CN1118568C - Anti P185/c-erbB-2 monoclonal anti-body hybridoma, prepn. method therefor, use as tumor detection - Google Patents

Abstract

The present invention relates to a monoclonal antibody hybridoma of antioncogene neu/c-erbB-2 product P185<neu/c-erbB-2>, a preparation method thereof, and an application in tumor detection. The present invention is characterized in that a cell surface epitope embedding method is used for immunization, namely that mice are immunized by using neu/c-erbB-2 transgenic NIH/3T3 cell T6-17 embedded within anti-NIH/3T3 antiserum of mice; the subclasses of single antibodies secreted by the obtained anti P185<neu/c-erbB-2> monoclonal antibody hybridoma are IgG1, and the single antibodies can specially combine with the outer membrane regions of the P185<neu/c-erbB-2> on cancer cell membranes, and have no achiasmate reaction with growth factor receptors and have no reaction with benign cell tissue; the obtained antibodies has strong conjugation specificity, and the positive staining on human breast cancer pathological section is membrane coloring and has low background, so the obtained antibodies can develop into a diagnostic reagent kit and be used for detecting the P185<neu/c-erbB-2> expression level of tumor tissue.

Description

Anti-P185neu/c-erbB-2 monoclonal antibody hybridoma, its preparation method and tumor detection application

The invention relates to the technical field of monoclonal antibody hybridoma in biology, its immunological preparation method, and tumor detection method.

The product of the Neu/c-erbB-2 oncogene, P185 ^{neu/c-crbB-2,} is a receptor tyrosine kinase. The American "Science" magazine (Science, 1989.244: 707-712) reported that using a specific antibody against p185 ^neu/c-erbB-2 , 688 cases of breast cancer patients and 120 cases of ovarian cancer were detected by immunohistochemical method Expression levels of p185 ^neu/c-erbB-2 in ovarian tissues of patients. Observe and compare the antibody-specific staining intensity of the cells in the tissue under a microscope, and divide the expression level in the tissue into four grades: ① negative to very weak; ② 1+; ③ 2+; Negative, the rest are different degrees of overexpression, that is, overexpression or high expression, and are judged as positive. According to this standard, over-expression of P185 ^neu/c-erbB-2 occurs in more than 30% of the tumor tissue cells of breast and ovarian cancer patients, and the tumors of such patients are highly malignant and the prognosis is poor. The US magazine Gene (Gene, 1995.159: 19-27) reviewed the results of a large number of clinical studies and pointed out that when the cell membrane P185 ^neu/c-erbB-2 is highly expressed, that is, overexoression, it indicates that the tumor has a high degree of malignancy and is prone to occur after surgery. Metastasis and recurrence. Therefore, it is of great significance to prepare monoclonal antibodies for clinical diagnosis of the expression level of P185 ^neu/c-erbB-2 in tumor tissues.

The existing method for preparing monoclonal antibody hybridoma mainly includes four steps of immunization, cell fusion, screening and cloning. After searching, it is known that the existing immune methods used in the preparation of anti-P185 ^neu/c-erbB-2 monoclonal antibody hybridoma process mainly contain the following three: A method, such as the American magazine "Oncogene" (oncogene, 1989, 4 : 543-548) reported subtractive immunization. However, this immunization method not only has many steps, but also reduces the immunity of animals due to the use of the immunosuppressant cyclophosphamide, making the animals susceptible to infection and death. For method B, please refer to the American magazine "Cancer Research" (Cancer Research, 1990, 50: 1550-1558). The immunization method involved in the article is to first use the mouse fibroblast NIH/ Mice were immunized four times with 3T3, then immunized twice with P185 ^neu/c-erbB-2 membrane extract purified by wheat germ agglutinin, and immunized with purified P185 ^neu/c-erbB-2 protein for the last time. This immunization method has cumbersome steps, the immunization time is nearly four months, and the membrane protein needs to be roughly extracted, which makes the operation complicated; the wheat germ agglutinin affinity chromatography column used and pure P185 ^neu/c-erbB-2 protein, etc. Reagents are expensive. Method C, such as the immunization method involved in the U.S. "Journal of Biological Chemistry" (The Journal of Biological Chemistry, 1992, 267: 15160-15167), is immunized with the human breast cancer cell line SKBR 3 that highly expresses P185 ^neu/c-erbB-2 , because the SKBR 3 cell membrane contains a variety of growth factor receptor proteins belonging to the same family as P185 ^neu/c-erbB-2 , this immunization method is prone to produce antibodies that cross-react with other growth factor receptor proteins, greatly Adding to the difficulty of screening anti-P185 ^neu/c-erbB-2 specific antibodies.

