CN104894073A - Hybridoma cell strain Zj/2D8 and application of hybridoma cell strain Zj/2D8 in detection of niacinamide-N-methylase antigen - Google Patents

Abstract

The invention discloses a hybridoma cell strain Zj/2D8 and application of the hybridoma cell strain Zj/2D8 in detection of a niacinamide-N-methylase antigen. The hybridoma cell strain Zj/2D8 is preserved in a China center for type culture collection (CCTCC) on October 22, 2013; the preservation number is CCTCC NO: C2013149; the preservation address is Wuhan university in Wuhan, China; the postal code is 430072. The hybridoma cell strain Zj/2D8 can efficiently secrete NNMT monoclonal antibody proteins, has very high antibody valence, good avidity and high purity for the NNMT antigen, can be applied to clinical test, can carry out immune histochemistry on pathological sections of different tissues, and detects the expression conditions of NNMT proteins in the tissues.

Description

The application of hybridoma cell strain Zj/2D8 and detection NNMT antigen thereof

(1) technical field

The present invention relates to a kind of protein immunization detection method, be specifically related to a kind of purposes that can resist the monoclonal antibody of NNMT albumen, the cell strain secreting this monoclonal antibody, preparation method and this monoclonal antibody.

(2) background technology

Malignant tumour is the disease of a class serious harm human health.According to World Health Organization's latest data display, annual global cancer new cases more than 1,000 ten thousand, dead more than 700 ten thousand, account for 12% of total death toll.Before the year two thousand twenty, will increase 1,500 ten thousand cancer patientss newly every year, moreover, the death toll of cancer also rises rapidly in the whole world, and the year two thousand thirty, this numeral may increase to 13,200,000.

The early diagnosis of tumour is the key for the treatment of and prevention of tumour, by stages closely related when the death of tumour and the danger of recurrence and first visit, and the susceptibility of chemotherapy, radiotherapy depends on enzyme in cancer cells or protein molecular to a certain extent.Biomarker is the signal of interest indication molecule representing this cytobiology state under cell is in specified conditions.In order to reduce the mortality ratio of tumour or improve the effect for the treatment of, discovery and the screening of tumour key organism standard substance are devoted in much research.Therefore, early diagnosis of tumor is sought, by stages or/and the tumor markers of instruction chemotherapy, the susceptibility of radiotherapy, prognosis and potential therapy target is the study hotspot of oncology always.

NNMT (NNMT, EC2.1.1.1) normal expression is in human liver organization, and its hetero-organization such as intestines, lung, kidney, placenta, heart, skeletal muscle, brain, pancreas are expressed very low or do not express.NNMT activity difference in the liver of different people is larger, active height depends on the concentration of NNMT in endochylema, the concentration of NNMT is relevant with the mRNA level in-site of NNMT, have nothing to do with gene polynorphisms, it has been generally acknowledged that the mechanism of NNMT process LAN is owing to regulating and controlling caused by abnormal after gene translation, may the activation such as hepatocyte neclear factor-1 (HNF-1), TGF-β 1, STAT3 be subject to.

The major function of NNMT is for donor with S-adenosine-L methionine(Met) (SAM), methylating of catalysis nicotinamide and pyridines material, form pyridinium salt, in normal liver cell, participate in the bio-transformation of medicine, be a kind of enzyme of catalysis medicine, xenobiotic metabolism.NNMT is the key enzyme maintaining nicotinamide balance, so can infer that increasing of NNMT can affect the function that nicotinamide participates in cell.Nicotinamide is the integral part of NAD molecule, produces NAD by salvage route, and the much basic of NAD participation cell may as ability metabolism, the corresponding opposing with damaging, the life-span etc. of cell.It is relevant that parkinsonism is considered to increase the nicotinamide metabolic disturbance caused with NNMT, in cerebral tissue, this enzymic activity excessively increases and causes parkinsonism by two approach, one is generate neurotoxic substance N-picoline (as N-methyl-4 phenylpyridine (MPP+)) activation to cause Mitochondria complex 1 impaired, and two are reduction of nicotinamide causes energy metabolism impairment.Nicotinamide can also suppress poly ADP polysaccharase (PARP), and PRAP regulates the integrity of maintainer gene group by many cells such as base excision repair, necrosis, apoptosis.Therefore have reason to infer that NNMT may participate in the multiple biological function of tumour cell, with the generation of tumour with develop relevant, maybe can impact, radiotherapeutic effect.If the effect of clear and definite NNMT in tumour cell, and illustrate the mechanism of action of NNMT in tumour or analyze module or the path of its downstream associated molecule composition, will further clear and definite NNMT as the value in treatment and prevention of tumour.

