CN1824789A - Preparation of testis specific protein 50 monoclonal antibody and polyclonal antibody and use - Google Patents

Abstract

The present invention relates to a kind of biological reagents for detecting testicular specific protein 50 related to genesis of malignant tumors of mammary carcinoma, etc, and discloses a preparation method of testicular specific protein 50 monoclonal antibody and polyclonal antibody. Besides, said invention also provides the application of these antibodies and their derivatives in preparation of medicine and diagnosis reagent. These antibodies can be used for clinically detecting TSP 50 level of malignant tumor patient, at the same time the described antibodies and their derivatives can be used as medicine for curing the diseases related to TSP50.

Description

Testes specificity albumen 50 monoclonal antibodies, Polyclonal Antibody Preparation and purposes

Technical field

What the present invention relates to is and closely-related testes specificity albumen 50 monoclonal antibodies of tumour and preparation method of polyclonal antibody such as mammary cancer, the purposes of these antibody or derivatives thereofs in preparation medicine and diagnostic reagent also is provided simultaneously, belonged to technical field of biological genetic engineering.

Background technology

Mammary cancer is women's common cancer, and sickness rate has the trend that rises year by year, and from the epidemiology development trend, mammary cancer more and more concentrates on the big city, leaps to the first place of women's malignant tumour.At present the diagnostic means for mammary cancer still is based on iconography and histopathological examination, and not only expense is higher but also can't realize early diagnosis.

The research prompting of relevant in recent years cancer genetics, in the generation of tumour and the vicious transformation process usually with the activation of oncogene with (or) inactivation of cancer suppressor gene.Testes specificity albumen 50 (testes specificprotein 50, hereinafter to be referred as: TSP50) be a nearest newfound proto-oncogene, its 385 amino acid whose albumen of encoding, and this albumen has similar structure to multiple serine protease, but its catalytic residue Ser is replaced by Thr, so it may be the serine/threonine protein enzyme of a specific type.Under normal circumstances it is expressed in people's the testis tissue, and only is present in the spermatogonium, and not in sperm, points out it to have important effect in the process that sperm produces.And in other healthy tissuess beyond the testis, this gene is not expressed (Yuan, L., Shan, J., De Risi, D., Broome, J., Levecchio, J., Gal, D., Vinciguerra, V., and Xu HP.Isolation of a novel gene, TSP50, by a hypomethylated DNA fragment in human breast cancer.Cancer Res.1999,59,3215-3221).But nearest studies show that, finds the unusual high expression level of TSP50 in the breast cancer tissue more than 90%, and (Shan, J. in this kind expression epithelial cell of being confined to cancerate, Yuan, L, Xiao, Q., Chiorazzi, N., Budman, D., Teichberg, S., and Xu, HP, TSP50, A possible protease in human testis, is activated in breast cancerepithelial cells.Cancer Res.2002,62,290-294).We infer that TSP50 is a new tumor-testis antigen (cancer testes antigen thus, CTA), play an important role in mammary cancer generation and sperm forming process as the serine/threonine protein enzyme, it will be the important target spot of breast cancer diagnosis and treatment.

Summary of the invention

The invention provides a kind of testes specificity albumen 50 monoclonal antibodies and preparation method of polyclonal antibody, purpose is to prepare mono-clonal and the polyclonal antibody of TSP50, set up stable, responsive ELISA double-antibody sandwich system, by the expression that detects TSP50 TSP50 relative diseases such as mammary cancer are carried out early diagnosis, and provide strong theoretical foundation for treatment of diseases such as mammary cancer and prognosis.

The present invention also provides testes specificity albumen 50 monoclonal antibodies and the purposes of polyclonal antibody in preparation medicine and diagnostic reagent.

Another object of the present invention is to and to be used for the location and the treatment of tumour behind this monoclonal antibody and the polyclonal antibody mark.

The present invention also uses the interaction of this antibody test TSP50 and other molecule, thus the function of research TSP50.

For reaching the object of the invention, the inventor according to the aminoacid sequence (seeing accompanying drawing 1 for details) of people TSP50 and with the homology of other serine protease relatively, the amino acid pro pool 156-206 amino acid of choosing in the TSP50 contact reacts functional zone synthesizes, these 50 amino acid whose little peptides are the special peptides of TSP50, and we are with its called after pep-50.We have designed the TSP50 Auele Specific Primer also according to the cDNA sequence of people TSP50 simultaneously, by the RT-PCR method, angle from people's testis tissue and have got the TSP50cDNA fragment, and connect into protokaryon and carrier for expression of eukaryon, express.With pep-50 and prokaryotic expression protein and carrier for expression of eukaryon as antigen immune mouse and rabbit respectively, preparation antibody.