The object of the present invention is to provide an anti-P185 ^neu/c-erbB-2 monoclonal that can specifically target P185 ^neu/c-erbB -2 and can be used for clinical diagnosis of P185 ^neu/c-erbB -2 expression level in tumor tissue Antibody hybridoma and its preparation method, and the application of the antibody secreted by the anti-P185 ^neu/c-erbB-2 monoclonal antibody hybridoma in tumor detection.

The anti-P185 ^neu/c-erbB-2 monoclonal antibody hybridoma of the present invention is characterized in that it includes three hybridomas respectively named A18, A21 and A22, and the antibodies A18, A21 and A22 secreted by them respectively, and their subtypes are all It is IgG1; it can specifically bind to P185 ^neu/c-erbB-2 on the cancer cell membrane, but has no cross-reaction with growth factor receptors, and does not bind to benign tissue cells, so it can be used to detect tumor tissue The expression level of P185 ^neu/c-erbB-2 ; wherein A21 and A22 can be compared with the DPLNNTTPVTG of the two polypeptides 99-109 from P185 ^neu/c-erbB-2 and the DLDDKGCPAEQR of 614-625 and the bovine serum albumin BSA The coupling product has specific binding.

The preparation method of the anti-P185 ^neu/c-erbB-2 monoclonal antibody hybridoma of the present invention includes four steps of immunization, cell fusion, screening and cloning, and is characterized in that the immunization adopts the cell surface epitope embedding method, namely: First, use the antiserum against NIH/3T3 cells that have not been transfected with the neu/c-erbB-2 oncogene to embed the P185 ^neu/c- Immunize mice with other antigens other than ^erbB-2 ; detect the antiserum titer of the immunized mice, select the mouse with the highest antiserum titer after the last immunization, and take spleen cells and myeloma cells SP2/0 for cell fusion ; After positive clones were screened by enzyme-linked immunoassay, cloned by limiting dilution method, continued to screen, repeated no less than 2 times, and obtained hybridomas capable of secreting anti-P185 ^neu/c-ereB-2 monoclonal antibodies.

The use of the anti-P185 ^neu/c-erbB-2 monoclonal antibody hybridoma of the present invention in tumor detection is characterized in that the antibody A18 secreted by the anti-P185 ^neu/c-erbB-2 monoclonal antibody hybridoma A18, A21 and A22 is used , A21 and A22 were used to detect the expression level of P185 ^neu/c-erbB-2 in tumor tissue. When the expression level of P185 ^neu/c-erbB-2 in the cell membrane was high, it indicated that the tumor was highly malignant and prone to metastasis and recurrence after operation.