In recent years, give financial aid to students with albumen and biochip technology compares different carcinoma tissue and cancer beside organism's difference time discovery NNMT unconventionality expression in multiple cancerous tissue.Compare with cancer beside organism with healthy tissues, NNMT is woven with process LAN in tumor group such as colorectal cancer, lung cancer, bladder cancer, cancer of the stomach, thyroid papillary carcinoma, clear cell carcinoma of kidney, oral squamous cell carcinoma, mammary cancer and pancreas, and raises in the serum of kinds of tumors patient.These research prompting NNMT and tumours occur closely related, and supposition NNMT may participate in the multiple biological function of tumour cell, with the generation of tumour with develop relevant, and maybe can impact, radiotherapeutic effect.

(3) summary of the invention

The present invention adopts NNMT albumen to prepare the cell strain of the anti-NNMT monoclonal antibody of energy stably excreting as immunogen immune mouse, then screened by the indirect elisa method of NNMT albumen and be further purified the clone obtaining and can secrete NNMT antibody and the monoclonal antibody extracted by affiliated clone, therefore the object of the invention is to provide the hybridoma cell strain of a strain efficient secretion anti-NNMT (NNMT) antibody, this cell strain is utilized to extract NNMT antibody, NNMT antibody is adopted to detect the NNMT antigen in tissue to be measured, and then determine the content of NNMT antigen in tissue to be measured, contribute to clinical diagnosis.

The technical solution used in the present invention is:

The invention provides a strain of hybridoma strain Zj/2D8, be preserved in China typical culture collection center, preservation date is on October 22nd, 2013, and deposit number is CCTCC NO:C2013149, and preservation address is Wuhan, China, Wuhan University, postcode: 430072.

The invention still further relates to a kind of described hybridoma cell strain Zj/2D8 and detect the application in NNMT (NNMT) antigen, the NNMT antibody in hybridoma cell strain Zj/2D8 is extracted in concrete described application, again according to antigen and antibody specific combination principle, utilize NNMT antigenic content in NNMT antibody test testing sample.

The extracting method of NNMT antibody of the present invention is: hybridoma cell strain Zj/2D8 is used the RPMI-1640 containing 10% foetal calf serum by (1), at 37 DEG C, containing 5%CO ₂cell culture incubator in enlarged culturing, collecting cell, is mixed with 0.4 × 10 with PBS (pH value is 7.4) ⁶the enchylema of/ml, then by the BALB/c mouse (being not limited to BALB/c mouse, mouse model well known in the art) of enchylema abdominal injection paraffin sensitization, often only inject 1ml, inoculate latter 9 days results ascites, 1200 revs/min centrifugal, removing precipitation, collects supernatant liquor;

(2) add pH 5.0, the 0.06mol/L sodium-acetate buffer of 2 times of volumes in the supernatant liquor collected to step (1), then adjust pH to 4.8 with 1mol/L HCl, make dilution supernatant liquor; Then add the sad ratio of 11 μ l in every milliliter of dilution supernatant liquor, it is sad dropwise to add in dilution supernatant liquor under stirring at room temperature, adds in 30 minutes, and 4 DEG C leave standstill 2 hours, and take out, centrifugal 30 minutes of 15000g, abandons precipitation; Supernatant is after 125 μm of nylon mesh are filtered, and filtrate adds the 0.01mol/L PBS of 1/10 volume, adjusts pH to 7.2 with 1mol/L NaOH; Ammonium sulfate to 45% saturation ratio (described 45% saturation ratio refers to mass concentration) is added at 4 DEG C, stir 30 minutes, leave standstill 1 hour, centrifugal 30 minutes of 10000g, abandon supernatant, precipitate with the PBS containing 137mmol/L NaCl+2.6mol/LKCl+0.2mmol/L EDTA for dialyzate is dialysed, 4 DEG C are spent the night, until use 10g/L BaCl ₂aqueous assay trapped fluid is without SO ₄ion, take out trapped fluid, centrifugal 30 minutes of 10000g, remove insoluble sediment, supernatant is NNMT antibody.

