CN1242425A - Anti P185/c-erbB-2 monoclonal anti-body hybridoma, prepn. method therefor, use as tumor detection - Google Patents

Abstract

The anti-cancer gene neu/c-erbB-2 product P185 ^neu/c-erbB-2 monoclonal antibody hybridoma of the present invention and its preparation method and application in tumor detection are characterized in that the cell surface epitope embedding method is used for immunization , immunize mice with neu/c-erbB-2 transgenic NIH/3T3 cells T6-17 embedded in mouse anti-NIH/3T3 antiserum; the obtained anti-P185 ^neu/c-erbB-2 monoclonal antibody hybridoma , the subclass of the secreted monoclonal antibody is IgG1, which can specifically bind to the extramembrane region of P185 ^neu/c-erbB-2 on the cancer cell membrane, but has no cross-reaction with growth factor receptors, and is compatible with benign tissues. The cells also had no reaction; the monoclonal antibody obtained had strong binding specificity, and the positive staining of human breast cancer pathological sections was membrane staining with low background, and could be developed into a diagnostic kit for clinical detection of P185 ^neu/c-erbB-2 in tumor tissues The expression level.

Description

Anti-P185 Neu/c-erbB-2Monoclonal antibody hybridoma its preparation method and lesion detection purposes

The present invention relates to monoclonal antibody hybridoma and immunology preparation method and lesion detection approach technical field in the biology.

The product P 185 of Neu/c-erbB-2 oncogene ^Neu/c-erbB-2It is a receptor tyrosine kinase.(Science, 1989.244:707-712) report have the mammary cancer more than 30% and the cell of ovarian cancer patient tumor tissues P185 to occur to U.S.'s " science " magazine approximately ^Neu/c-erbB-2Overexpression, such patient's malignancy of tumor degree height, poor prognosis.(Gene 1995.159:19-27) points out behind a large amount of clinical study results of summary, as cytolemma P185 U.S.'s " gene " magazine ^Neu/c-erbB-2During high expression level, the grade malignancy height of this tumour is described, postoperative easily takes place to shift and recurrence.Therefore prepare P185 in the clinical diagnosis tumor tissues ^Neu/c-erbB-2The monoclonal antibody tool of expression level has very important significance.

The existing method for preparing monoclonal antibody hybridoma mainly comprises immunity, cytogamy, screening and four steps of clone.Learn the anti-P185 of existing preparation by retrieval ^Neu/c-erbB-2Used immunization method mainly contains following three kinds in the monoclonal antibody hybridoma process: the A method, and as USA Magazine " oncogene " (oncogene, 1989,4:543-548) Bao Dao subtraction immunization.But this immunization method not only operation steps is many, and owing to used the immunosuppressor endoxan, has reduced the immunological competence of animal, and the animal easy infection is caused death.The B method, can referring to USA Magazine " tumor research " (Cancer Research, 1990,50:1550-1558), the l cell NIH/3T3 immune mouse that the immunization method that relates in the literary composition changes the Neu/c-erbB-2 oncogene for elder generation uses four times is used the P185 through the wheat germ agglutinin purifying again ^Neu/c-erbB-2The P185 of purifying is used in film extract immunity twice for the last time ^Neu/c-erbB-2Albumen carries out immunity.This immunization method complex steps, immune for up to nearly four months, and to slightly carry membranin, complicated operation; Wheat germ agglutinin affinity column that uses and pure P185 ^Neu/c-erbB-2Reagent such as albumen cost an arm and a leg.The C method, as U.S.'s " journal of biological chemistry " (The Journal ofBiological Chemistry, 1992, the immunization method that 267:15160-15167) relates to is used high expression level P185 ^Neu/c-erbB-2Human breast cancer cell strain SKBR3 carry out immunity because contain multiple and P185 on the SKBR3 cytolemma ^Neu/c-erbB-2The growth factor receptors proteinoid that belongs to same family, this immunization method are easy to generate the antibody that cross reaction is arranged with other growth factor receptors proteinoid, have increased the anti-P185 of screening greatly ^Neu/c-erbB-2The difficulty of specific antibody.