Compared with the prior art, the present invention has the following advantages and specificity:

The monoclonal antibodies A18, A21 and A22 secreted by the anti-P185 ^neu/c-erbB-2 hybridoma of the present invention were adopted indirect immunoperoxidase staining method, immunofluorescence method, flow cytometry (FACS) method and immune The results showed that these antibodies could specifically bind to P185 ^neu/c-erbB-2 ; among them, it was detected that monoclonal antibodies A21 and A22 could bind to two polypeptides 99-109 from P185 ^neu/c-erbB-2 DPLNNTTPVTG and 614-625 DLDDKGCPAEQR specifically bind to the coupling product of bovine serum albumin (note: the binding specificity of A18 has not been determined yet). Immunohistochemical staining was performed on breast cancer tissue and benign breast tumor tissue using the monoclonal antibodies A18, A21 and A22 secreted by the three hybridomas of the present invention, and the test results showed that the positive staining of the three antibodies had good membrane localization, And it is closely related to tumor grade. The positive rate of P185neu ^/c-erbB-2 is on the rise in breast cancer with poorly differentiated tumor tissue and more lymph node or hematologic metastasis. Accordingly, these monoclonal antibodies can be developed into diagnostic kits for detecting the expression level of P185 ^neu/c-erbB-2 in tumor tissues, which has the significance of clinical diagnosis and guiding the selection of treatment options.

The preparation method of the anti-P185 ^neu/c-erbB-2 monoclonal antibody hybridoma of the present invention has the following advantages compared with the prior art due to the adoption of the immunization method of the cell surface region epitope embedding method: In other words, this method has the advantages of simple operation, fast and easy operation; compared with method B, this method has the advantages of simple and easy to obtain reagents and low price; compared with method C, this method can obtain secreted The difficulty of the target antibody hybridoma is greatly reduced; because the method used in the present invention only needs to be immunized with two kinds of cells successively without further purification of membrane protein, so the operation process is simplified and the preparation cost is relatively cheap; and because it does not need to use immunosuppressant Cyclophosphamide, so that it is not easy to kill the immunized animals; and after using anti-NIH/3T3 antiserum combined with T6-17 after the P185 ^neu/c-erbB-2 gene transfer, the specificity of immunity can be greatly improved and the screening target hybridization can be reduced. tumor difficulty.

The antibodies A18, A21 and A22 secreted by the anti-P185 ^neu/c-erbB-2 monoclonal antibody hybridomas A18, A21 and A22 obtained by the method of the present invention are used to detect the expression of P185 ^neu/c-erbB-2 on tumor tissue The expression level, the specificity of the monoclonal antibody is strong, the positive staining is membrane staining, and the background is low, it is suitable for immunohistochemical detection of clinical pathological sections, and may also be developed to the detection of patient serum.

The embodiment of the present invention is given as an example as follows:

1. Preparation of anti-P185 ^neu/c-erbB-2 monoclonal antibody hybridoma:

The preparation process mainly includes four steps of immunization, cell fusion, screening and cloning.

The specific operation can be divided into the following five steps:

1. Immunization with cell surface epitope embedding method:

Take eight 6-8-week-old BaLb/C mice, collect mouse fibroblasts NIH/3T3, wash, suspend in buffer for immunization, the dose is 0.5×10 ⁶ to 5×10 ⁷ /time / mouse, the number of immunizations should be three or four times; the mouse antiserum is taken, which is the mouse anti-NIH/3T3 antiserum; the NIH/3T3 cell T6-17 transgenic neu gene is collected and used 1:20 to 1:100 diluted mouse anti-NIH/3T3 antiserum was suspended; after incubation overnight, cells were collected and resuspended in buffer;

Another eight BaLb/C mice of 6-8 weeks old were injected with T6-17 cells incubated with NIH/3T3 antiserum, and the number of immunizations was not less than twice, with a dose of 0.5×10 ⁶ to 5×10 ⁷ / times/mouse;

