CN108864272B - Mammaglobin A epitope and application thereof - Google Patents

Mammaglobin A epitope and application thereof Download PDF

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CN108864272B
CN108864272B CN201810715786.3A CN201810715786A CN108864272B CN 108864272 B CN108864272 B CN 108864272B CN 201810715786 A CN201810715786 A CN 201810715786A CN 108864272 B CN108864272 B CN 108864272B
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mam
breast cancer
epitope
antibody
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冯晓燕
杨锡琴
张贺秋
刘志强
李江雪
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Institute of Pharmacology and Toxicology of AMMS
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Abstract

The invention discloses a mammaglobin A epitope and application thereof, belonging to the technical field of immunology. The amino acid sequence of the mammaglobin A epitope is shown as a sequence table SEQ ID NO. 1. The mammary globin A epitope is transmembrane Mam-A epitope, and a monoclonal antibody targeting cell membrane Mam-A is screened and identifies the epitope. The monoclonal antibody of the Mam-A based on the cell membrane localization has higher specificity and affinity than the existing commercial monoclonal antibody of the Mam-A based on the cell membrane localization. Therefore, the breast cancer immunodetection and targeted therapy based on the antibody have higher specificity. The monoclonal antibody of the invention has higher affinity and specificity than commercial antibodies, so the specificity of breast cancer detection and the targeting property and effectiveness of breast cancer treatment can be improved obviously.

Description

Mammaglobin A epitope and application thereof
Technical Field
The invention belongs to the technical field of immunology, and particularly relates to a mammaglobin A epitope and an application thereof.
Background
Breast cancer is the most common malignancy in women. According to the statistics data related to the international cancer research center, the new breast cancer cases worldwide account for 22.7 percent of the female malignant tumor diseases worldwide every year, the death cases account for 14.1 percent of all female malignant tumor deaths, and the harm of the breast cancer exceeds the cervical cancer, so the breast cancer becomes the first tumor disease threatening the female health. Therefore, the development of early diagnosis and new treatment methods of breast cancer are significant for improving the life quality of patients and prolonging the life cycle of patients.
In the first-line treatment of breast cancer, methods such as chemotherapy, radiotherapy, surgery, targeted therapy and the like are mostly adopted. However, since most breast cancer patients are accompanied by lymph node metastasis, surgical resection cannot be completely cured, often accompanied by chemical adjuvant therapy. Many chemical drugs have been demonstrated to have good antitumor ability in vitro, but have not achieved clinically the desired antitumor effect. Traditional chemotherapy is administrated by intravenous injection, and the medicine reaches the tumor part through blood circulation, so that the activity of the medicine is greatly reduced, meanwhile, the medicine has no specific binding capacity to the tumor, and the actual action concentration of the medicine at the tumor part is lower. In order to obtain better treatment effect, the concentration of the drug at the tumor part must be increased by increasing the dosage of the drug, so that the toxic and side effects of the drug are increased. Therefore, the targeting drug preparation can improve the administration concentration of the focal area and reduce the toxic and side effects, is a promising novel administration method, and receives more and more attention in recent years.
Mammaglobin A (Mam-A) is glycoprotein containing 93 amino acids, has mammary gland specificity, is generally up-regulated in breast cancer patients, has a positive rate of over 70 percent according to research reports, and is recognized as a potential ideal diagnosis target of breast cancer. Research shows that the diagnosis efficiency of the Mam-A is better in the aspects of early breast cancer diagnosis, metastasis, in-situ breast cancer and metastatic breast cancer differentiation and the like. Screening research on in-situ breast cancer shows that the positive rate of Mam-A in early primary breast cancer is more than 70%, and the Mam-A is not expressed in non-breast cancer tissues such as breast fibroadenoma and the like. The lung migration and chest migration specimen diagnosis research of the breast cancer shows that the positive rate of the Mam-A is higher than that of the liquid protein 15 of the vesicular disease, which shows that the diagnosis efficiency is good.