The mono-clonal NNMT antibody that hybridoma Zj/2D8 provided by the invention produces all has high-caliber antibody titer for NNMT antigen, therefore the active fragments of mono-clonal NNMT antibody, its active fragments or its conservative mutation body and mark or its conservative mutation body, or its arbitrary combination all can be used for preparing the reagent, medicament or the test kit that detect NNMT antigen.The reagent, medicament or the test kit that detect NNMT antigen prepared of the present invention preferably adopts double-antibody method to detect NNMT antigen.

For active fragments or its conservative mutation body of mark of the present invention, refer to biomarker well known in the art or chemical labeling.Such as enzyme labelling, fluorescent mark (such as fluorescein-labelled) or chemiluminescent labeling.Preferably, described enzyme labelling is horseradish peroxidase, pyruvate kinase or glucose oxidase enzyme labelling.

Monoclonal antibody of the present invention is preferably humanized monoclonal antibody, and (humanized antibody mainly refers to that mouse resource monoclonal antibody is with gene clone and the transformation of DNA recombinant technology, the antibody of again expressing, its Most amino-acids sequence behaviour source sequence replaces, the avidity of basic reservation parent mouse monoclonal antibody and specificity, again reduce its heterology).

Compared with prior art, beneficial effect of the present invention is mainly reflected in:

(1) monoclonal hybridoma strain Zj/2D8 provided by the invention can efficient secretion (referring to that in 1.3 of embodiment 1 nutrient solution supernatant dilutes 1000 times and detects emiocytosis afterwards to the antibody in supernatant liquor also for positive) NNMT monoclonal antibody protein.

(2) the NNMT monoclonal antibody that the present invention extracts from monoclonal hybridoma strain Zj/2D8 has very high (1:1000000) antibody titer to NNMT antigen, good avidity and higher purity, point out this monoclonal antibody to can be applicable to detect the scientific research field of NNMT antigen.

(2) the NNMT monoclonal antibody extracted from monoclonal hybridoma strain Zj/2D8 of the present invention can be applicable to clinical detection, by carrying out immunohistochemical methods to the pathological section of different tissues, detects the expression of NNMT albumen in this tissue.

(4) accompanying drawing explanation

Fig. 1 is Zj/2D8,1E7 and 2F8 sterling antibody SDS-PAGE electrophorogram, M:Marker, 1:Zj/2D8 sterling antibody, 2:1E7 sterling antibody, 3:2F8 sterling antibody.

Fig. 2 is the subclass of Zj/2D8,1E7 and 2F8 cell strain secretory antibody, light chain type figure, A be Zj/2D8, B be 1E7, C are 2F8C.

Fig. 3 is Zj/2D8 sterling antigen gel scintigram.

Fig. 4 is the avidity response curve figure of Zj/2D8,2F8 and 1E7 cell strain sterling antibody.

Fig. 5 is the immunoblotting of Zj/2D8 cell strain monoclonal antibody, 1:GST albumen, 2:NNMT-GST fusion rotein, 3:NNMT albumen.

Fig. 6 is the immunoblotting of 10 synthesis small peptides.

Fig. 7 is the immunoblotting after colorectal cancer cell lines and positive cell strain HT-29 cell NNMT slow virus disturb.

Fig. 8 is the immunohistochemical methods (× 100) of transplanted tumor in nude mice HT-29 cell (A) and the rear HT-29 cell (B) of NNMT interference.

(5) embodiment

Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:

Percentage concentration of the present invention, if do not have special indicating, is all mass concentration, and solvent is all water.

Embodiment 1: the acquisition of hybridoma cell line, the screening of monoclonal antibody, preparation and qualification

1, the preparation of monoclonal antibody

The monoclonal antibody hybridoma cell strain Zj/2D8 of the anti-NNMT of the present invention is prepared according to ordinary method known in the art, main monoclonal antibody hybridoma cell strain Zj/2D8 (the common Kohler and MIlrtein preparing the anti-NNMT of humanization form with reference to the Principles and ways described in United States Patent (USP) 5585089, Nature 256:495-96,1975), following reagent will be mentioned in method:

(1) coating buffer: Na ₂cO ₃1.5g, NaHCO ₃2.9g, NaN ₃0.2g, is dissolved in 1L water, adjusts pH to 9.6.

(2) confining liquid: 1% bSA.

(3) washings: 0.5mlTween-20 is dissolved in 1000ml 1 × PBS.