2. Measure the titer of mouse antiserum by enzyme-linked immunoassay (ELISA):

Collect several flasks of overgrown T6-17 cells, suspend them with hypotonic buffer (10mM Tris, 10mMKCl, 5mM EDTA, pH8.0), incubate at 4°C for half an hour, sonicate, centrifuge at 18,000rpm, take the supernatant, and measure its total protein Content, diluted to 200ug/ml, is the antigen; add 50ul/hole to the microtiter plate, and coat overnight at 4°C; fill the well with blocking buffer (containing 0.25% bovine serum albumin (BSA) and 0.05% Tween (Tween) in phosphate buffer solution (PBS)), placed at room temperature for more than 2 hours; use the same treated NIH/3T3 cells as a negative control; discard the blocking buffer, wash with distilled water, add each 50ul/well of mouse antiserum was used as the primary antibody, incubated at 37°C for 1.5 hours; after washing with distilled water for 3 times, sheep anti-mouse IgG-horseradish peroxidase (HRP) was added as the enzyme-labeled secondary antibody, 37 Incubate at ℃ for 1 hour; wash with distilled water for 3 times, add OPD to develop color, measure absorbance (OD) value at 492nm; if T6-17 is used as antigen, OD ₄₉₂ > 1.0, and when NIH/3T3 is used as antigen, OD ₄₉₂ < 0.2 The mice with the highest titer of antiserum (>1:10 ⁴ ) were selected, and the same treated T6-17 cells were used for final immunization.

3. Carry out cell fusion according to conventional methods:

Myeloma cells SP2/0 were revived about one week before fusion, and fresh medium was changed one day before fusion. On the day of fusion, the spleen cells of the mice after the last immunization were taken, washed with D-hanks buffer, and SP2/0 were collected, mixed so that the ratio of spleen cells:SP2/0 was 5:1, and washed again. Add 1ml of sterilized 50% polyethylene glycol 4000 (PEG4000, dissolved in serum-free RPMI-1640 medium) dropwise to the cells within 1 minute, and after standing for 10 minutes, suspend the cells in HAT selection The culture medium is divided into 96-well cell culture plates and cultured for about two weeks until the hybridoma cell clones are visible to the naked eye.

4. Screen positive clones by ELISA:

According to the above-mentioned ELISA method, one of the primary antibodies was changed to hybridoma supernatant, and positive hybridoma cells secreting anti-P185 ^neu/c-erbB-2 antibodies were screened out.

5. Carry out monoclonalization according to the conventional limiting dilution method:

Dilute the hybridoma cells in the positive wells at about 1:10000 and add them to the cell culture plate to keep the cloning rate at 1/3 to 1/2; after the clones grow up, use the ELISA method to determine the clones after dilution. The positive rate was selected, and the clone with the strongest reaction was selected to continue to dilute, and repeated several times until the positive rate reached 100%. The hybridoma secreting the monoclonal antibody against P185 ^neu/c-erbB-2 is the target product to be obtained in this experiment.

In this experiment, three hybridomas secreting monoclonal antibodies against P185 ^neu/c-erbB-2 were obtained. According to the clone numbers, they were named A18, A21, and A22, and the antibodies they secreted were also named A18, A18, and A22 respectively. A21 and A22.

2. Characterization of monoclonal antibodies A18, A21 and A22

1. The above three strains of hybridomas A18, A21 and A22 obtained by screening positive clones by ELISA method were respectively amplified and implanted into the peritoneal cavity of 8-week-old BaLb/C mice. After ascites developed, the ascites was taken out, which was crude produced monoclonal antibodies.

2. Determination of the obtained antibody subclasses by ELISA detection kit:

Add 50ul of 20ug/ml rabbit anti-mouse IgG subclass antibodies (such as rabbit anti-mouse IgG1 antibody, rabbit anti-mouse IgG2a antibody, rabbit anti-mouse IgG2b antibody) to each well of the microtiter plate, and place in a humid box at room temperature. Incubate for 2 hours or overnight; remove the supernatant, fill the wells with 3% BSA/PBS, block at room temperature for 2 hours or overnight; wash three times with PBS, add 50ul of various hybridoma supernatants and ascites, and warm at room temperature in a wet box Incubate for 2 hours, discard the supernatant, wash three times with PBS, add goat anti-rabbit IgG-horseradish peroxidase, incubate at room temperature for 2 hours, add OPD substrate solution to develop color, measure OD value at 492nm. Results The subclasses of antibodies A18, A21 and A22 were all IgG1.