The Mam-A is also recognized as a potential molecular target for treating breast cancer, and has important application prospect. However, the distribution of the epitope on the cell membrane is unknown and the corresponding recognition antibody is lacked, so that the related research is long-standing. In 2009, researchers first confirmed that a large amount of Mam-a exists in a transmembrane form, a part of the Mam-a is distributed in a cell membrane and can be recognized by a specific antibody through bioinformatics technology and experimental means, and reported that currently, only one commercial monoclonal antibody mAb304(MA512638) capable of recognizing Mam-a on the cell membrane; this is the first report on the distribution of Mam-a epitopes on cell membranes and their use as immune-directed drug delivery. In 2011, Mam-a is used as a target, and researchers use the Mam-a antibody coupled with a near infrared dye for in vivo tracking of breast cancer, which indicates that Mam-a can be used as a molecular target for targeted drug delivery for treatment of breast cancer. In the current research of targeting therapy of Mam-A for breast cancer, only one monoclonal antibody with cell membrane recognition capability is reported. The shortage of commercially recognized cell membrane Mam-A seriously hinders the application research in the future.
Disclosure of Invention
In order to solve the problem of lack of specificity of breast tissue in clinical diagnosis and targeted therapy of breast cancer, the invention provides a novel mammaglobin A epitope, the amino acid sequence of which is KELLQEFIDD, and the epitope is shown as a sequence table SEQ ID NO. 1.
The mammaglobin A epitope provided by the invention can be applied to preparation of a mammaglobin A antibody.
Further, the antibody is a monoclonal antibody.
The invention also provides a monoclonal antibody of the mammaglobin A, which identifies the mammaglobin A epitope.
In order to select the antigen epitope with higher activity, the invention uses Biosun biological software to predict the Mam-A antigen epitope and clone and express the Mam-A full-length antigen and each antigen epitope segment antigen.
The method comprises the steps of preparing monoclonal antibodies by adopting full-length Mam-A immune mice, screening and identifying corresponding antigen epitopes identified by different monoclonal antibodies by an ELISA method based on antigen epitope segment antigens, identifying the location of Mam-A proteins identified by different monoclonal antibodies in cells on a cellular level according to the immunofluorescence staining characteristics of the different monoclonal antibodies to breast cancer cells, obtaining the antigen epitopes, and primarily screening and determining the monoclonal antibodies of the Mam-A targeting cell membranes.
The mammaglobin A epitope and the monoclonal antibody thereof can be prepared by a chemical synthesis or biosynthesis method.
The invention also provides the application of the antigen epitope or the monoclonal antibody in preparing a reagent or a medicament for preventing or treating diseases caused by abnormal expression of the mammaglobin A.
Furthermore, the invention also provides application of the epitope or the monoclonal antibody in preparation of a reagent or a medicament for detecting, preventing or treating breast cancer.
In the application, the reagent is an immunohistochemical detection reagent, an immunofluorescence detection reagent, an ELISA detection reagent and a breast cancer circulating tumor cell capture reagent.
The invention also provides a targeted composition for treating breast cancer, which comprises the monoclonal antibody.
The composition also comprises azithromycin hydrochloride.
The composition also comprises PLGA nano-particles, and the PLGA nano-particles are combined with the monoclonal antibody.
The preparation method of the targeted composition for treating breast cancer comprises the following steps:
and (3) carrying out EDC/NHS treatment on the PLGA nano-particles, activating surface carboxyl, and combining with the amino group of the monoclonal antibody to obtain the nano-particles coupled with the monoclonal antibody. And adding doxorubicin hydrochloride into the PLGA oil phase, and uniformly mixing to prepare the drug-loaded particles (mAb-NP) of the coupled monoclonal antibody.
The invention has the beneficial effects that:
the invention provides a transmembrane Mam-A epitope, and a monoclonal antibody of a targeting cell membrane Mam-A is screened, and the monoclonal antibody can identify the epitope. The monoclonal antibody of the Mam-A based on the cell membrane localization has higher specificity and affinity than the existing commercial monoclonal antibody of the Mam-A based on the cell membrane localization. Therefore, the breast cancer immunodetection and targeted therapy based on the antibody have higher specificity. The monoclonal antibody of the invention has higher affinity and specificity than commercial antibodies, so the specificity of breast cancer detection and the targeting property and effectiveness of breast cancer treatment can be improved obviously.