(4) TMB nitrite ion (comprising substrate colour developing A liquid and B liquid), substrate colour developing A liquid: sodium-acetate 13.6g, citric acid 1.6g, 30% hydrogen peroxide 0.3ml, distilled water adds to 500ml; Substrate colour developing B liquid: disodium ethylene diamine tetraacetate 0.2g, citric acid 0.95g, glycerine 50ml, get 0.15g tetramethyl benzidine (TMB) and be dissolved in 3ml DMSO, distilled water adds to 500ml.Respectively get 50 μ l during use to mix.

(5) stop buffer: 2.0mol/L H ₂sO ₄the aqueous solution.

1.1, immunogenic preparation

Immunogen is GST-NNMT fusion rotein and NNMT albumen, utilizes genetic engineering technique to prepare NNMT albumen: be encoded to gi:12652950 people NNMT cDNA sequence according to NCBI RiboaptDB, the PCR primer of synthesis NNMT gene,

P1(sense):3′-CG'GGATCC'ATGGAATCAGGCTTCACCTCC-5′；

P2(antisense):3′-G'CTCGAG'AATTGAGGTCACAGGCATCACAG-5′。

In the 5' end of P1 and 3' end design BamHI and the XhoI site of P2.With liver c DNA for template (it is consistent that sequence and above-mentioned NCBI RiboaptDB are encoded to gi:12652950 people NNMT cDNA sequence), pcr amplification NNMT fragment is carried out under primer P1, P2 effect, reaction parameter is 94 DEG C of denaturation 2min, 94 DEG C of 20s, 60 DEG C of 30s, 72 DEG C of 45s.After 20 circulations, add 0.8 μ l Taq enzyme immediately, 72 DEG C extend 10min, 4 DEG C of preservations.PCR primer is after glue reclaims, T-A is cloned into PGEM-Teasy carrier, obtain pGEM-T-Easy-NNMT plasmid, plasmid is cut with BamHI and XhoI enzyme after sequence verification, reclaim DNA fragmentation, be connected to p GEX-4T-2 carrier, build pGEX-4T-2/NNMT plasmid, be transformed in intestinal bacteria BL-21-STAR (DE3) to express GST-NNMT fusion rotein, obtain recombination bacillus coli BL-21-STAR (DE3)-GST-NNMT, after 37 DEG C of cultivation 2h, 3h are induced at 30 DEG C with the IPTG of final concentration 0.1mM, 4 DEG C, centrifugal 10 minutes of 5000rpm, collect thalline, use scientz-II D ultrasonic cell disruptor, at 100 watts, ultrasonic 10 seconds, stop 10 seconds, ultrasonication thalline under the condition of 60 circulations, 13000rpm is after centrifugal 3 minutes, leave and take supernatant liquor, by supernatant liquor through Glutathione Sepharose 4B (purchased from American Pharmacia company) affinity chromatography column purification GST-NNMT albumen.Then in GST-NNMT albumen, add zymoplasm (every 1mg albumen adds 10U zymoplasm) makes its abundant enzyme cut, again through Glutathione Sepharose 4B (purchased from American Pharmacia company) affinity column, room temperature jog 30 minutes, GST albumen is fully combined with affinity column, collect effluent liquid, centrifugal 5 minutes of 500g, collect supernatant, only containing NNMT albumen in supernatant, obtain the NNMT albumen after purifying.Measure protein concentration by the red method of adjacent benzenetricarboxylic acid, 10%SDS-PAGE electrophoresis and Western-blot identify NNMT albumen, only containing NNMT albumen in supernatant liquor.

1.2, animal immune: immune programme for children is: initial immunity gets BALB/C female mice in 8 week age, after NNMT albumen and Freund's complete adjuvant equal-volume being mixed, at the subcutaneous multi-point injection of mouse back, dosage is every mouse 140 μ g.After 30 days, after again mixing with NNMT albumen and Freund's incomplete adjuvant equal-volume, at the subcutaneous multi-point injection of mouse back, dosage is every mouse 140 μ g.NNMT albumen and the immunity of Freund's incomplete adjuvant equal-volume homomixture is used afterwards more once, totally three times, as booster immunization every 10 days.Finally carry out first three sky of cytogamy, NNMT albumen and physiological saline equal-volume mix pneumoretroperitoneum and inject, and dosage is every mouse 80 μ g.Freund's incomplete adjuvant consists of whiteruss and lanolin mixes, and volume components is than being 2:1, and Freund's complete adjuvant consists of in Freund's incomplete adjuvant and adds bacille Calmette-Guerin vaccine (concentration 5mg/ml).