Antibody with above-mentioned antigen-immunized animal preparation comprises monoclonal antibody and polyclonal antibody.The acquisition of TSP50 polyclonal antibody is to be antigen with pep-50 and prokaryotic expression protein and carrier for expression of eukaryon, with eukaryotic vector during as antigen, directly with the recombinant plasmid injection animal quadriceps muscle of thigh that contains the TSP50 gene, pep-50 and prokaryotic expression protein add during as antigen fully and incomplete Freund's adjuvant immune animal repeatedly, comprise rabbit, mouse, rat, sheep etc., obtain to combine active serum with natural TSP50.The MONOCLONAL ANTIBODIES SPECIFIC FOR method, be to adopt hybridoma technology (Kohler G.and Milstein C.Nature 1975,256:495) preparation mouse anti TSP50 monoclonal antibody, but be not limited to mouse, also available rat (Rat Hybridomas and RatMonoclonal Antibodies P75-115,265-270, Bazin H.CRC Press, Inc.Florida1990).The how anti-and monoclonal antibody of above-mentioned gained detects its biologic activity by the ELISA method.First usefulness is synthesized little peptide pep-50 wrapper sheet 4 μ g/ml, and the second usefulness prokaryotic expression protein wrapper sheet, 5 μ g/ml detect homemade antibody with the ELISA method of routine again and the two all has association reaction.The subclass of surveying 3 strain monoclonal antibodies through indirect elisa method is respectively IgG1, IgM, IgG2b.Tiring of antibody is respectively 1: 128000, and 1: 64000,1: 96000.The results are shown in Figure 2.How anti-tiring is 1: 2000 to gained, the results are shown in Figure 3.

The present invention also comprises from above monoclonal antibody deutero-antibody.In the present invention, the what is called antibody of deriving is meant any molecule, particularly chimeric antibody, single-chain antibody (ScFv) and the Fab fragment of the idiotype that comprises monoclonal antibody of the present invention.Chimeric antibody can be according to Morrison etc., bacteriology magazine (J.Bacteriol.), 159:870 (1984); Neberger etc., nature (Nature) 312:604-608 (1984); Takeda etc., nature, the described technology of 314:452-454 (1985) obtains.The Fab fragment that contains antibody antibody uniqueness of the present invention can produce by any technology well known by persons skilled in the art.For example, these fragments include but not limited to: F (ab ') ₂Fragment, the fragment that can produce by pepsin digested antibody; Can be by reduction F (ab) ₂The Fab fragment that segmental disulfide linkage obtains, and can handle the Fab fragment that antibody obtains by papoid and reductive agent.

Target of the present invention also is from said monoclonal antibody deutero-ScFv.This single-chain antibody can pass through patent US4946778, and technology described in US5132405 and the US5476786 obtains.

The invention still further relates to the nucleotide sequence of the encoding gene that contains single-chain antibody that said monoclonal antibody is derived.Being applied among the patent application WO94/29446 of the preparation method of this sequence and expression in vivo antibody stated.

The invention still further relates to the nucleotide sequence of these ScFv of coding or contain of the application of their carrier at the pharmaceutical composition of preparation treatment or Surgery Treatment human or animal body.The invention still further relates to the pharmaceutical composition that contains carrier (particularly virus vector) as defined above or nucleotide sequence.

The invention still further relates to the application of antibody in the preparation medicine as defined above.The present invention relates more specifically to the application of described antibody in the medicine of preparation treatment or prevention excess proliferative disease.Antibody of the present invention can be made targeted drug with various toxin, isotropic substance or drug coupling, combine with the specificity of TSP50 by antibody and medicine entrained on the antibody, toxin or isotropic substance are taken to the position of TSP50 unconventionality expression, as the neoplasm position, thereby reach the treatment disease relevant with the TSP50 unconventionality expression.

The invention still further relates to and contain the antibody of the present invention for the treatment of significant quantity and the optional pharmaceutical composition of blended medicine acceptable carrier with it, described amount can in and the TSP50 activity.

Antibody of the present invention can also be used for identifying or quantitative assay TSP50 albumen as diagnostic reagent.In fact, proved TSP50 albumen overexpression in tumour cell, and this overexpression constitutes the sign in the cell proliferation phenomenon.Therefore these antibody can be used in particular for diagnosing the excess proliferative disease that causes the TSP50 protein overexpression.