The purpose of this invention is to provide a kind of energy specificity at P185 ^Neu/c-erbB-2And can be used for clinical diagnosis tumor tissues P185 ^Neu/c-erbB-2The anti-P185 of expression level ^Neu/c-erbB-2Monoclonal antibody hybridoma and preparation method thereof, and this anti-P185 ^Neu/c-erbB-2The purposes of monoclonal antibody hybridoma excretory antibody on lesion detection.

The anti-P185 of the present invention ^Neu/c-erbB-2Monoclonal antibody hybridoma is characterized in that comprising the three strains hybridoma of called after A18, A21 and A22 respectively, and their 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, thereby can both be used for detecting tumor tissues P185 ^Neu/c-erbB-2Expression level; Wherein A21 and A22 can be respectively with from P185 ^Neu/c-erbB-2The DPLNNTTPVTG of two sections polypeptide 99-109 and the DLDDKGCPAEQR of 614-625 have specificity to combine with the coupled product of bovine serum albumin BSA.

The anti-P185 of the present invention ^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-erbB-2The hybridoma of monoclonal antibody.

The anti-P185 of the present invention ^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, as cytolemma P185 ^Neu/c-erbB-2During high expression level, the grade malignancy height of this tumour is described, postoperative easily takes place to shift and recurrence.

The present invention compared with prior art has the following advantages and specificity:

With the anti-P185 of the present invention ^Neu/c-erbB-2Hybridoma is distinguished excretory monoclonal antibody A18, A21 and A22 and is adopted IIP staining, immunofluorescence technique, flow cytometer (FACS) method and immunoprecipitation method to analyze, the result show these antibody can both with P185 ^Neu/c-erbB-2The specificity combination; Wherein record monoclonal antibody A21 and A22 can with from P185 ^Neu/c-erbB-2The DPLNNTTPVTG of two sections polypeptide 99-109 and the DLDDKGCPAEQR of 614-625 combine (annotate: the binding specificity of A18 is undetermined also) with the coupled product specificity of bovine serum albumin.Excretory monoclonal antibody A18, A21 and A22 have carried out immunohistochemical staining to breast cancer tissue and optimum breast tumor tissues respectively to use three strain hybridomas of the present invention, detected result shows that the positive staining of this three strain antibody has good film location, and it is closely related with tumor grade, tumor tissues breaks up difference and more lymphoglandula is arranged or the mammary cancer of blood road transfer, its P185 ^Neu/c-erbB-2Positive rate is in rising trend.In view of the above as can be known, these monoclonal antibodies can develop into diagnostic kit and are used for detecting tumor tissues P185 ^Neu/c-erbB-2Expression level, have clinical diagnosis and instruct to select the meaning of treatment plan.

The anti-P185 of the present invention ^Neu/c-erbB-2The preparation method of monoclonal antibody hybridoma owing to adopted the immunization method of cell surface zone epi-position entrapping method, has the following advantages compared with prior art: for the A method, and advantage simple to operate, that easily go fast that present method has; For the B method, present method have used reagent be simple and easy to, cheap advantage; For the C method, the difficulty that present method can make screening obtain secretion purpose antibody hybridoma reduces greatly; Because method used in the present invention only need be successively immune with two kinds of cells, and the membranin of need not further purifying, so operating process is simplified, preparation cost is relatively cheap; And owing to must not use the immunosuppressor endoxan, thereby be difficult for making immune animal to cause death; And with the antiserum(antisera) of anti-NIH/3T3 in conjunction with changeing P185 ^Neu/c-erbB-2Behind the T6-17 behind the gene, can improve the specificity of immunity greatly and reduce the difficulty of screening purpose hybridoma.

The anti-P185 that adopts the inventive method to obtain ^Neu/c-erbB-2Antibody A 18, A21 and A22 that monoclonal antibody hybridoma A18, A21 and A22 are secreted are used to detect P185 on the tumor tissues ^Neu/c-erbB-2Expression level, the high specificity of its monoclonal antibody, positive staining are that film is painted, background is low, the immunohistochemical methods that is suitable for clinical pathology section detects, and also may develop into patients serum's detection.