1.3, cytogamy: get immune BALB/C mouse spleen cell and SP2/0 myelomatosis (purchased from Chinese Academy of Sciences's cell bank) in proportion (5:1) merge, centrifugal 5 minutes of 1200rpm, abandons supernatant.At 37 DEG C, in 1 minute, add 50%PEG (0.8ml every 10 to cell precipitation ⁸splenocyte), leave standstill fusion 90 seconds, centrifugal 5 minutes of 1200rpm, abandons supernatant, adds 20ml HAT and selects nutrient solution (containing 1640 substratum 400ml in every 500ml HAT selection nutrient solution, foetal calf serum 100ml, 50 × HAT 10ml (sigma)), spread 96 porocyte culture plates, adopt HAT to select nutrient solution to cultivate hybridoma (normal cell cultures, 37 DEG C, 5%CO ₂cell cultures incubator in), select what survive to be namely hybridoma.BALB/C mice splenocyte and SP2/0 myeloma cell complete cytogamy, and cell confluency is 96.7%.Indirect elisa method within 7th day, is adopted to detect cell conditioned medium, to screen positive cell.Indirect elisa method is as follows: NNMT albumen coating buffer is diluted to 0.025 μ g/ml, every hole 100 μ l coated elisa plate, and GST-NNMT albumen coating buffer is diluted to 0.04 μ g/ml, and every hole 100 μ l coated elisa plate, 4 DEG C of placements are spent the night.Next day, washings washs 3 times, adds confining liquid about 350 μ l/ hole, 37 DEG C, 1 hour, pats dry after washings washing.Every hole adds hybridoma supematant 100 μ l, if the positive serum controls (serum of immunized mice, how anti-), negative serum control (serum of non-immune mouse), blank control wells (1 × PBS) is set simultaneously, 37 DEG C of reactions are after 2 hours, washings detersive enzyme target, add 100 μ l rabbit anti-mouse IG-HRP (Cell signaling Technology) (diluting with PBS1:4000), 37 DEG C are reacted detersive enzyme target after 1 hour, add TMB nitrite ion, 37 DEG C are developed the color 15 minutes, add stop buffer termination reaction, spectrophotometer (BIO RAD, Model 680, Japan) OD is read on ₄₅₀absorbancy.The primary dcreening operation positive rate of fused cell is 62.4%.Anti-NNMT titre high (reaching 1:1000) is selected and the hybridoma cell strain 21 of anti-GST-NNMT titre very low (lower than 1:10) is routine in about 100 routine primary dcreening operation positives, further cloning is (by the cell picking mono-clonal in a positive hole to enlarged culturing in 96 orifice plates, carry out above-mentioned qualification again, until the cell in this hole pick out complete in positive, be monoclonal antibody strain) until positive rate reaches 100%, thus screen monoclonal antibody hybridoma cell strain Zj/2D8, hybridoma cell strain 1E7 and hybridoma cell strain 2F8, hybridoma cell strain 1E7 is preserved in China typical culture collection center, preservation date on August 31st, 2011, deposit number CCTCC NO:C201167, preservation address is Wuhan, China Wuhan University, postcode 430072, hybridoma cell strain 2F8 is preserved in China typical culture collection center, preservation date on January 15th, 2015, and deposit number CCTCC NO:C201506, preservation address is Wuhan, China Wuhan University, postcode 430072.

As a result, the OD value of positive serum controls is 2.192, and the OD value of negative serum control is 0.101, blank well OD value is 0.097, and through screening, the OD value corresponding to object cell strain obtained is as shown in table 1.

Table 1 cell strain of monoclonal antibody screening cell conditioned medium OD value

The 3 strain of hybridoma strains filtered out thus all can secrete the antibody of anti-NNMT albumen and OD value is greater than 1.4.And the OD value of anti-GST-NNMT is all very low, almost consistent with the OD value of blank well.Wherein the anti-NNMT albumen OD value of hybridoma cell strain Zj/2D8 is apparently higher than hybridoma cell strain 1E7 and hybridoma cell strain 2F8, and the OD value of anti-GST-NNMT is lower.