Drawings
FIG. 1 shows the Mam-A targeting of monoclonal antibodies recognizing different epitopes.
FIG. 2 shows immunohistochemical detection of breast cancer tissue by using cell membrane Mam-A targeting monoclonal antibody mAb-6.
FIG. 3 shows immunofluorescence identification of mAb-6 labeled immunoliposome captured breast cancer cells.
FIG. 4 shows the targeting detection of mAb-6 modified PLGA drug-loaded particles.
Figure 5 is a statistical graph of the positive cell rate of Ki67 detection.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 preparation, screening and characterization of anti-Mam-A monoclonal antibodies
1. Mam-A antigen epitope analysis and clone expression
The Biosun biological software is used for analyzing the distribution situation of the epitope of Mam-A, and the amino acid sequence section with high peak value and good antigenic activity is screened, and the result shows that the epitope is mainly distributed in 4 sections, namely an A section (20-52aa), a B section (43-70aa), a C section (56-82aa) and a D section (69-93 aa). Deducing each segment gene according to the dominant codon of the escherichia coli, synthesizing full-length (1-93aa) and each segment antigen gene, and expressing by utilizing a prokaryotic expression system to obtain the corresponding antigen.
2. Preparation, purification and subtype identification of Mam-A monoclonal antibody
The whole-length Mam-A antigen plus an equal amount of Freund's complete adjuvant was injected into the back and the abdominal cavity of 50. mu.g/mouse, and 2 nd and 3 rd immunizations were performed at 4 th and 8 th weeks. Fusing myeloma cells and spleen cells according to a conventional hybridoma technology, screening positive clones by using an enzyme linked plate coated with a Mam-A full-length antigen by adopting an indirect ELISA method, and storing clone strains. Mouse Monoclonal IgG Purification was performed using the Mel Gel Monoclonal IgG Purification Kit (Thermo). The antibody subtype identification is carried out by adopting a mouse monoclonal antibody subtype identification kit of HBT company. A total of 14 anti-Mam-A monoclonal antibodies were obtained, and the results are shown in Table 1.
TABLE 1 identification and characterization of anti-Mam-A monoclonal antibodies
Figure BDA0001717418870000041
3. Identification of epitopes recognized by monoclonal antibodies
Diluting the four Mam-A antigen fragments and the Mam-A full-length antigen to 5ug/ml by using a coating solution, adding 100ul of antigen solution into each hole, incubating at 37 ℃ for 1h, standing overnight at 4 ℃, washing, and patting to dry. Adding 100ul of sealing solution into each well, sealing at room temperature for 1h, washing, and drying. Each monoclonal antibody was administered at a ratio of 1: the 1000 ratios were diluted with antibody dilutions. The coated enzyme-linked plate was taken, 100ul of diluted antibody was added to each well, incubated at 37 ℃ for 1h, washed and patted dry. HRP-labeled secondary goat anti-mouse antibody was labeled with 1: 1000 dilution, 100ul per well, incubation at 37 ℃ for 1h, washing and patting dry. 100ul of color developing solution is added into each hole, and the reaction is carried out for 15 minutes at room temperature in a dark place. 100ul of chromogenic stop solution is added into each hole, and the OD value is measured by an enzyme-labeling instrument at 450 nm.
The results are shown in table 1, and the 14-strain monoclonal antibodies can be classified into five types according to the different recognition epitopes: monoclonal antibody mAb-1, 2, 3, 4 only recognizes the A section, and the epitope is 20-52 aa; mAb-5 and mAb-6 recognize both the A and B segments, and the epitope is located in the A, B coincidence region, 43-52 aa; the monoclonal antibody mAb-7, 8, 9 only recognizes the B segment, and the epitope is 43-70 aa; mAb-10, 11, 12, 13 recognizes both the B and C segments, and the epitope is located in the B, C coincidence region, 56-70 aa; the monoclonal antibody mAb-14 only recognizes the C segment, and the epitope is 56-82 aa. The detection result also shows that the commercial mammaglobin monoclonal antibody mAb304(MA512638 of PIERCE) only recognizes the D segment, and the epitope is 69-93 aa.