Embodiment of the present invention is exemplified below:

One, anti-P185 ^Neu/c-erbB-2The preparation of monoclonal antibody hybridoma:

Preparation process mainly comprises immunity, cytogamy, screening and four steps of clone.

Concrete operations can be divided into following five steps:

1, carry out immunity with cell surface epi-position entrapping method:

Getting 6-8 week product is eight of the mouse of BaLb/C, and collection l cell NIH/3T3 after the washing, is suspended in and carries out immunity in the damping fluid, and dosage is 0.5 * 10 ⁶To 5 * 10 ⁷/ time/mouse, immune time is advisable with three times or four times; Get mouse resisting anteserum, be mouse anti NIH/3T3 antiserum(antisera); Collect the NIH/3T3 cell T6-17 that changes the neu gene, with the mouse-anti NIH/3T3 antiserum(antisera) suspension of dilution in 1: 20 to 1: 100; After being incubated overnight, collecting cell, resuspending is in damping fluid;

Other gets eight of the BaLb/C mouse in 6-8 week, and injection is through the T6-17 cell of NIH/3T3 antiserum(antisera) incubation, and immune time is no less than twice, and dosage is 0.5 * 10 ⁶To 5 * 10 ⁷/ time/mouse;

2, measure the titre of mouse resisting anteserum with enzyme-linked immunosorbent assay (ELISA):

Collect the several bottles of T6-17 cells that cover with, with hypotonic buffer liquid (5mMEDTA pH8.0) suspends, 4 ℃ of incubation half an hour for 10mMTris, 10mMKCl, ultrasonication, 18000rpm is centrifugal, gets supernatant, surveys its total protein content, is diluted to 200ug/ml, is antigen; Be added in the enzyme plate by the 50ul/ hole, 4 ℃ of bags are spent the night; Fill it up with sealing damping fluid (phosphoric acid buffer (PBS) that contains 0.25% bSA (BSA) and 0.05% tween (Tween)) in the hole, room temperature is placed more than 2 hours; With the same NIH/3T3 cell of handling as negative control; Discard the sealing damping fluid, with after the distillation washing, the antiserum(antisera) 50ul/ hole that adds each mouse respectively was anti-as one, 37 ℃ of incubations 1.5 hours; After the distillation washing 3 times, add sheep anti mouse IgG-horseradish peroxidase (HRP), 37 ℃ of incubations 1 hour as ELIAS secondary antibody; After the distillation washing 3 times, add the OPD colour developing, 492nm surveys absorbancy (OD) value; If OD when being antigen with T6-17 ₄₉₂＞1.0, and OD when being antigen with NIH/3T3 ₄₉₂＜0.2, promptly positive; Select the highest the mouse (＞1:10 of antiserum(antisera) titre ⁴), carry out the last immunity with the T6-17 cell of handling equally.

3, carry out cytogamy according to a conventional method:

Merge recovery myeloma cell SP2/0 about the last week, renewing bright substratum the day before yesterday merging.Merging the same day, getting the spleen cell of mouse after the last immunity,, collecting SP2/0, mixing, making that splenocyte: SP2/0 is 5: 1, once more washing with the washing of D-hanks damping fluid.With gone out 50% the Macrogol 4000 (PEG4000 of bacterium of 1ml, be dissolved in the serum-free RPMI-1640 substratum) in 1 minute, drip in cell, after leaving standstill 10 minutes, cell suspension is selected substratum in HAT, divide and be added in the 96 porocyte culture plates, cultivate about two weeks, as seen until the hybridoma cell clone naked eyes.

4, carry out the positive colony screening with the ELISA method:

By above-mentioned ELISA method, will be wherein one anti-ly change the hybridoma supernatant into, filter out the anti-P185 of secretion ^Neu/c-erbB-2The positive hybridoma cell of antibody.