1.4, ascites preparation: by hybridoma cell strain Zj/2D8 enlarged culturing (normal cell cultures, 37 DEG C, 5%CO ₂cell cultures incubator in), be mixed with 0.4 × 10 with PBS after collecting cell ⁶/ ml enchylema, by the BALB/c mouse of enchylema abdominal injection paraffin sensitization (paraffin sensitization refer to abdominal injection 1ml paraffin/only), often only injects 1ml, inoculate latter 9 days results ascites, 1200 revs/min centrifugal, removing precipitation, collect supernatant liquor, be upper strata monoclonal antibody ascites.

1.5, antibody purification: add 2 parts of sodium-acetate buffers (0.06mol/L, pH 5.0) in 1 part of supernatant liquor (prepared by step 1.4), adjusts pH to 4.8 with 1mol/L HCl, makes dilution supernatant liquor.Add the sad ratio of 11 μ l in every milliliter of dilution supernatant liquor, it is sad dropwise to add in dilution supernatant liquor under stirring at room temperature, adds in 30 minutes, and 4 DEG C leave standstill 2 hours, take out 15000g centrifugal 30 minutes, abandon precipitation; Supernatant filters after (125 μm) through nylon mesh, and filtrate adds the 0.01mol/L PBS of 1/10 volume, adjusts pH to 7.2 with 1mol/L NaOH; At 4 DEG C, add ammonium sulfate to 45% saturation ratio, stir 30 minutes, leave standstill 1 hour; Centrifugal 30 minutes of 10000g, abandons supernatant; Precipitate with the PBS containing 137mmol/L NaCl+2.6mol/L KCl+0.2mmol/L EDTA for dialyzate (molecular weight cut-off of dialysis tubing is for 14000), dialyse with the dialyzate of 50-100 times of precipitation volume, 4 DEG C are spent the night, change dialyzate therebetween more than 3 times, until with the BaCl of 10g/L ₂aqueous assay trapped fluid is without SO ₄ion.Take out trapped fluid, centrifugal 30 minutes of 10000g, removes insoluble sediment, and supernatant is the NNMT monoclonal antibody of purifying, after determined by ultraviolet spectrophotometry protein content, adds 0.02%NaN ₃packing saves backup.Ascites crude product is through 4% sad extraction and 50% ammonium sulfate precipitation, and main foreign protein, as albumin etc. is almost all removed.Assorted band after SDS-PAGE electrophoresis showed (Fig. 1) purifying except the heavy chain (50KD) of antibody and light chain (25KD) band is less, shows that purification effect is better.

1.6, the qualification of antibody

1.6.1, the subclass of antibody of hybridoma secretion and light chain type, the IsoQuick Kits for Mouse Monoclonal Isotyping of sigma-aldrich company is used to detect: (antibody or the Zj/2D8 of this experiment preparation hereinafter mentioned all refer to 1., 5 purification gained) 1.5 purification gained Zj/2D8 secrete NNMT antibody, for IgG2a κ class (in Fig. 2 A), identical with 1E7 hybridoma cell strain type (in Fig. 2 B), but be IgG2b κ class different (in Fig. 2 C) from the antibody that 2F8 secretes.

1.6.2, the purity of sterling antibody and molecular weight: 1.5 purification gained NNMT antibody are after SDS-PAGE electrophoresis and gel image scanning are analyzed, and antibody purity is 91.4% (Fig. 3).Light chain molecule amount is about 25KD, and heavy chain molecule amount is about 50KD, conforms to the characteristic of antibody molecule.

1.6.3, sterling antibody titer and avidity analysis: 1.5 purification gained NNMT antibody, 1E7,2F8 cell strain sterling antibody are after indirect ELISA is analyzed, antibody titer is all more than 100000, but the NNMT antibody that ZJ/2D8 is separated is higher than the antibody that 1E7 with 2F8 (as table 2 and Fig. 4) is separated.Concrete grammar is as follows: NNMT albumen coating buffer is diluted to bag enzyme plate after 0.025 μ g/ml, 4 DEG C are spent the night.Add confining liquid in enzyme plate, close after 1 hour for 37 DEG C, washings washs.After washing pats dry, add NNMT antibody, the antibody of 1E7 separation, the antibody (10mg/ml) of 2F8 separation that Zj/2D8 is separated respectively, by 1:100 ~ 1:10 ⁷10 times of ratios (table 2) and 2 times of ratio (Fig. 4) amount of dilution hand-holes, 37 DEG C washing 2 hours after, add 100 μ l bis-anti-rabbit against murine HRP-IgG and wash after 1 hour.Last TMB nitrite ion develops the color 15 minutes, adds stop buffer termination reaction, measures the OD value (table 2) of wavelength 450nm.Show purified after, tiring of antibody is increased to some extent, and maintains the immunocompetence of antibody.