The present invention thereby also relate to as defined above antibody as the application of diagnostic reagent and the immunodiagnosis kit that contains described monoclonal antibody and polyclonal antibody.This test kit includes but not limited to double antibody sandwich method ELISA detection kit, can be used for detecting but be not limited to the proteic amount of TSP50 in the various liquid samples such as tissue homogenate, cell pyrolysis liquid, cell exocrine liquid, serum, is that its test kit of example comprises with the double antibody sandwich method:

(1) standard substance: mammary cancer marker molecule TSP50 protein solution 400 μ l, its concentration is 0.1mg/ml.

(2) monoclonal antibody: mouse anti TSP50 monoclonal antibody 50 μ l, antibody concentration is 0.1mg/ml, can dilute 1000 times during application.

(3) polyclonal antibody: the anti-TSP50 polyclonal antibody 50 μ l of rabbit, antibody concentration is 1mg/ml, can dilute 1000 times during application.

(4) enzyme labelled antibody: goat anti-rabbit igg antibody 50 μ l (available from promega company) can dilute 1000 times during application.

Test kit comprises mentioned reagent at least, for convenience simultaneously, and the enzyme plate of also can packing into, concentrated cleaning solution, concentrated confining liquid, developer A, developer B, stop buffer, semi-logarithmic coordinate paper etc.

Described concentrated cleaning solution is a phosphoric acid buffer, and confining liquid is that BSA-phosphate buffered saline buffer, developer A are 1% phosphorus phenylenediamine (OPD), and developer B is a hydrogen peroxide, and stop buffer is a 2M sulfuric acid.

The making of typical curve when above-mentioned breast cancer related protein TSP50 can be used for measuring, this protein solution can be diluted to different concns 100 μ g/ml, 10 μ g/ml, 1 μ g/ml, 100ng/ml, 10ng/ml, 1ng/ml with phosphate buffered saline buffer, six points are as the X-coordinate of curve, with OD492 mean value is ordinate zou, the drawing standard curve.

The using method of this test kit is as follows: wrap by 96 hole enzyme plates with TSP50 monoclonal antibody 0.1-5 μ g/ml, 4 ℃ are spent the night, the BSA of inferior daily 1-5% seals, 1-4 hour after scouring also adds the TSP50 albumen of different concns, 37-43 ℃ acts on 0.5-4 hour, add the anti-TSP50 polyclonal antibody of rabbit then, 37-43 ℃ acts on 0.5-4 hour, PBST washing back adds the goat anti-rabbit igg of HRP mark, 37-43 ℃ acts on 0.5-4 hour, PBST washing back adds chromogenic substrate OPD, and 2MH is used in lucifuge reaction 5-10 minute ₂SO ₄Termination reaction is surveyed the OD492 value with microplate reader.

Aforesaid monoclonal antibody and polyclonal antibody can also with that add lustre to, fluorescence, biotinylated, radioactive or other mark couplings.These antibody can be used for the immune labeled method of immunohistochemistry, flow cytometry, are used in the known diagnosis test kit of radioimmunoassay (RIA) or immunoenzymatic assay (ELISA) and any kind, also can be used for location or killing tumor cells.

Description of drawings

Fig. 1 has shown the aminoacid sequence of people TSP50, has underscore partly to be synthetic 50 peptide positions

Fig. 2 has shown tiring of three strain monoclonal antibodies.A is 1E9B11, and B is 3F7B11, and C is 2B11D9

Fig. 3 has shown that rabbit against human T SP50 polyclonal antibody tires

Fig. 4 detects the proteic expression of TSP50 with the ELISA method.Be the typical curve of TSP50 detection kit shown in the figure, and with irrelevant Protein G ST as negative control, prove the specificity of this test kit, the susceptibility of this test kit can reach 10ng/ml.

Embodiment

Except that the above, the present invention also comprises other feature and advantage from following examples, and these embodiment should be considered as to explanation of the present invention rather than to the restriction of its scope.

Embodiment 1

The TSP50 MONOCLONAL ANTIBODIES SPECIFIC FOR

1. get 6 of female BALB/C mice in age in 6-8 week,, add adjuvant and (use Freund's complete adjuvant for the first time with antigen TSP50 (50 μ g/ only/time), full freund adjuvant toos many or too much for use later on) abdominal injection, every At intervals of two to three weeks immunity once is total to immune 3-5 time, and the spleen fusion was got in last immunity in back 5 days.