4. Mam-A targeting of monoclonal antibodies recognizing different antigen epitopes
Selecting representative antibodies from the five monoclonal antibodies, namely mAb-1, mAb-6, mAb-7, mAb-10 and mAb-14, as primary antibodies to perform an immunofluorescence experiment, and detecting the distribution of Mam-A recognized by the antibodies in cells, wherein the specific operation is as follows:
and (3) recovering the breast cancer cell line ZR75-1 containing the GFP reporter gene marker, taking a 24-pore plate, and preparing a cell climbing sheet. After the cell density of each well reached 70-80%, the medium was removed, washed 2 times with PBS buffer, 400ul of 4% paraformaldehyde was added to each well, fixed at room temperature for 15 minutes, and washed 3 times with PBS for 5 minutes each. The closed solution was sealed at room temperature for 1 h. The 5 antibodies were diluted with antibody dilutions. 250ul of diluted antibody was added to each well and incubated for 2h at room temperature. PBS was washed 3 times for 5 minutes each. AF 594-labeled goat anti-mouse secondary antibody was diluted with antibody at a rate of 1: 100 dilutions were made in 250ul per well, incubated for 1h at room temperature, and washed 3 times with PBS for 5min each. DAPI was diluted with PBS at 1: 1000 dilutions were added to 250ul per well and incubated for 15min at room temperature. PBS was washed 3 times for 5 minutes each. Taking out the cover glass, sealing the cover glass with glycerol, and observing under laser confocal condition.
ZR75-1 cells were transfected with lentivirus and expressed Green Fluorescent Protein (GFP) in the cytoplasm to visualize staining. DiI staining (red) was used as a cell membrane positive staining control to determine membrane-targeting antibodies. The goat anti-mouse secondary antibody labeled with AF594 showed red fluorescence, and the nuclear position was determined by DAPI staining. By confocal laser observation, the results are shown in FIG. 1, and only mAb-6 is typically cell membrane stained compared with DiI staining; mAb304 is a commercial mAb targeting Mam-A on the cell membrane, but its cell membrane staining is not typical and the staining intensity is weaker than that of mAb-6. The corresponding epitope of mAb-6 is 43-52aa, i.e., the amino acid sequence is KELLQEFIDD.
5. BIAcore kinetic affinity analysis of interactions between antigens and antibodies
The BIAcore binding experiment was used to detect antibody-antigen interactions, and the kinetic data are shown in Table 2. ka represents the binding rate constant, the binding speed of the reaction macromolecule; kd represents the dissociation rate constant, reflecting the dissociation rate of molecular complexes; KD denotes the dissociation equilibrium constant (KD ═ KD/ka). The ka values show that the binding rate constant of mAb-6 to the full-length protein of Mam-A is significantly higher than that of mAb304, indicating that the mAb-6 reacts with the Mam-A antigen most rapidly within the same time span. Meanwhile, the dissociation equilibrium constant of the mAb-6 and the Mam-A full-length protein is the lowest, which indicates that the binding strength between the mAb-6 and the Mam-A antigen is the highest. From the above results, the affinity of mAb-6 for the Mam-A antigen fragment was significantly higher than that of the commercial antibody mAb 304.
TABLE 2 BIAcore kinetic affinity analysis of antigen-antibody interactions
Figure BDA0001717418870000061
By way of example 1, we obtained 14 monoclonal antibodies against human Mam-a and screened monoclonal antibody mAb-6 specifically binding to membrane-localized Mam-a, mAb-6 being typically cell membrane-colored, significantly better than commercial mAb304, and also having significantly higher affinity for the Mam-a antigen than commercial antibody mAb 304.