The monoclonal antibody that table 2 Zj/2D8,1E7 and 2F8 extract is to NNMT albumen indirect method antibody titer result

Result is with the logarithm of antibody concentration for X-coordinate, and OD value is ordinate zou, draws the mensuration curve (as Fig. 4) of Affinity to MoAbs.Antibody concentration (unit μ g/ml) when Zj/2D8 cell strain reaches maximum combined 50% is 0.11 μ g/ml, all lower than the 0.22 μ g/ml of 0.34 μ g/ml and 2F8 of 1E7, show that the relative affinity of antibody secreted by Zj/2D8 cell strain is all higher than 1E7 and 2F8 strain, its avidity can be satisfied with all kinds of detection reagent of production and test kit.

1.7, antibodies specific qualification (adopting Western-blot)

1.7.1, method

A. polyacrylamide gel electrophoresis (SDS-PAGE)

(1) by table preparation polyacrylamide 15% separation gel.Final step adds TEMED, after mixing, separation gel is injected ready glass gels mould carefully, will stay the space of 1.5-2cm above for concentrating glue.To add water covering at top layer, make interface smooth, and prevent the oxygen in air from playing restraining effect to gel polymerisation, ambient temperatare puts about 30 minutes, makes it polymerization.After being polymerized, the water inclined on separation gel, blots the remaining liquid on gel top as far as possible with thieving paper.

(2) concentrate glue by table preparation 5%, add glass gels mould after mixing, plug comb, ambient temperatare puts 30 minutes.Gel electrophoresis plate is put into electrophoresis chamber, is immersed in 1 × Tris glycine running buffer, carefully removes comb.

(3) sample is prepared: get sample-loading buffer 8 μ l, 1M DTT 2 μ l, protein sample 10 μ l, be placed in the centrifuge tube of 0.5ml, boiling water bath is placed on ice after 10 minutes at once.

(4) loading: each swimming lane uses micro sample adding appliance application of sample 10 μ l respectively.

(5) switch on power, 35mA electrophoresis 10 ~ 15 minutes, makes the quick swimming of protein to separation gel upper strata and is condensed into an arrowband; Voltage changes 25mA into, continues electrophoresis about 1.5 hours, until tracer dye is close to the bottom of gel.

B. transferring film and development

(1) get 4 with running gel filter paper of the same size and a polyvinylidene fluoride filter membrane (PVDF, Polyvinylidene-Fluoride), with methyl alcohol soak 15 minutes transparent to filter membrane.

(2) two layers of filter paper is placed on fixing transfer device, the polyacrylamide gel that own electrophoresis is good is placed on filter paper, then PVDF filter membrane is placed on polyacrylamide gel, PVDF filter membrane covers two layers of filter paper again, each step all to be rushed bubble, then stationary installation with glass stick.

(3) whole device is placed in electrophoresis chamber, note making gel coat be positioned at negative pole, filter membranous layer is positioned at positive pole, adds the transfering buffering liquid of precooling in electrophoretic blotting groove.

(4) switch on power, 250mA transferase 12 hour.

(5) close: rocked at room temperature 60 minutes in confining liquid.

(6) add primary antibodie: primary antibodie (anti-NNMT monoclonal antibody) confining liquid dilutes, join the rear primary antibodie 2ml (volume ratio 1:12000) of dilution, put into by film wherein, 4 DEG C of rotations are spent the night.

(7) film is embathed 3 times by confining liquid room temperature, each 10 minutes.

(8) add two to resist: by two anti-rabbit anti-mouse (IgG-HRP) (Cell signaling Technology), dilute with confining liquid 1:5000, join the rear two anti-2ml of dilution, film is put into wherein, rocked at room temperature 2 hours.

(9) band is embathed 3 times, each 10 minutes with embathing liquid chamber temperature.

(10) develop after adding ECL developing solution.

Result identifies monoclonal antibody through Western-blot, on PVDF (Polyvinylidene-Fluoride) film, only have NNMT-GST fusion rotein (55KD) and the corresponding molecular weight region of NNMT albumen (29KD) swimming lane to occur an obvious specific band, and do not occur respective strap (as shown in Figure 5) at GST albumen (26KD) swimming lane.