5, carry out mono-clonalization according to the limiting dilution assay of routine:

With the hybridoma in the positive hole by dilution in about 1: 10000 after, add in the Tissue Culture Plate, make cloning efficiency remain on 1/3 to 1/2; After treating that the clone grows up, measure, calculate the positive rate of dilution rear clone, choose the strongest clone of reaction and proceed dilution, repeat repeatedly to reach 100% until positive rate with the ELISA method.Obtain secreting anti-P185 ^Neu/c-erbB-2The hybridoma of monoclonal antibody, be the purpose product that this experiment will obtain.

This experiment obtains three strains and secretes anti-P185 ^Neu/c-erbB-2The hybridoma of monoclonal antibody, according to clone's numbering, with its called after A18, A21 and A22 respectively, their excretory antibody also corresponding called after A18, A21 and A22 respectively.

Two, the character of monoclonal antibody A18, A21 and A22 is identified

1, above-mentioned three strain hybridoma A18, the A21 that obtains by ELISA method screening positive clone and A22 are increased respectively back plants in the abdominal cavity of BaLb/C mouse in 8 weeks, treat long ascites after, take out ascites, be rough monoclonal antibody.

2, measure gained antibody subclass with the ELISA detection kit:

Anti-various mouse IgG subclass antibody (as rabbit anti-mouse igg 1 antibody, rabbit anti-mouse igg 2a antibody, the rabbit anti-mouse igg 2b antibody) 50ul of rabbit that in each hole of enzyme plate, adds 20ug/ml respectively, room temperature incubation 2 hours or spend the night in wet box; Remove supernatant, fill it up with 3%BSA/PBS in the hole, room temperature was sealed 2 hours or was spent the night; It is inferior to give a baby a bath on the third day after its birth with PBS, adds cleer and peaceful ascites 50ul on the various hybridomas, and the room temperature incubation is 2 hours in the wet box, abandons supernatant, and it is inferior to give a baby a bath on the third day after its birth with PBS, adds goat anti-rabbit igg-horseradish peroxidase, and room temperature incubation 2 hours adds the OPD substrate solution and develops the color, and 492nm surveys the OD value.The subclass that the result records antibody A 18, A21 and A22 is IgG1.

3, use the IIP staining to gained monoclonal antibody A18, A21 and A22 and cell surface P185 ^Neu/c-erbB-2Specificity in conjunction with detecting:

NIH/3T3, T6-17, SKBR3, MCF7 (are the low P185 that expresses ^Neu/c-erbB-2Human breast cancer cell), NE91 (being the NR-6 cell of reincarnation growth factor receptor body EGFR gene, high expression level EGFR on its cytolemma) cell reaches respectively on the cover glass in the little culture dish, continue to cultivate 2-3 days; Take out cover glass, wash twice, with 3% ice-cold formalin fixed 10 minutes with PBS; Each Dropwise 5 0ul dilutes with the sealing damping fluid on cover glass ascites A18, A21 and A22, room temperature was placed 1-1.5 hour in wet box; PBS washes 3 times, 5 minutes/time; Dropwise 5 0ul goat anti-mouse igg-HRP (1: 1000), wet box incubation 1 hour; It is inferior to give a baby a bath on the third day after its birth with PBS, 5 minutes/time; Drip DAB colour developing liquid, the room temperature incubation is about 10 minutes; Mirror is observed down, find that T6-17 and SKBR3 have strong film positive reaction for these three kinds of ascites, and NIH/3T3, MCF7 and NE91 is reactionless, illustrate hybridoma A18, A21 and A22 excretory antibody A 18, A21 and A22 all with the P185 that is expressed on the cytolemma ^Neu/c-erbB-2The film outskirt specificity combination is arranged; Because the cell NE91 of high expression level EGFR does not have and combines on these a few strain monoclonal antibodies and the cytolemma, they and EGFR no cross reaction are described.