Example 2 immunohistochemical pathological detection of breast cancer tissue
After being dried, pathological paraffin sections of breast cancer patients are immersed in dimethylbenzene (I), dimethylbenzene (II), absolute alcohol, 95% alcohol and 70% alcohol in sequence. And after dewaxing, soaking the mixture in clean water for washing once for 5 min/time. Soaking and cleaning the slices in distilled water for 5 minutes, and putting the box with the citric acid working solution in a microwave oven on a high fire for 3 minutes. The slices are immersed in citric acid working solution for 13 minutes with medium and low fire. Taking out the box cover from the microwave oven, taking down the box cover, and placing the box cover in a ventilation position for cooling. Cutting machineThe tablets were washed 1 time 5 min/time with TBS. After TBS was spun off, 3% H was added dropwise2O2The solution was treated in a wet box for 15 minutes at room temperature. Sections were washed 3 times 3 min/time with TBS. The TBS solution was spun off and the blocking solution was added dropwise and blocked at 37 ℃ for 30 minutes. After the confining liquid is thrown off, the same amount of pre-confining antibody is dripped to cover the whole specimen tissue. Slides were placed in wet boxes and incubated overnight at 4 ℃. TBS 3 times, 3 min/time. And (4) dripping secondary antibody working solution after the slide is completely thrown and wiped dry, and putting the slide in a wet box for incubation at 37 ℃ for 20 minutes. TBS 3 times, 3 min/time. After being thrown and wiped completely, the HRP-labeled anti-mouse IgG polymer working solution is dripped into the wet box and incubated for 20 minutes at 37 ℃. TBS 3 times, 3 min/time. After the TBS is thrown off, the slide is dripped with DAB developing solution and placed in a wet box for 10 minutes at room temperature in a dark place. And (5) soaking and cleaning the mixture for 5 minutes by using clear water after the reaction is stopped. And (3) spin-drying the glass slide, immersing the glass slide into hematoxylin, dyeing for 3 minutes, and washing for 20 seconds by using clear water. The slides were immersed in 1% HCl for 3s and washed 1 time with water. The reaction solution was rewound with 1% aqueous ammonia for about 3 minutes, and washed with water 1 time. Dehydrating with 80%, 95%, 100% gradient alcohol for 2min, and treating with xylene for 7 min. And (5) sealing the neutral resin.
The result is shown in figure 2, the cell membrane Mam-A targeting monoclonal antibody mAb-6 can specifically identify breast cancer tissues and can be used for pathological detection of the breast cancer tissues.
EXAMPLE 3 immunofluorescence detection method for Breast cancer
And (3) recovering the GFP reporter gene labeled breast cancer cell line ZR75-1, taking a 24-well plate, and preparing a cell slide. After the cell density of each well reached 70-80%, the medium was removed, washed 2 times with PBS buffer, 400ul of 4% paraformaldehyde was added to each well, fixed at room temperature for 15 minutes, and washed 3 times with PBS for 5 minutes each. The closed solution was sealed at room temperature for 1 h. Antibody mAb-6 was diluted with antibody at 1: 100 dilution. 250ul of diluted antibody was added to each well and incubated for 2h at room temperature. PBS was washed 3 times for 5 minutes each. AF 594-labeled goat anti-mouse secondary antibody was diluted with antibody at a rate of 1: 100 dilutions were made in 250ul per well, incubated for 1h at room temperature, and washed 3 times with PBS for 5min each. DAPI was diluted with PBS at 1: 1000 dilutions were added to 250ul per well and incubated for 15min at room temperature. PBS was washed 3 times for 5 minutes each. Taking out the cover glass, sealing the cover glass with glycerol, and observing under laser confocal condition.
The result is shown in figure 1, the cell membrane Mam-A targeting monoclonal antibody mAb-6 can specifically identify breast cancer cells and can be used for immunofluorescence detection of the breast cancer cells.
