CN106188288A - Anti-S100P protein monoclonal antibody and application thereof - Google Patents

Abstract

The present invention relates to biological technical field, disclose a kind of hybridoma cell strain (deposit number is CGMCC No.9575), and monoclonal antibody UMAB24 that thus hybridoma cell strain produces.The invention still further relates to monoclonal antibody UMAB24 and prepare the application in the immune detection instrument detecting S100P albumen, immunohistochemical kit containing monoclonal antibody UMAB24, and the application that monoclonal antibody UMAB24 is in preparation is used for the test kit of marked tumor.Monoclonal antibody of the present invention can be combined with S100P protein-specific, and with other albumen no cross reactions intracellular, significantly improve S100P protein immunization detection specificity and reliability.

Description

Anti-S100P protein monoclonal antibody and application thereof

Technical field

The present invention relates to biological technical field, being specifically related to can the monoclonal anti of specific bond S100P albumen Body UMAB24, produce described monoclonal antibody cell strain and apply the diagnostic method of this antibody and Purposes.

Background technology

S100P is belonging to a little molecular calcium associated proteins of S100 family, initially finds in Placenta Hominis because of it And named S100P, there are typical two EF hand-type structures in S100 calbindin family, extensively join Live with cell proliferation, cell differentiation, cytoskeleton composition, immunoreation and Extracellular Matrix Secretion The process such as dynamic.

S100P is the secretory protein of a kind of small-molecular-weight, can express in nucleus and cytoplasm, it is also possible to It is secreted into extracellular.S100P initially synthesizes with unactivated state, the most then due to reasons such as calcium bindings It is activated into the dimeric state of activation, also can serve as extracellular outside born of the same parents at intracellular accomodation of activities Signaling molecule.Secret out of the S100P outside born of the same parents can be combined, by work by ligand binding site outer with the born of the same parents of RAGE Change downstream ERK/MAPK signal path and control relevant expression of target gene, regulate cell proliferation；Intracellular S100P albumen can participate in the reconstruct of cytoskeleton with Ezrin protein binding and affect cell adhesion and fortune Dynamic, it is also possible to the degraded that ubiquitination path CacyBP/SIP protein binding participates in β-catenin, it is achieved right Tumor existence and the regulation and control of transfer.No matter being outside intracellular or born of the same parents, S100P can be thin by affecting tumor The propagation of born of the same parents, existence, action edge and aggressivity affect the pernicious performance of tumor.

S100P expression in the various tissues of people is different, and the expression in placenta tissue is the highest, secondly It is esophageal tissue, in Stomach duodenum, Colon and rectum, prostate and leukocyte, has the expression of moderate, But the expression in jejunum, ileum, ovary, spleen and thymus is relatively low.Numerous studies show that S100P is multiple Tumor tissues all has obvious high expressed, such as transitional cell carcinoma of bladder, nonsmall-cell lung cancer, gastric cancer, Cancer of pancreas, breast carcinoma, carcinoma of prostate, colorectal cancer, ovarian cancer, cervical cancer etc..S100P take part in carefully Born of the same parents' paraplasm, malignant change of cell, the mobility of tumor cell strengthen, the infiltration tumor such as power enhancing occurs and Development pathological process, and to Partial tumors by stages, prognosis and drug susceptibility relevant.Along with in recent years Carry out people to its deep understanding and research, it is believed that S100P has the value of potential tumor mark, leads at present The auxiliary diagnosis of carcinoma of prostate to be used for, bladder cancer etc..

The most generally by S100P in immunohistochemistry (IHC) Pathological experiment detection tumor cell Expression situation.The monoclonal antibody that core is specific binding S100P of IHC experiment, its performance excellent The bad sensitivity that directly decide whole detection and specificity.Therefore, a kind of binding specificity is developed relatively The high monoclonal antibody for S100P albumen has great importance.

Summary of the invention

In view of this, it is an object of the invention to provide the higher S100P albumen of a kind of binding specificity Monoclonal antibody, and in preparation application in the immune detection instrument detecting S100P albumen.

The invention provides a kind of hybridoma cell strain, be preserved in Chinese microorganism strain preservation conservator Meeting common micro-organisms center (referred to as CGMCC), preservation date is on August 22nd, 2014, preservation Numbered CGMCC No.9575.

Present invention also offers monoclonal antibody UMAB24 of a kind of specific binding S100P albumen, by Above-mentioned hybridoma cell strain produces.