4, detect gained monoclonal antibody and P185 with immunoprecipitation ^Neu/c-erbB-1Proteic specific combination:

At first, reagent preparation:

(1)4mM?Na ₃VO ₄；

(2)50mM?NaF；

(3) cell washing damping fluid: contain 0.4mM Na ₃VO ₄PBS with 5mM NaF;

(4) cell lysis buffer solution: contain 1%NP40,1% Deoxycholic Acid and receive salt, 0.1% dodecyl sulphur

Acid sodium, 0.15M NaCl, 0.01M sodium hydrogen phosphate salt (pH7.4), 1% proteolytic enzyme suppress

Agent Tracyrol (Aprotinin 200U/ml), 1mM PMSF, 2mM EDTA, 10mM

NaF, 10mM trisodium phosphate, 0.4mM Na ₃VO ₄With 10mM C ₂H ₄INO transfers pH at last

To 8.0;

(5) gel detergent liquid: pH contains 0.5M NaCl, 0.4mM Na among 7.4 the PBS ₃VO ₄,

0.1%NP40,10mM NaF and 10mM trisodium phosphate.

The concrete operations step is as follows:

The SKBR3 cell grow on the 15cm culture dish converge after, wash on ice twice with the cell washing damping fluid, press the 1.5mL/ ware and add cell lysis buffer solution, 4 ℃ of incubations 30 minutes are scraped cell to the centrifuge tube of 1.5mL, at 4 ℃ with 14,000 rev/min centrifugal 15 minutes, get supernatant, survey protein concentration, transfer to 1-3mg/mL and be used for immunoprecipitation; Each adds 2ul ascites A18, A21 and A22, behind the mixing, places on ice 1-2 hour, adds the gel Sepharose that 40uL is connected with protein A, rocks 1 hour at 4 ℃, again with 7500 rev/mins of low-speed centrifugals 3 minutes; Gel is given a baby a bath on the third day after its birth inferior with gel detergent liquid; Abandon supernatant, add 20ulSDS-polyacrylamide gel electrophoresis sample buffer, boiled 3 minutes, in cooled on ice, the centrifugal pearl that gets rid of down carries out the SDS-polyacrylamide gel electrophoresis with supernatant, concentrated glue is gum concentration 5%, and separation gel is a gum concentration 7.5%, and electrotransfer is to nitrocellulose filter; With sealing damping fluid closing membrane; How anti-adding with the anti-P185 of rabbit of dilution in 1: 5000,37 ℃ of incubations 1.5 hours; Washing adds goat-anti rabbit-HRP, 37 ℃ of incubations 1 hour; Washing, the fluorogenic substrate colour developing was with X-ray sheet exposure 30 seconds; The result shows that this three strains monoclonal antibody institute bonded albumen is that molecular weight is the P185 of 185kd ^Neu/c-erbB-2Albumen.

5, detect P185 on gained monoclonal antibody and the cytolemma with the FACS method ^Neu/c-erbB-2The specificity combination:

Preparation FACS damping fluid: the PBS that contains 0.5%BSA and 0.05% sodium azide;

Collect SKBR3 cell and NIH/3T3 cell respectively, each sample is 10 ⁵Cell is with the washing of FACS damping fluid; In each sample, add 2ul ascites and 4ulFACS damping fluid, ice bath 30 minutes; Wash with the FACS damping fluid; In each sample, add sheep anti-mouse igg-fluorescein (FLTC) 0.5ul and 40ulFACS damping fluid, ice bath 30 minutes; With after the FACS damping fluid washing, detect its fluorescence intensity with flow cytometer, wherein the SKBR3 cell has hyperfluorescence, and the fluorescence of NIH/3T3 cell is very weak, meanwhile illustrated these three kinds of ascites all with the SKBR3 cytolemma on P185 ^Neu/c-erbB-2The specificity combination, and with do not express P185 ^Neu/c-erbB-2The debond of NIH/3T3 cell.

6, monoclonal antibody A21, A22 and derive from P185 ^Neu/c-erbB-2The binding characteristic of the polypeptide of molecule:

Will be with glutaraldehyde from P185 ^Neu/c-erbB-2Two sections polypeptide, be the DPLNNTTPVTG of 99-109 and the DLDDKGCPAEQR of 614-625, after the bovine serum albumin coupling, be coated on the enzyme plate as antigen, recording ascites A21 and A22 and they with the ELISA method has positive reaction, thereby explanation A21, A22 can have specificity to combine with these two sections polypeptide.

