonal antibody of 4Galb1-4Glcb.
3. it is according to claim 2 detection or treatment people's carcinoma of endometrium kit, it is characterised in that described Dan Ke
Grand antibody is preserving number for CGMCC No.11796 hybridoma cell strains are secreted.
4. it is according to claim 3 detection or treatment people's carcinoma of endometrium kit, it is characterised in that described reagent
Box also includes detection reagent container, described for detecting that the monoclonal antibody of GAB31 tumor markerses is contained in described appearance
In device.
5. it is according to claim 4 detection or treatment people's carcinoma of endometrium kit, it is characterised in that described detection
The detection reagent of people's Tumor Markers for Endometrium Carcinoma GAB31 also includes carrier, and described monoclonal antibody is coupled to the load
On body.
6. it is according to claim 5 detection or treatment people's carcinoma of endometrium kit, it is characterised in that described carrier
It is magnetic microsphere.
7. it is according to claim 6 detection or treatment people's carcinoma of endometrium kit, it is characterised in that described reagent
The box also dyeing liquor including endometrial carcinoma cell, described dyeing liquor is Rui Shi Jim Sa pathological stainings, described dyeing liquor
It is similarly positioned in cell therefor.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201620583217.4U CN206270347U (en) | 2016-03-08 | 2016-06-15 | A kind of kit for detecting or treating people's carcinoma of endometrium |
| PCT/CN2017/088502 WO2017215638A1 (en) | 2016-06-15 | 2017-06-15 | Marker and antibody of human endometrial cancer and use thereof |
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| CN201620176110 | 2016-03-08 | ||
| CN2016201761108 | 2016-03-08 | ||
| CN201620583217.4U CN206270347U (en) | 2016-03-08 | 2016-06-15 | A kind of kit for detecting or treating people's carcinoma of endometrium |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105859872A (en) * | 2016-03-08 | 2016-08-17 | 李翀 | Marker of human endometrial cancer, antibody and application of antibody |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105859872A (en) * | 2016-03-08 | 2016-08-17 | 李翀 | Marker of human endometrial cancer, antibody and application of antibody |
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Effective date of registration: 20170726 Address after: Langfang Ruixue road 065099 city of Hebei province Anci District Dragon River High-tech Industrial Zone No. 29, No. 14 building two layer Patentee after: Hebei Boshengdasi Biological Technology Co. Ltd. Address before: 100101 Beijing city Chaoyang District Datun Road No. 15 room 1617 Patentee before: Li Li |
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Effective date of registration: 20190925 Address after: Room 102, Unit 102, Unit 31, Unit 4, District 4, Langfang Pipeline Bureau, Hebei Province, 065000 Patentee after: Wang Hong Address before: 065099 Second Floor of No. 14 Workshop, 29 Ruixue Road, Longhe High-tech Industrial Zone, Anci District, Langfang City, Hebei Province Patentee before: Hebei Boshengdasi Biological Technology Co. Ltd. |
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