3. The specific binding of the obtained monoclonal antibodies A18, A21 and A22 to P185 ^neu/c-erbB-2 on the cell surface was detected by indirect immunoperoxidase staining method:

NIH/3T3, T6-17, SKBR3, MCF7 (human breast cancer cells with low expression of P185 ^neu/c-erbB-2 ), NE91 (NR-6 cells with transgrowth factor receptor EGFR gene, on the cell membrane Highly expressing EGFR) cells were transferred to the coverslips in small culture dishes, and continued to culture for 2-3 days; the coverslips were taken out, washed twice with PBS, and fixed with ice-cold 3% formalin for 10 minutes; Add 50ul of ascites A18, A21 and A22 diluted with blocking buffer to each slide, place in a humid chamber at room temperature for 1-1.5 hours; wash with PBS 3 times, 5 minutes each time; add 50ul of goat anti-mouse IgG-HRP dropwise (1:1000), incubate in a wet box for 1 hour; wash three times with PBS, 5 minutes/time; add DAB chromogenic solution dropwise, and incubate at room temperature for about 10 minutes; observe under the microscope, it is found that for these three kinds of ascites, T6-17 and SKBR3 had a strong membrane positive reaction, while NIH/3T3, MCF7 and NE91 had no reaction, indicating that the antibodies A18, A21 and A22 secreted by hybridomas A18, A21 and A22 were all related to P185 ^neu/c expressed on the cell membrane ^-erbB-2 had specific binding to the extramembrane region; since these monoclonal antibodies did not bind to NE91 cells with high expression of EGFR on the cell membrane, it indicated that they had no cross-reaction with EGFR.

4. Use immunoprecipitation to detect the specific binding of the obtained monoclonal antibody to P185 ^neu/c-erbB-2 protein:

First, prepare the reagents:

( ₁ ) 4mM _Na3VO4 ;

(2) 50mM NaF;

(3) Cell washing buffer: PBS containing 0.4mM Na ₃ VO ₄ and 5mM NaF;

(4) Cell lysis buffer: containing 1% NP40, 1% sodium deoxycholate, 0.1% sodium dodecylsulfonate, 0.15M NaCl, 0.01M sodium hydrogen phosphate (pH7.4), 1% Protease inhibitor Tracyrol (Aprotinin 200U/ml), 1mM PMSF, 2mM EDTA, 10mM NaF, 10mM sodium pyrophosphate, 0.4mM Na ₃ VO ₄ and 10mM C ₂ H ₄ INO, finally adjust the pH to 8.0;

(5) Gel washing solution: PBS with pH 7.4 contains 0.5M NaCl, 0.4mM Na ₃ VO ₄ , 0.1% NP40, 10mM NaF and 10mM sodium pyrophosphate.

The specific operation steps are as follows:

After the SKBR3 cells grow to confluence on a 15cm culture dish, wash twice with cell washing buffer on ice, add cell lysis buffer at 1.5mL/dish, incubate at 4°C for 30 minutes, scrape the cells into a 1.5mL centrifuge In the tube, centrifuge at 14,000 rpm for 15 minutes at 4°C, take the supernatant, measure the protein concentration, and adjust it to 1-3 mg/mL for immunoprecipitation; add 2ul ascites A18, A21 and A22 each, mix well, and store on ice Place on the top for 1-2 hours, add 40uL gel Sepharose with protein A, shake at 4°C for 1 hour, then centrifuge at 7500 rpm for 3 minutes at low speed; wash the gel three times with gel washing solution; discard the supernatant , add 20ul SDS-polyacrylamide electrophoresis sample buffer, boil for 3 minutes, cool on ice, centrifuge and shake off the beads, perform SDS-polyacrylamide electrophoresis on the supernatant, the concentration gel is 5% gel concentration, and the separation gel is gel Concentration 7.5%, electrotransfer to nitrocellulose membrane; block membrane with blocking buffer; add rabbit anti-P185 polyclonal antibody diluted 1:5000, incubate at 37°C for 1.5 hours; wash, add goat anti-rabbit-HRP, 37 Incubate at ℃ for 1 hour; wash, develop color with fluorescent substrate, and expose to X-ray film for 30 seconds; the results show that the protein bound by these three monoclonal antibodies is P185 ^neu/c-erbB-2 protein with a molecular weight of 185kd.