2. prepare the immune mouse spleen cell suspension.

3. get mouse thymus, the preparation feeder layer cells.

4. recover and the cultivation myeloma cell.

5. merge: with myeloma cell and immunocyte with 1: 7 mixed after, 1ml50%PEG is dropwise added in the cell mixing, add in 1 minute, left standstill 1 minute, add serum free medium 5ml then, add in 5 minutes, other adds the 25ml serum-free medium.800rpm, centrifugal 8 minutes, abandon supernatant, add the complete RPMI-1640 that contains HAT, add 96 well culture plates, every hole 100 μ l behind the mixing, cultivate in 37 ℃ of carbonic acid gas incubators, 1 week hybridoma cell clone occurred later, in time carried out ELISA and detected, screens, and picking strong positive clone carries out cloning.

6. the detection of hybridoma and cloning

With TSP50 albumen coated elisa plate, concentration is 5 μ g/ml, 100 μ l/ holes, and 4 ℃ are spent the night; Inferior daily 1%BSA shrouding, 37 ℃, 1 hour; Discard confining liquid, add hybridoma supernatant to be measured, 100 μ l/ holes, 37 ℃ were reacted 2 hours; Wash plate 3 times with the PBS that contains 0.1%Tween-20; The goat anti-mouse igg that adds the HRP mark, 100 μ l/ holes, 37 ℃, 1 hour; The PBS of 0.1%Tween-20 adds OPD colour developing 5 minutes, 2M H after washing plate 3 times ₂SO ₄Termination reaction.Select the positive cell hole, get hybridoma, carry out cloning with limiting dilution assay.Screening, cloning again cloned three times at least continuously afterwards, gets positive colony, enlarged culturing.

7. preparation ascites

With the hybridoma enlarged culturing, carry out frozen.Get female BALB/C mice in 4～8 ages in week simultaneously, in the injecting fluid paraffin 0.5ml of abdominal cavity elder generation, after 3～14 days, the inoculation hybridoma is observed the belly size every day and is analyzed the ascites production.No. 12 syringe needles are inserted ascitogenous mouse left lower abdomen, access ascites with test tube, centrifugal 5 minutes of 1500rpm/min, it is frozen standby to draw ascites.Frozen in liquid nitrogen with adding frozen storing liquid in the cell precipitation.

Embodiment 2

Purification of Monoclonal Antibodies

The ascites that will contain monoclonal antibody with 50% ammonium sulfate precipitation is once used 33% ammonium sulfate precipitation secondary again, and the desalination of dialysing then is further purified with DEAE-Mierocrystalline cellulose chromatography method again.

1) DEAE-cellulose activation: the distilled water of getting 50 times of amounts of a certain amount of DEAE-Mierocrystalline cellulose adding fully soaks, room temperature left standstill 30 minutes, to go up 1/3 gently outwells, the fine particles that floats in the supernatant is removed thereupon, the 0.5N NaOH that adds 20 times of amounts fully stirred 20 minutes, be statically placed in the refrigerator and soaked 1 hour, abandon supernatant liquor, extremely neutral with distilled water flushing, fully soaked 1 hour with 0.5N HCl, still be washed till neutrality, with the PB liquid of pH6.3,0.0175M abundant balance more than at least 24 hours in refrigerator with distilled water.

2) dress post: be 2cm generally with the post bore, high 50cm, the Mierocrystalline cellulose that above-mentioned balance is good once adds in the post with suction pipe, makes the interior uniformity up and down of post, opens end opening adjusting flow velocity and is advisable with 20 of per minutes.

3) go up sample: after installing post, PB liquid with pH6.3,0.0175M fully washes, open end opening and mix up flow velocity, when cylinder liquid retains about 0.5cm height, close end opening, sample is slowly added around post jamb with suction pipe, open end opening after adding, sample enters the PB liquid abundant wash-out of the interior back of post with pH6.3,0.0175M.

4) sample collection: sample enters beginning wash-out and collection in the post.According to preparing some test tubes, every pipe is collected 110 during collection.With 20% sulphosalicylic acid inspection, allly contain proteic pipe, put into refrigerator immediately, after all having collected, it is concentrated that protein part is put into dialysis tubing.

Embodiment 3

The evaluation of immunoglobulin (Ig)

1. determining the protein quantity: survey the purifying protein optical density value with ultraviolet spectrophotometer, calculate protein content.