Example 4 double antibody sandwich ELISA serum immunodetection method for breast cancer
The enzyme-linked plates were coated with mAb-6 antibody at 100. mu.l/well overnight at 4 ℃. Blocking with 3% BSA-PBS, 120. mu.l/well, 6 hours at room temperature, removing the blocking solution, and drying at room temperature for later use. Adding 50 mul of serum to be detected into each hole, adding 50 mul of biotin labeled antibody, fully mixing, incubating for 1 hour at 37 ℃, and washing the plate for 5 times. Add 100. mu.l of avidin-labeled horseradish peroxidase, incubate at 37 ℃ for 30min, wash the plate 5 times. A. Mu.l of each solution B was added, incubated at 37 ℃ for 10min, and 50. mu.l of the stop solution was stopped to measure the OD450nm value.
The result of double antibody sandwich detection on the serum of a breast cancer patient shows that 52 of 60 cases of breast cancer serum are positive, the sensitivity is 86.7 percent, and only 2 of 60 cases of normal control serum are positive, and the specificity is 96.7 percent.
Example 5 methods for Breast cancer circulating tumor cell Capture and identification
The breast cancer cell line ZR75-1 cell suspension or collected breast cancer patient anticoagulated blood is centrifuged at 1000 Xg for 10 min. The supernatant was carefully removed and placed in an EP tube, and an equal amount of PBS was added thereto and mixed well. Adding 30 μ L of cell membrane Mam-A targeting monoclonal antibody mAb-6 labeled immune lipid magnetic ball, incubating at room temperature for 30min, and mixing 1 time every 10 min. The EP tube was inserted into a magnetic separation rack and adsorbed for 5min, the supernatant was aspirated and washed 3 times with PBS. Adding 30 μ L of DAPI staining solution and 10 μ L of Mam-A-FITC (mAb-13) staining solution, mixing, staining in dark for 15min, and washing with PBS for 3 times. Adding 30 mu L of deionized water into an EP tube for resuspension, uniformly coating the mixture on an anti-falling glass slide, and observing the mixture under a fluorescence microscope after the liquid drops to be dry.
The result is shown in figure 3, the cell membrane Mam-A targeting monoclonal antibody mAb-6 labeled immune lipid magnetic beads can capture breast cancer tumor cells. mAb-6 is a labeled antibody and mAb-13 is an identifying antibody.
Example 6 Targeted therapeutic Effect of mAb-6 modified PLGA drug-loaded particles on Breast cancer
1. Targeting of mAb-6 modified PLGA (polylactic-co-glycolic acid) drug-loaded particles to breast cancer cells
200 μ l (100mg/mL) PLGA nanoparticles were centrifuged, resuspended in EDC/NHS activation buffer and incubated with shaking at room temperature for 1 h. After centrifugation, the cells were resuspended in 200. mu.l of PBS buffer, 10. mu.l (1mg/mL) of monoclonal antibody mAb-6 was added, and incubated overnight at 4 ℃. Washing with PBS buffer solution for 2 times, resuspending with 200 μ l of blocking solution, blocking at room temperature for 1h, and storing at 4 deg.C to obtain coupled monoclonal antibody nanoparticles. 0.01g of chemotherapeutic drug doxorubicin hydrochloride (Dox) is weighed and added into PLGA oil phase to be mixed evenly, and the drug-carrying particles (mAb-NP-Dox) of the coupling monoclonal antibody are prepared.
ZR75-1 cells are transmitted into a 24-pore plate for normal culture, when the cell density reaches about 60%, single Dox, PLGA drug-loaded particles (NP-Dox) and mAb-6 modified PLGA drug-loaded particles (mAb-NP-Dox) are respectively added, incubation is carried out for 1h at 37 ℃, cell nuclei are stained, and observation is carried out under a fluorescence microscope. The drug-loaded particles are red fluorescence, and the cell nucleus is blue fluorescence.