The preparation method of monoclonal antibody of the present invention is as follows:

(1) structure of recombinant expression carrier: according to S100P ORF nucleotide sequence (S100P ORF Nucleotide sequence as shown in SEQ ID NO.1,285bp；S100P aminoacid sequence such as SEQ ID NO.2 Shown in) design primer carry out PCR amplification, gene both sides introduce respectively restriction endonuclease sites SgfI and MluI, inserts expression vector pCMV6-Entry, builds S100P recombinant expression plasmid；

(2) expression and purification of S100P recombiant protein: S100P recombinant expression plasmid is converted HEK293T, cracks centrifuging and taking supernatant, DDK affinity chromatograph column purification, it is thus achieved that the S100P restructuring of purification Albumen；

(3) screening of monoclonal antibody and preparation: use the S100P recombiant protein immunity of above-mentioned purification BALB/c mouse, takes Mouse spleen cells and merges with SP2/0 cell, and limiting dilution assay obtains Dan Ke Grand, ELISA method screening positive hybridoma cell, it is thus achieved that the hybridization of anti-S100P specific antibody can be secreted Tumor cell strain, named UMAB24, hypotype is accredited as IgG1；Antibody is prepared by serum-free medium, S100P monoclonal antibody is obtained by affinity chromatograph column purification.Respectively by Western Blot, immunity The sensitivity of groupization this monoclonal antibody of experimental verification and specificity.

Use OriGene high-density protein chip that the specificity of said monoclonal antibody is tested further Card:

10,000 HEK293T cell protein process LAN cracking are comprised on OriGene high-density protein chip Thing, every kind of protein lysate has the repetition of two copies on chip.Protein lysate by trace at nitre On acid cellulose film.The location of each clock protein lysate can be accurately positioned by Excel file. On protein chip, albumen is divided into 40 sub-matrixes, and each sub-matrix has some references, by referring to, can With the content of albumen on quantitative each chip point, monitor the repeatability of each immunoreation data, Yi Jiding The direction of position positive signal.

Described monoclonal antibody UMAB24 and said chip are hybridized and determine positive signal position by the present invention Point, result shows: this stated clearly monoclonal antibody UMAB24 specific binding S100P albumen, and With other albumen no cross reactions.

Present invention also offers monoclonal antibody UMAB24 in preparation for the immunity detecting S100P albumen Application in detection instrument.

Specifically, described immune detection instrument is test kit, chip or reagent paper.

In the particular embodiment, the invention provides a kind of immunologic combined detection reagent kit, including above-mentioned Monoclonal antibody UMAB24, can detect the expression situation of S100P in histiocyte.

Present invention also offers said monoclonal antibody preparation answering in the test kit of marked tumor With.

Wherein said tumor specifically refers to the propagation of tumor cell and the closely-related tumor of expression of S100P, Include but not limited to non-small cell carcinoma, gastric cancer, cancer of pancreas, breast carcinoma, carcinoma of prostate, colorectal cancer, Ovarian cancer, cervical cancer or bladder cancer.

Compared with prior art, (deposit number is CGMCC to the invention provides a kind of hybridoma cell strain No.9575), monoclonal antibody UMAB24 that and thus hybridoma cell strain produces.The present invention also carries Supply monoclonal antibody UMAB24 preparation answering in the immune detection instrument detecting S100P albumen With, the immunohistochemical kit containing monoclonal antibody UMAB24, and monoclonal antibody UMAB24 In preparation application in the test kit of marked tumor.Monoclonal antibody of the present invention can be with S100P Protein-specific combine, and with other nearly 10000 kinds of other albumen no cross reactions on chip, significantly carry The specificity of high S100P protein immunization detection and reliability, be widely used in the mark for various tumors Note.

Preservation information

Classification And Nomenclature for the hybridoma cell strain UMAB24 of preservation is: S100P mouse monoclonal antibody Hybridoma cell strain；

Depositary institution's full name: China Committee for Culture Collection of Microorganisms's common micro-organisms center；

Depositary institution is called for short: CGMCC；

Depositary institution address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences's microorganism is ground Study carefully institute；

Preservation date: on August 22nd, 2014；

Deposit number: CGMCC No.9575.

Accompanying drawing explanation

In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to reality Execute the required accompanying drawing used in example or description of the prior art to be briefly described.