2. the mensuration of immunoglobulin subclass: with antibody purified pH7.4, the PBS of 0.01M is diluted to 4 μ g/ml, adds 96 hole enzyme plates, every hole 100 μ l, and 4 ℃ are spent the night; Discard coating buffer next day, every hole adds 200 μ l2%BSA-PBS, and 43 ℃ were sealed 1 hour; Discard confining liquid, wash twice, add the antibody of the anti-rat immune globulin subclass of various rabbits, hatched 1 hour for 43 ℃ with washings; Discard liquid in the hole, give a baby a bath on the third day after its birth time with washings, add the HRP-goat anti-rabbit antibody, hatched 1 hour for 43 ℃, discard the liquid hole in, give a baby a bath on the third day after its birth time with washings, add colour developing liquid, room temperature lucifuge reaction 10 minutes is with microplate reader survey OD492 value.

3. titration: the immunoglobulin (Ig) of purifying is carried out doubling dilution, measure with indirect ELISA and tire, method is measured with serum antibody titer.

Embodiment 4

The TSP50 Polyclonal Antibody Preparation

1. immunizing rabbit

1) preparation water-in-oil antigen emulsion: the 5ml freund's adjuvant is drawn in the 10ml syringe, (final concentration is 15～30mg/ml) to put in another 10ml syringe with equivalent antigen and bacille Calmette-Guerin vaccine, two syringes connect with communicating vessels, the suction mixing, until being mixed into oyster white, extraction difficult splashes into till the water surface do not loose.

2) immune animal: select the healthy rabbits of 2～2.5kg for use,, be total to injections of antigens emulsion 2ml (only containing antigen 50mg/) for the first time with intracutaneous multi-point injection method.Intracutaneous multiple spot immunity once more after three weeks, back in intravenous injection soluble antigen 100mg every two weeks again, this is a reinforced immunological.

2. examination blood

Strengthen back 7 days ear veins of injection and get blood, separation of serum is surveyed it with indirect elisa method and is tired, and method is with the mensuration of mice serum antibody titer.

3. blood sampling

Select once full blood-collecting method for use, from the aseptic bloodletting of carotid artery, separation of serum adds an amount of sanitas, the packing bottle, and cryogenic refrigerator is preserved.

4.IgG extraction

With 50% ammonium sulfate precipitation once, 33% ammonium sulfate precipitation twice carries out purifying to rabbit anteserum with the DEAE chromatography then.Method is seen purifying antibody.

5. the mensuration of tiring

The IgG that measures purifying with indirect elisa method tires, and method is identical with the detection of serum antibody titer.

Embodiment 5

Use horseradish peroxidase-labeled antibody

Horseradish peroxidase 8mg is dissolved in the 1ml distilled water, adds 200 μ l, 0.1M metaperiodic acid sodium solution at room temperature left standstill mixed solution 30 minutes.Utilize 1mM acetate buffer (pH4.5), make enzyme solution 4 ℃ of following dialysed overnight.Add 100 μ l, 0.2M carbonate buffer solution (pH9.5) is 9.5 to regulate the pH value.

On the other hand, 8mg is dissolved in 0.1M phosphate buffered saline buffer (pH7.4) with the anti-people TSP50 of purifying antibody, in, utilize 0.01M sodium carbonate buffer (pH9.5) 4 ℃ of following dialysed overnight.The peroxidase that obtains is mixed with antibody, at room temperature static 2.5 hours, add Sodium Borohydride, descended static 2 hours at 4 ℃.With resulting peroxidase labeled antibodies, utilize phosphate buffered saline buffer, promptly obtain the enzyme labelled antibody binding substances 4 ℃ of dialysed overnight, add equal-volume glycerine-20 ℃ preservation, select suitable extent of dilution during use.

Embodiment 6

The using method of TSP50 protein ELISA detection kit

Test kit is formed:

1. standard substance: mammary cancer marker molecule TSP50 protein solution 400 μ l, its concentration is 0.1mg/ml

2. monoclonal antibody: mouse anti TSP50 monoclonal antibody 50 μ l, antibody concentration is 0.1mg/ml, it is 1: 1000 that ELISA tires

3. polyclonal antibody: the anti-TSP50 polyclonal antibody 50 μ l of rabbit, antibody concentration is 1mg/ml, it is 1: 1000 that ELISA tires

4. enzyme labelled antibody: it is 1: 1000 that goat anti-rabbit igg antibody 50 μ l (available from promega company), antibody ELISA tire

Test kit comprises mentioned reagent at least, for convenience simultaneously, and the enzyme plate of also can packing into, concentrated cleaning solution, concentrated confining liquid, developer A, developer B, stop buffer, semi-logarithmic coordinate paper etc.