5. Use the FACS method to detect the specific binding of the obtained monoclonal antibody to P185 ^neu/c-erbB-2 on the cell membrane:

Prepare FACS buffer: PBS containing 0.5% BSA and 0.05% sodium azide;

SKBR3 cells and NIH/3T3 cells were collected separately, each sample was 10 ⁵ cells, washed with FACS buffer; 2ul ascites and 4ul FACS buffer were added to each sample, and ice bathed for 30 minutes; washed with FACS buffer; Add goat anti-mouse IgG-fluorescein (FITC) 0.5ul and 40ul FACS buffer to each sample, and ice-bath for 30 minutes; after washing with FACS buffer, use flow cytometry to detect its fluorescence intensity, in which SKBR3 cells have a strong Fluorescence, while the fluorescence of NIH/3T3 cells is very weak. At the same time, it shows that these three kinds of ascites all specifically bind to P185 ^neu/c-erbB-2 on the SKBR3 cell membrane, but not to those that do not express P185 ^{neu/c-erbB- 2} NIH/3T3 cells do not bind.

6. The binding properties of monoclonal antibodies A21, A22 and peptides derived from P185 ^neu/c-erbB-2 molecules:

Two peptides from P185 ^neu/c-erbB-2 , DPLNNTTPVTG at 99-109 and DLDDKGCPAEQR at 614-625, were coupled to bovine serum albumin with glutaraldehyde and coated on a microtiter plate as an antigen , ascitic fluid A21 and A22 were positively reacted to them by ELISA method, thus indicating that A21 and A22 can specifically bind to these two polypeptides.

3. Determination of the optimal working concentration of monoclonal antibodies A18, A21 and A22 for the detection of P185 ^neu/c-erbB-2 expression level in human breast cancer tissue by immunohistochemical method:

Use the ascites of the above-mentioned A18, A21 and A22 monoclonal antibodies at a titer of 1:50, 1:100, 1:200, 1:400, and 1:800 for the paraffin pathological sections of known P185-positive human breast cancer tissue. The streptavidin peroxidase (SP) method was used for immunohistochemical staining, and the obvious cell membrane staining was used as the positive standard of P185 to test the optimal working concentration. Namely: deparaffinize the paraffin sections to water, wash with PBS; add dropwise methanol containing 3% H ₂ O ₂ , incubate at room temperature for 10 minutes; wash with PBS, add 10% non-immune animal serum dropwise, and incubate at room temperature For 10 minutes, absorb excess liquid; dilute ascites A18, A22 at 1:800, and A21 at 1:600, drop onto the slices, incubate at 37°C for 60 minutes or overnight at 4°C; wash 3 times with PBS, 5 minutes /time; add biotin-labeled goat anti-mouse secondary antibody and incubate at room temperature for 10 minutes; wash 3 times with PBS, 5 minutes/time; drop streptavidin-HRP solution, incubate at room temperature for 10 minutes; wash with PBS, add Freshly prepared DAB solution, developed color for 3-10 minutes, rinsed with tap water; counterstained with hematoxylin, sealed with neutral gum, and observed the staining results of ^pathological sections under a microscope. If the background is weak, the corresponding maximum dilution is the best working concentration: 1:800 for A18 and A22, and 1:600 for A21.