Fig. 1 shows embodiment 1 cloning site design such as figure, and wherein shading part is ORF district；

Fig. 2 shows embodiment 2S100P albumen Western blot testing result figure, detects with anti-DDK The expression in HEK293T cell of the S100P albumen, wherein swimming lane L is the HEK293T of transfection empty carrier Cell pyrolysis liquid be the testing result of antigen, swimming lane R be the HEK293T of transfection pCMV6-S100P plasmid The testing result of cell pyrolysis liquid antigen；

Fig. 3 shows embodiment 2S100P protein SDS-PAGE result figure；

Fig. 4 shows that (one resists for S100P monoclonal antibody embodiment 4 Bladder Cancer ImmunohistochemistryResults Results figure UMAB24)；

Fig. 5 shows that (one resists for S100P monoclonal antibody embodiment 4 human placenta ImmunohistochemistryResults Results figure UMAB24)；

Fig. 6 shows that (one resists for S100P monoclonal antibody embodiment 5OriGene protein chip qualification result figure UMAB24,1:100；Two resist donkey anti-Mus IgG, 1:500 for Alexa 647-labelling).

Detailed description of the invention

Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete Describe, it is clear that described embodiment is only a part of embodiment of the present invention rather than all wholely Embodiment.Based on the embodiment in the present invention, those of ordinary skill in the art are not making creativeness The every other embodiment obtained under work premise, broadly falls into the scope of protection of the invention.

Embodiment 1, the structure of S100P recombinant expression plasmid

With the cDNA clone plasmid SC116407 of Origene company S100P as template, design two and draw Thing also introduces restriction enzyme site SgfI and MluI respectively, is cloned into expression vector pCMV6-Entry, sets up S100P recombinant expression plasmid RC201533.Cloning site designs as shown in Figure 1.

Embodiment 2, the expression and purification of S100P recombiant protein

1, transfection HEK293T cell: HEK293T cell reaches with 1:3 and continues in culture dish to cultivate； Take 7.5mLDMEM (serum-free and antibiotic) in 50mL pipe, add 300 μ LPEI MegaTran 1.0 mixing；Add 75 μ g S100P recombinant plasmid dnas in mixing liquid, mix and stand 30 minutes； Take respectively 515 μ L in each culture dish in 37 DEG C of 5%CO₂Incubator is cultivated.After transfecting 24 hours, Every ware cell adds 25 μ L2M sodium butyrates to final concentration 5mM.

2, cell lysis: after transfecting 48 hours, carry out cell cracking.Suck culture medium, add 1mLPBS Rinse, suck PBS.Add 1mL lysis buffer, before using, add protease inhibitor PI And PMSF.Being placed in ice chest and vibrate on shaking table, collect and obtain lysate in all culture dishs, 4 degree are centrifuged, Collect supernatant.

3, DDK affinity chromatograph column purification: with 0.45 μM, after 33mm pvdf membrane filter filter centrifugation Lysate supernatant and proceed to 15mL pipe, add the Beads 1mL that mixes, after sealing, put into 360 degree In vortex mixer, combine 2 hours in 4 DEG C；Take out 15mL pipe, lysate is poured into BIO-RAD chromatography In post, and catch and penetrate liquid, penetrate liquid sampling WB detection after dripping to the greatest extent, see Fig. 2；Rush with lysis buffer Wash post material 1-2 time, rinse Beads 3 times with TBST again after dripping to the greatest extent, with 0.1M Glycine pH3.5 after dripping to the greatest extent Eluting, for the first time 200 μ L, drip and do not collect to the greatest extent, and second and third each 500 μ L, 250 μ L for the third time receive Collect to a 1.5mL Tube, and be rapidly added NaH₂PO₄(pH=11.0) pH7.0 it is neutralized to left The right side, often pipe adds glycerol extremely final concentration of 10%, Tween-80 to final concentration of 0.1%.After purification S100P albumen SDS-PAGE identifies, sees Fig. 3.

From Fig. 2 result, tag antibody anti-DDK can detect transfection pCMV6-S100P plasmid HEK293T cell pyrolysis liquid in S100P albumen (R), and band is single special, molecular size range It is consistent with expection, shows that S100P albumen is correctly expressed.

From Fig. 3 result, through DDK affinity chromatograph column purification, Glycine eluting can get high-purity The S100P albumen of degree.

Embodiment 3, the preparation of S100P monoclonal antibody and screening

According to standard method with the S100P protein fragments of the purification of restructuring generation for B6/C57 mice (Beijing Vital River Experimental Animals Technology Co., Ltd.) carries out immunity.Concrete grammar is as follows:

1, animal immune: purified S100P antigen with complete Freund's adjuvant emulsifying, use subcutaneous or Lumbar injection method immunity 6-8 week old BALB/c mouse, immunizing dose is 50 μ g/, after being spaced two weeks Carrying out second time immunity, with incomplete Freund's adjuvant emulsifying, immunizing dose is 50 μ g/.After immunity twice Take tail blood and measure serum titer with ELISA method gradient dilution；Booster immunization is determined whether, choosing according to result Take the highest mice of antibody titer and carry out cell fusion.