Described concentrated cleaning solution is a phosphoric acid buffer, and confining liquid is that BSA-phosphate buffered saline buffer, developer A are 1% phosphorus phenylenediamine (OPD), and developer B is a hydrogen peroxide, and stop buffer is a 2M sulfuric acid.

Using method:

1. various concentrated solutions are diluted to application concentration, are about to 100 times (concentration is 0.01M during detection) of concentrated cleaning solutions dilution specifically; 10 times (BSA concentration is 1% during detection) of confining liquid dilution

2. prepare different dilution standard substance: the washings with dilution is diluted to 100 μ g/ml, 10 μ g/ml, 1 μ g/ml, 100ng/ml, 10ng/ml, 1ng/ml with standard substance.

3. with the mouse-anti TSP50 monoclonal antibody of purifying, be diluted to 0.4 μ g/ml, wrap by 96 hole enzyme plates with 0.01M PBS damping fluid (pH 7.4), 100 μ l/ holes, 4 ℃ are spent the night.

4. discard antigen coated liquid, with 1%BSA sealing, 43 ℃, 30 minutes.

5. abandon confining liquid, with 0.1% Tween20-PBS washed twice, each 5 minutes.

6. add different dilution TSP50 albumen, add testing liquid simultaneously, 100 μ l/ holes, 43 ℃, 30 minutes.

7. with 0.1% Tween20-PBS washing three times, each 5 minutes.

8. add the anti-TSP50 polyclonal antibody of rabbit, 100 μ l/ holes, 43 ℃, 30 minutes.

9. with 0.1% Tween20-PBS washing three times, each 5 minutes.

10. adding goat anti-rabbit igg, 100 μ l/ holes, 43 ℃, 30 minutes.

11. with 0.1% Tween20-PBS washing three times, each 5 minutes.

12. add the OPD chromogenic substrate, room temperature reaction 5 minutes.

13. every hole adds 50 μ l 2M H ₂SO ₄Termination reaction, microplate reader is surveyed the OD492 value.

Make typical curve according to TSP50 standard substance measurement result, from then on all samples to be checked can find its corresponding value on the typical curve according to its OD492 value, and this numerical value is the content of TSP50 in this sample.

Embodiment 7

Immunohistochemical methods detects the expression of TSP50 in the mammary cancer

Get mammary cancer and cancer beside organism's paraffin section, 0.3%H is used in conventional dewaxing entry ₂O ₂-methyl alcohol soaked 30-40 minute, and PBS washing three times is sealed with 0.1% gel again, 30-60 minute, discard unnecessary confining liquid, add TSP50 polyclonal antibody (also available monoclonal antibody), 37 ℃ were spent the night in 1 hour or 4 ℃, give a baby a bath on the third day after its birth time with PBS, each 5 minutes, add the goat anti-rabbit igg (anti-ly then being the HRP-rabbit anti-mouse igg herein) of HRP mark then with monoclonal antibody as one, room temperature 1 hour, it is inferior to give a baby a bath on the third day after its birth with PBS, each 5 minutes, adds freshly prepared DAB (0.5mg/ml)+H ₂O ₂(1 μ l/ml), 3-5 minute, conventional dehydration, mounting, microscopy.

Embodiment 8

With TSP50 antibody TSP50 is carried out the epi-position structural analysis

Monoclonal antibody screening phage 15 peptide storehouses (can buy commercialization peptide storehouse) with TSP50, its concrete grammar is: increased in the peptide storehouse earlier, join then to have wrapped and hatched in the culture dish of anti-TSP50 monoclonal antibody, submission phage surface can with the little peptide of antibodies will with the antibody mortise, to there be the bonded phage to wash away, the phage of wash-out and antibodies is also increased, phage after the amplification is dropped into the next round screening again, 3-4 time so repeatedly, by the peptide storehouse is increased, eluriated and screens, obtain positive colony.The gained positive colony is screened with the competitive ELISA method, find and TSP50 competition and monoclonal antibody bonded clone, concrete grammar is: use the monoclonal antibody coated elisa plate, BSA sealing back adds TSP50 albumen and gained phage, be control group with independent adding TSP50 albumen hole simultaneously, the TSP50 polyclonal antibody that adds horseradish peroxidase-labeled then, the OPD colour developing, measure the OD492 value, find and with the phage of TSP50 competition and antibodies and to extract its DNA and to check order, find out its conserved sequence, this conserved sequence is the epi-position of the TSP50 that infers in theory, further determines the epi-position structure of TSP50 again by computer simulation.