4. Clinical tumor detection test

Immunohistochemical staining of 40 human breast cancer pathological tissue sections and 12 benign breast tissue sections in the Affiliated Hospital of Anhui Medical University using the monoclonal antibody secreted by the obtained anti-P185 ^neu/c-erbB-2 monoclonal antibody hybridoma by the above method , the results showed that positive staining had good membrane localization and was strongly correlated with tumor grade. Taking A18 as an example, the P185 ^neu/c-erbB-2 staining of the benign mammary duct lobules were all negative, and 33 cases had lymph node or blood metastasis in breast cancer tissue; the positive rate of P185 ^neu/c-erbB-2 was in order of 57% (4/7) of negative lymph nodes, 62% (8/15) of lymph node metastasis < 4 pieces; 69% (9/13) of lymph node metastasis > 4 pieces and hematologic metastasis. The positive rate of P185 ^neu/c-erbB-2 was 50% (6/12) in the histological grade I, and 64% (18/28) in the histological grade II-III, showing that the tumor tissue was poorly differentiated, with relatively The positive rate of P185neu ^/c-erbB-2 in breast cancer with multiple lymph nodes or hematologic metastases is on the rise. The results of clinical experiments show that P185 ^neu/c-erbB-2 can be used as one of the markers of breast cancer diagnosis, degree of differentiation and biological behavior, and at the same time show that the anti-P185 ^neu/c-erbB-2 monoclonal antibody hybridoma of the present invention Secreted monoclonal antibodies A18, A21 and A22 have the effect of actually detecting P185 ^neu/c-erbB-2 in tumor tissue; these monoclonal antibodies can be developed into a diagnostic kit for detecting P185 ^neu/c-erbB-2 in tumor tissue level of expression.

Claims (3)

1, a kind of anti-P185 ^Neu/c-erbB-2Monoclonal antibody hybridoma is characterized in that comprising the three strains hybridoma of called after A18, A21 and A22 respectively; They are excretory antibody A 18, A21 and A22 respectively, and its subclass all is IgG1; Can both specificity ground in conjunction with the P185 on the cancer cell membrane ^Neu/c-erbB-2, and with the growth factor receptors no cross reaction, with benign tissue cell also debond; Wherein A21 and A22 can be respectively with from P185 ^Neu/c-erbB-2The DPLNNTTPVTG of two sections polypeptide 99-109 have specificity to combine with the DLDDKGCPAEQR of 614-625 with the coupled product of bovine serum albumin.

2, a kind of anti-P185 ^Neu/c-erbB-2The preparation method of monoclonal antibody hybridoma, comprise immunity, cytogamy, screening and four steps of clone, it is characterized in that described immunity employing cell surface epi-position entrapping method, that is: use the anti-antiserum(antisera) embedding of not changeing the NIH/3T3 cell of neu/c-erbB-2 oncogene to change on the NIH/3T3 cell T6-17 surface of neu/c-erbB-2 oncogene earlier except that P185 ^Neu/c-erbB-2Immune mouse again behind other antigen in addition; Detect the antiserum(antisera) titre of immune mouse, select the highest mouse last immunity of antiserum(antisera) titre after, get spleen cell and myeloma cell SP2/0, carry out cytogamy; After pressing the enzyme-linked immunosorbent assay screening positive clone again, clone, continue screening, repeat to be no less than 2 times, obtain to secrete anti-P185 with limiting dilution assay ^Neu/c-crbB-2The hybridoma of monoclonal antibody.

3, anti-P185 ^Neu/c-erbB-2The purposes of monoclonal antibody hybridoma on lesion detection is characterized in that using anti-P185 ^Neu/c-erbB-2Monoclonal antibody hybridoma A18, A21 and A22 excretory antibody A 18, A21 and A22 detect P185 in the tumor tissues ^Neu/c-erbB-2Expression level, P185 on cancer cell membrane ^Neu/c-crbB-2During high expression level, the grade malignancy height of this tumour is described, postoperative easily takes place to shift and recurrence.