2, cell merges: myeloma cell uses the sp2/0 that BALB/c originates, and is in logarithm raw during fusion For a long time；Take immune mouse spleen, make lymphocyte single cell suspension；Mouse spleen lymphocyte and bone Myeloma cells mixes with 1:5-1:10, drips 50%PEG (PH 8.0) 1mL of 37 DEG C, cannot add completely Full culture medium and remaining stop buffer, be centrifuged after abandoning supernatant and add the suspension mixing of HAT culture medium, MC constant volume To 50mL, it is dispensed in 3.5cm culture dish, is put in wet box, be placed in 37 DEG C, 5%CO₂Constant temperature is trained Support in case and cultivate.

3, screen and clone: in merging 7-10 days, selecting cell clone, using S100P purification of Recombinant egg Carry out ELISA test in vain.Labeled cell strain number.Positive porocyte is carried out limiting dilution, the most limited Within after dilution 5-6 days, measuring ELISA value, the monoclonal hole that picking OD280 positive value is higher carries out limited dilute Release, until ELISA measures 96 orifice plates and entirely hardens fruit for the positive.The picking positive is worth high monoclonal and determines strain. Its corresponding plate cell strain that merges is UMAB24.

4, the preparation of cell conditioned medium monoclonal antibody and purification: by cell strain UMAB24 with containing 15% serum DMEM culture medium culturing is cultivated in 10cm culture dish, spreads cultivation to about 4 × 10⁷Time individual, 800rpm from Heart 5min, abandons supernatant and is transferred to by cell in 2L rolling bottle, adds serum-free medium, makes cell close Degree is about 3 × 10⁵Individual/ml.After continuing to cultivate 1～2 week, (this when cell mortality reaches 60%-70% Time cell density be about 1-2 × 10⁶Individual/ml), collect cell suspension 6000rpm high speed centrifugation 20min, Taking supernatant, affinity chromatography carries out supernatant purification, selects corresponding post material, monoclonal antibody according to antibody die mould UMAB24 hypotype is IgG1, uses protein G to be purified.Monoclonal antibody concentration after purification measures, divides Fill, be saved in 4-8 DEG C.

Embodiment 4, monoclonal antibody UMAB24 are an anti-SABC detection

(1), experimental technique:

1, take Bladder Cancer block respectively and placenta tissue block carries out paraffin embedding, use Finesse tissue Microtome is cut into slices, and tissue thickness is 4 μm.

2, dewaxing and aquation: analytical pure dimethylbenzene 3 × 10min, dehydrated alcohol 3 × 10min, 95% second Alcohol 5min, 85% ethanol 5min, 75% ethanol 5min, deionized water soaks 3min × 3 time

3, antigen retrieval buffers (0.01M, pH6.0 sodium citrate buffer) pressure cooker high pressure hot repair is added Multiple 2min, when pressure cooker temperature is down to about 90 DEG C, opens pressure cooker, takes out specimen, the coldest But to room temperature.Deionized water soaks 3min × 3 time.

4, using 3% hydrogen peroxide deactivation tissue endogenous peroxydase, room temperature stands 10min.Go from Sub-water soaking 5min × 3 time.

5, plus confining liquid (PBS+5% defatted milk powder+5% Normal Goat Serum), 60min is hatched for 37 DEG C.

6, remove confining liquid, do not rinse, addition S100P monoclonal antibody (UMAB24), thinner ratio: 1:150, Use confining liquid is diluted.It is placed in wet box, hatches 60min for 37 DEG C.PBST (0.1%Tween-20) Wash 2 times, wash 5min every time.PBST (0.02%Tween-20) washs 1 time, washs 5min every time.

7, dropping Polink-test kit 2 (Catlog No.D37-15) reagent 1,37 DEG C hatches 10-20 Minute.PBS is used to wash 3 times, each 5min.Dropping Polink-2 test kit (Catlog No.D37-15) Reagent 2, hatches 10-20 minute for 37 DEG C, uses PBS to wash 3 times, each 5min.

8, application DAB solution (Zhong Shan Golden Bridge ZLI-9019) colour developing, colour developing 3～10min.Distilled water Washing.

9, haematoxylin redyeing nucleus 2min, distilled water rinses, 1% hydrochloric acid differentiation.Distilled water rinsing 3 Secondary, room temperature stands 1min.

10, dehydration and transparent: 75% ethanol 5min, 100% ethanol 5min x 3 times, 85% ethanol 5min, 95% ethanol 5min, 100% ethanol 3 × 5min；Dimethylbenzene 3 × 5min, neutral gum mounting.