Embodiment 9

With TSP50 antibody TSP50 is carried out Position Research in the cell

To cultivate good MCF-7 MCF-7 cell inoculation in 12 well culture plates that are placed with cover glass, every hole 5 * 10 ⁵Individual cell, 37 ℃, CO ₂The incubator overnight incubation, abandon supernatant, wash secondary with PBS, fix 30 minutes with 4% Paraformaldehyde 96 room temperature then, it is inferior to give a baby a bath on the third day after its birth with PBS, the methyl alcohol effect 20 minutes that adds ice bath again, add TSP50 monoclonal antibody (or polyclonal antibody) after 1 hour with the sealing of 1%BSA room temperature, 4 ℃ are spent the night, the inferior daily PBS back of giving a baby a bath on the third day after its birth time adds sheep anti-mouse antibody (or the goat anti-rabbit antibody of FITC mark, gather around one anti-and decide according to institute), 4 ℃ of effects 2 hours, with cover glass seal to the slide glass the back with the burnt phase microscope observation of copolymerization TSP50 in intracellular location situation.

Embodiment 10

The medicinal radiative nuclear element of TSP50 mediated monoclonal antibody ¹³¹Locate in the body of I to mammary cancer

Get female nude mice, respectively inject the well-grown human breast carcinoma continuous cell line of 0.2ml MCF-7 cell suspension (cell count 8 * 10 at its belly mammary gland both sides fat pad ⁶Individual), preparation mice with tumor model.

Will with known Idogen or chloramine-t method mark medicinal radiative nuclear element ¹³¹I (but also mark ⁹⁹Tc ^m, ¹²³I reaches ¹¹¹In) TSP50 monoclonal antibody 1E9B11 injects through mouse tail blood vessel.After 24,48,60,72,84,96 hours, detect with the radio immuno imaging method ¹³¹The I traget antibody is the video picture situation in the tumor bearing nude mice body, and the result shows after artery injects radiolabeled antibody, and the video picture rate is to rise in the cancer district in 90.9%, 48 hour to gather, and 96 hours the most clear, and T/NT is 6.2.

Embodiment 11

With TSP50 antibody affinity purification TSP50 albumen from the TSP50 protein extract

(1) preparation of affinity column (Zhao Qiang etc., journal of Beijing Medical University 1990,22:307)

The spissated TSP50 monoclonal antibody of purifying (or polyclonal antibody) and PBS and binding buffer liquid dialysed overnight, slow liquid for several times.

In the ratio of 10-30g antibody/g carrier, crosslinked as required amount takes by weighing CNBr-sepharose4B, soaks with 1mM HCl, 4 ℃, 15 minutes, uses 1mM HCl suction filtration, washing then, to remove protective material.It is neutral getting express developed to pH with yellow soda ash, the sodium bicarbonate binding buffer liquid of 0.1M, mixes and add an amount of binding buffer liquid immediately with antibody 20g, and shaking table stirs at a slow speed, and 4 ℃ are spent the night.Suction filtration is also measured anti-body contg in the suction filtration liquid.Yellow soda ash, sodium bicarbonate binding buffer liquid suspension dress post with 0.1M are washed till no antibody and flow out (OD280＜0.01), measure and flow out the antibody total amount.Soak the post bed with the 1M thanomin, sealing residue reactive group.Wash the post secondary with the circulation of 0.1M acetate buffer solution and binding buffer liquid, wash post with 0.1M glycine-hydrochloride buffer again, be dormant state after washing post with 0.01M PBS at last.

(2) affinitive layer purification TSP50 albumen

Get the TSP50 protein crude extract administration of eucaryon or prokaryotic expression, slowly add in the affinity column, treat that it all enters and clamp the affinity column outlet behind the post bed and made albumen and post effect 30 minutes, be washed till OD280＜0.01 with PBS, be washed till OD280＜0.01 with 2.5M NaCl again, use the glycine-hydrochloride buffer wash-out of 2 times of column volumes at last, collect the protein liquid that washes, and immediately with 1M NaHCO ₃Extremely neutral in the liquid with pH.