Three, adopt immunohistochemical methods method mensuration monoclonal antibody A18, A21 and A22 to be used for human breast carcinoma tissue P185 ^Neu/c-erbB-2The best effort concentration that expression level detects:

Ascites with above-mentioned A18, A21 and A22 monoclonal antibody, titre with 1: 50,1: 100,1: 200,1: 400,1: 800, known P185 male human breast carcinoma tissue paraffin pathological section is dyeed with streptavidin peroxidase (S-P) method immunohistochemical methods, painted with tangible cytolemma as the P185 positive criteria, test best effort concentration.That is: with paraffin section de-waxing to water, wash with PBS; Dropping contains 3%H ₂O ₂Methyl alcohol, room temperature incubation 10 minutes; After washing with PBS, drip 10% non-immune serum, room temperature incubation 10 minutes is inhaled and is removed unnecessary liquid; With 1: 800, A21 dripped in section with dilution in 1: 600 with ascites A18, A22, and 37 ℃ of incubations spent the night in 60 minutes or 4 ℃; Wash 3 times 5 minutes/time with PBS; Add biotin labeled sheep anti mouse two anti-room temperature incubations 10 minutes; PBS washes 3 times, 5 minutes/time; Drip streptavidin-HRP solution, room temperature incubation 10 minutes; Wash with PBS, add the DAB solution of fresh configuration, developed the color 3-10 minute, the tap water flushing; Hematorylin is redyed, neutral gum mounting, microscopic examination staining pathologic section result, high expression level P185 in the section ^Neu/c-erbB-2Position film dyeing clear and background is weak, pairing greatest dilution is that best effort concentration: A18 and A22 are 1: 800, A21 is 1: 600.

Four, clinical tumor detects test

Adopt aforesaid method with the anti-P185 of gained ^Neu/c-erbB-2Monoclonal antibody hybridoma excretory monoclonal antibody has carried out immunohistochemical staining to 40 routine human breast carcinoma histopathologic slides of affiliated hospital of Medical University Of Anhui and 12 routine optimum mammary tissue sections, the result shows that positive staining has good film location, and closely related with tumor grade.With A18 is example, the P185 of optimum breast duct leaflet ^Neu/c-erbB-2It is all negative to dye, and what the record of lymphoglandula or blood road transfer condition was arranged in the breast cancer tissue is 33 examples; P185 ^Neu/c-erbB-2Positive rate is followed successively by lymphoglandula negative patient 57% (4/7), nodus lymphoideus transferring rate＜4 piece person 62% (8/15); Nodus lymphoideus transferring rate＞4 piece and blood road transferrer 69% (9/13).P185 ^Neu/c-erbB-2Positive rate is 50% (6/12) histology I level person, is 64% (18/28) histology II-III level person, shows the mammary cancer that the tumor tissues differentiation is poor, have more lymphoglandula or blood road to shift, its P185 ^Neu/c-erbB-2Positive rate is in rising trend.The clinical experiment presentation of results P185 ^Neu/c-erbB-2Can the anti-P185 of the present invention be described simultaneously as one of sign of breast cancer diagnosis, differentiation degree and biological behaviour ^Neu/c-erbB-2Monoclonal antibody hybridoma excretory monoclonal antibody A18, A21 and A22 have actual detected tumor tissues P185 ^Neu/c-erbB-2Effect; These monoclonal antibodies can develop into diagnostic kit and be used for detecting tumor tissues P185 ^Neu/c-erbB-2Expression level.

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-erbB-2The hybridoma of monoclonal antibody.

3, anti-P185 ^Neu/e-erbB-2The purposes of monoclonal antibody hybridoma on lesion detection is characterized in that using anti-P18 ^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-erbB-2During high expression level, the grade malignancy height of this tumour is described, postoperative easily takes place to shift and recurrence.