11, microscopy, is shown in Figure 4 and 5.

(2), experimental result:

From Fig. 4 result, S100P albumen has expression in Bladder Cancer, but at normal bladder tissue In not do not express.From Fig. 5 result, S100P albumen normal expression in placenta tissue.Show Dan Ke Grand antibody UMAB24 is capable of identify that the S100P albumen in Bladder Cancer, placenta tissue.

Embodiment 5, the specific detection of monoclonal antibody UMAB24

10,000 HEK293T cell protein process LAN cracking are comprised on OriGene high-density protein chip Thing, every kind of protein lysate has the repetition of two copies on chip.Protein lysate by trace at nitre On acid cellulose film.The location of each clock protein lysate can be accurately positioned by Excel file. On protein chip, albumen is divided into 40 sub-matrixes, and each sub-matrix has some references, by referring to, can With the content of albumen on quantitative each chip point, monitor the repeatability of each immunoreation data, Yi Jiding The direction of position positive signal.Below for using OriGene albumen (OriGene Cat PA100001) chip Carry out the experimental technique of UMAB24 Identification of the antibodies experiment:

1, a protein chip is placed in 50mL centrifuge tube, uses 40mL deionized water infiltration core Sheet, is placed on shaking table, mixed at room temperature 30 minutes.Discard deionized water, use 10mLPBST balance Chip.Room temperature treatment 10 minutes.

2, in 50mL centrifuge tube, add 40mL5% skim milk (being diluted with PBST) to be placed in On shaking table, room temperature is closed 30 minutes.

3, using confining liquid (5% skim milk) to dilute an anti-UMAB24, thinner ratio is classified as 1: 100。

4, pasting in laboratory table by clean sealed membrane, dropping 250-300 μ L mono-resists on sealed membrane.

5, protein chip is extracted out from confining liquid, face down the one of protein chip NC film, from chip While contact antibody, slowly slide, rely on surface tension of liquid, antibody will slowly infiltrate chip NC Film, until whole NC film infiltrates in an anti-solution.Whole operating process is avoided producing bubble.By chip Move on under 4 DEG C of environment, stand, an anti-overnight incubation.Chip is added a cover culture dish lid, it sticks one Open wet paper towel, cause antibody to evaporate to prevent from hatching for a long time.

6, chip was moved in 50mL centrifuge tube in second day, use PBST rinsing chip twice, remove Unnecessary antibody.Use 40mL PBST (0.1%Tween-20) washing chip, be placed on shaking table mixing Uniformly, wash three times, wash 5min every time.

7, confining liquid (5% skim milk) is used to dilute the donkey anti-Mus IgG of two anti-Alexa 647-labellings, dilute The ratio of releasing is 1:500.

8, according to above-mentioned steps 4, step 5 carries out two and anti-hatches operation.Incubated at room 1 hour.At core Hide with aluminium-foil paper above sheet, to prevent signal bleaching.

9, according to above-mentioned steps 6, PBST is used to wash chip.

10, deionized water rinsing chip is used, to remove remaining salinity and denaturant.

11, drying at room temperature chip, it is ensured that chip is completely dried.

12, chip scanner is used to read fluorescence signal.

13, the site of chip direction and positive signal is determined according to BSA-Cy3 and BSA-Cy5.

14, corresponding protein lysate ID is found out according to positive signal site, according to lysate database information, Finding corresponding protein name, NCBI typing number (accession number), protein I D, albumen is big The information such as little.Result is shown in Fig. 6.

Fig. 6 result shows, only has S100P albumen and be combined with antibody UMAB24 on protein chip, table Bright antibody UMAB24 of the present invention can be combined with S100P protein-specific.

Claims (7)

1. a monoclonal antibody UMAB24 of specific binding S100P albumen, deposit number be The hybridoma cell strain of CGMCC No.9575 produces.

2. a hybridoma cell strain, its deposit number is CGMCC No.9575.

3. monoclonal antibody as claimed in claim 1 is examined for the immunity detecting S100P albumen in preparation Application in survey instrument.

Application the most according to claim 3, described immune detection instrument is test kit, chip or examination Paper.

5. an immunologic combined detection reagent kit, including the monoclonal antibody described in claim 1.

6. monoclonal antibody as claimed in claim 1 is preparation answering in the test kit of marked tumor With.

Application the most according to claim 6, described tumor be non-small cell carcinoma, gastric cancer, cancer of pancreas, Breast carcinoma, carcinoma of prostate, colorectal cancer, ovarian cancer, cervical cancer or bladder cancer.