The result is shown in fig. 4, the breast cancer cells cannot be targeted by adding Dox alone, a small amount of Dox can be seen to adhere to the cells by PLGA drug-loaded particles (NP-Dox), and a large amount of Dox can be seen to adhere to the cells by mAb-6 modified PLGA drug-loaded particles (mAb-NP-Dox), which indicates that the mAb-6 antibody modified PLGA drug-loaded particles have good targeting property and can deliver the therapeutic drug to the breast cancer cells in a targeted manner.
2. Targeted inhibition effect of mAb-6 modified PLGA drug-loaded particles on breast cancer cell proliferation
PLGA (poly (lactic-co-glycolic acid)) drug-loaded particles (NP-Dox) carrying chemotherapeutic drug Dox and mAb-6 modified PLGA drug-loaded particles (mAb-NP-Dox) carrying chemotherapeutic drug Dox are respectively incubated with ZR75-1 breast cancer cells for 12h, then normal culture medium is replaced, and detection is carried out after 48 h. Ki67 immunohistochemistry was used to detect cell proliferation, and the statistics of positive cell rate are shown in FIG. 5. The graph shows that the positive cell rate of the mAb-6 modified PLGA drug-loaded particle group (mAb-NP-Dox) is significantly lower than that of the PLGA drug-loaded particle group (NP-Dox) which is not modified by the monoclonal antibody, which indicates that the mAb-6 modified PLGA drug-loaded particle has a more significant targeted inhibition effect on breast cancer cell proliferation.
The above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Sequence listing
<110> military medical research institute of military science institute of people's liberation force of China
<120> mammaglobin A epitope and application thereof
<130> 1
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 10
<212> PRT
<213> Artificial Sequence
<220>

Claims (5)

1. An epitope peptide of mammaglobin A, the amino acid sequence of which is shown in a sequence table SEQ ID NO. 1.
2. Use of the mammaglobin A epitope peptide of claim 1 for the preparation of an antibody to mammaglobin A.
3. The use of claim 2, wherein the antibody is a monoclonal antibody.
4. Use of the epitope peptide according to claim 1 for the preparation of a reagent for detecting breast cancer.
5. The use of claim 4, wherein the reagent is an immunohistochemical detection reagent, an immunofluorescence detection reagent, an ELISA detection reagent, a breast cancer circulating tumor cell capture reagent.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998007753A1 (en) * 1996-08-19 1998-02-26 Abbott Laboratories Mammaglobin, antibodies thereto, and their use for detecting diseases of the breast
WO2000073338A1 (en) * 1999-05-28 2000-12-07 Corixa Corporation Compositions and methods for the therapy, diagnosis and monitoring of breast cancer
CN1766634A (en) * 2005-06-13 2006-05-03 上海晶泰生物技术有限公司 Method for detecting mammaglobin and kit therefor
CN101974088A (en) * 2010-11-03 2011-02-16 中国人民解放军军事医学科学院基础医学研究所 Breast globin monoclonal antibody and application thereof in breast cancer diagnosis

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030045468A1 (en) * 1999-05-28 2003-03-06 Corixa Corporation Compositions and methods for the therapy, diagnosis and monitoring of breast cancer

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998007753A1 (en) * 1996-08-19 1998-02-26 Abbott Laboratories Mammaglobin, antibodies thereto, and their use for detecting diseases of the breast
WO2000073338A1 (en) * 1999-05-28 2000-12-07 Corixa Corporation Compositions and methods for the therapy, diagnosis and monitoring of breast cancer
CN1766634A (en) * 2005-06-13 2006-05-03 上海晶泰生物技术有限公司 Method for detecting mammaglobin and kit therefor
CN101974088A (en) * 2010-11-03 2011-02-16 中国人民解放军军事医学科学院基础医学研究所 Breast globin monoclonal antibody and application thereof in breast cancer diagnosis

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Mammaglobin-Based Strategies for Treatment of Breast Cancer;Peter S. Goedegebuure等;《Current Cancer Drug Targets》;20041231;第531-542页 *
乳腺珠蛋白在乳腺癌中的研究进展;胡园园 等;《现代肿瘤医学》;20170930;第25卷(第17期);第2836-2839页 *

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