Embodiment 12

The medicinal radiative nuclear element of TSP50 mediated monoclonal antibody ¹³¹I is to the therapeutic action of mammary cancer

Get female nude mice, respectively inject the well-grown human breast carcinoma continuous cell line of 0.2ml MCF-7 cell suspension (cell count 8 * 10 at its belly mammary gland both sides fat pad ⁶Individual), preparation mice with tumor model.

Will with known Idogen or chloramine-t method mark medicinal radiative nuclear element ¹³¹I (but also mark ⁹⁹Tc ^m, ¹²³I reaches ¹¹¹In) TSP50 monoclonal antibody 1E9B11 injects through mouse tail blood vessel.After 1,3,5,7,14 day, observe with the calculated value of CT ¹³¹The I traget antibody is to the influence of tumor bearing nude mice tumor growth, and the result shows that the tumour of 90.3% mice with tumor is dwindled, and wherein its tumour of 81.1% mice with tumor is dwindled more than 50%.

Embodiment 13

Passive inoculation TSP50 antibody is to the tumor growth restraining effect of mammary cancer mice with tumor

Get female nude mice, respectively inject the well-grown human breast carcinoma continuous cell line of 0.2ml MCF-7 cell suspension (cell count 8 * 10 at belly mammary gland both sides fat pad ⁶Individual), preparation mammary cancer mice with tumor model.With the TSP50 monoclonal antibody 1E9B11 of purifying and polyclonal antibody divide high (2mg/kg), in (0.8mg/kg), low dosage (0.2mg/kg) be expelled in the lotus knurl model mice body, establish rabbit igg contrast and physiological saline contrast simultaneously, injection once weekly, put to death animal after 21 days, measure the knurl volume change and carry out pathological examination, observe the antitumor action of the TSP50 antibody of passive inoculation.The result is as shown in table 1, and the tumour inhibiting rate of large, medium and small dosage monoclonal antibody is respectively: 76.0%, 65.4%, 54.1%; The tumour inhibiting rate of polyclonal antibody is respectively: 80.2%, 75.4%, 61.3%.Antibody group and physiological saline group and rabbit igg group comparison knurl volume weight all have significant difference (P＜0.001).

The anti-breast cancer effect of table 1:TSP50 antibody

Grouping	Dosage (mg/kg)	Number of animals	Tumor weight (g) (X ± S)	Tumor control rate
					0.9%NaCl rabbit igg monoclonal antibody 1E9B11 monoclonal antibody 1E9B11 monoclonal antibody 1E9B11 polyclonal antibody polyclonal antibody polyclonal antibody	0.2ml/10g 2.0 2.0 0.8 0.2 2.0 0.8 0.2	15 13 11 13 12 15 12 13	4.520±1.09 4.431±1.13 1.095±1.138* Δ 1.564±1.354* Δ 2.074±1.725* Δ 0.895±1.024* Δ 1.112±1.293* Δ 1.749±.692* Δ	1.9％ 76.0％ 65.4％ 54.1％ 80.2％ 75.4％ 61.3％

^*Expression and 0.9%NaCl group compares that there were significant differences, p＜0.001

^ΔExpression compares with the rabbit igg group that there were significant differences, p＜0.001

Claims (9)

1. MONOCLONAL ANTIBODIES SPECIFIC FOR method at human testicle specificity protein white 50, it is characterized in that it is the maturation protein immune mouse of testes specificity albumen 50 of little peptide, protokaryon and eukaryotic expression that utilizes the testes specificity albumen 50 of synthetic, prepares monoclonal antibody by hybridoma technology.

2. Polyclonal Antibody Preparation method at human testicle specificity protein white 50, it is characterized in that it be utilize synthetic testes specificity albumen 50 little peptide, protokaryon and eukaryotic expression testes specificity albumen 50 the maturation protein immune animal and obtain polyclonal antibody.

Among the claim 1-2 each prepared antibody in the 50 diseases related application of diagnosing of testes specificity albumen.

4. each antibody among the claim 1-2 is used to detect testes specificity albumen 50 structures and 50 variations of testes specificity albumen.

5. each antibody among the claim 1-2 is used for reaching in testes specificity albumen 50 bodies location that distributes in the cell.

6. the purposes that each antibody among the claim 1-2 is used for purifying testes specificity albumen 50.

7. each antibody among the claim 1-2 is used to prepare the medicine purposes diseases related with testes specificity albumen 50.

With each antibody among the claim 1-2 as the diseases related passive immunization preparation of testes specificity albumen 50.

9. the diseases related diagnostic kit of a testes specificity albumen 50 is characterized in that it comprises the antibody of one of claim 1-2 or the two method preparation.

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