1.8, antibody antigen Epitope Identification

1.8.1, method

The synthesis of a.NNMT small peptide: according to NNMT protein amino acid sequence (mesgftskdt ylshfnprdy lekyykfgsr hsaesqilkh llknlfkifc ldgvkgdlli digsgptiyq llsacesfke ivvtdysdqn lqelekwlkk epeafdwspv vtyvcdlegn rvkgpekeek lrqavkqvlk cdvtqsqplg avplppadcv lstlcldaac pdlptycral rnlgsllkpg gflvimdalk ssyymigeqk fsslplgrea veaavkeagy tiewfevisq sysstmanne glfslvarkl srpl) and structural analysis, entrusts Zhongtai Bio-Chem. Co., Ltd., Hangzhou's design and chemosynthesis 10 small peptides (N1-N10) (table 3).

B.Western-blot and Elisa method qualification antibody recognition epitope: utilize Elisa method to carry out epitope qualification, using 10 small peptides of synthesis as envelope antigen, using as negative control, carry out according to indirect Elisa method (as 1.6.3), 3rd article of small peptide of result display Zj/2D8 group is positive, illustrate that the epitope that Zj/2D8 identifies is N3 (SGPTIYQLLSACESFKEIVVTD), and 1E7 and 2F8 is all corresponding with it without response small peptide.Utilize Western-blot to verify simultaneously, after these small peptides are carried out SDS-PAGE electrophoresis, be transferred on NC film, use 3 strain monoclonal antibodies as primary antibodie respectively, sheep anti mouse HRP-IgG resists as two, through DAB colour developing, and result consistent with Elisa experimental result (Fig. 6).Because Zj/2D8 has clear and definite antigen recognition epi-position, and 1E7 and 2F8 does not also have clear and definite antigen recognition epi-position, so Zj/2D8 is more suitable for producing all kinds of detection reagent and test kit, for all kinds of detection and research.

10, table 3 synthesis short peptide sequence information

Title	Zero position	Sequence	Final position
				N1	8	KDTYLSH	14
N2	33	AESQILKHLLKNLFKIFCLDGVKGDLLID	61
				N3	64	SGPTIYQLLSACESFKEIVVTD	85
N4	106	DWSPVVTYVCD	116
				N5	181	RNLGSLLKPGGFLVIMD	179
N6	199	LKSSYYM	197
				N7	210	KFSSLPLGR	218
N8	220	AVEAAVKE	227
				N9	233	EWFEVISQS	241
N10	251	GLFSLVARK	259

1.10, the mensuration of antibody-secreting stability

By 3 strain of hybridoma of preparation frozen 6 months and recovery in 12 months, growth conditions is good, with indirect Elisa method qualification, and OD _450nmvalue is 1.3 ~ 2.0, shows that the ability of secretory antibody is all stable.

Embodiment 2: monoclonal antibody is applied to immunohistochemical methods and detects clinical samples (adopting two step indirect methods)

1, the anti-NNMT monoclonal antibody that this experiment is produced can be applied to the immunohistochemical experiment of clinical pathology sample.The NNMT monoclonal antibody (preparing gained after 1.5 purifying in embodiment 1) that we select hybridoma cell strain Zj/2D8 to be separated carries out immunohistochemical experiment to different tissues.First primary antibodie (specific antibody--NNMT monoclonal antibody) is combined with corresponding tissue antigen, form immune complex, be combined with two anti-(antibody of enzyme labelling) specific antibodies in mixture again, form Ag-Ab-hrp-antibody complex, finally use substrate chromogenic reagent.Concrete grammar is as follows:

(1) the HT-29 cell (purchased from Chinese Academy of Sciences's cell bank) after paraffin NNMT interference transplants nude mice, the tissue slice of transplanted tumor takes off cured to water (general term, paraffin is sloughed exactly with dimethylbenzene-raw spirit, then through raw spirit 70% alcohol, 50% alcohol is transitioned into water successively).

(2) 1 × PBS rinse, each 5 minutes, totally 3 times.

(3) 3% hydrogen peroxide methanol solution effects 20 minutes, block endogenous peroxidase activity.

(4) 1 × PBS rinse, each 5 minutes, totally 3 times.

(5) confining liquid closes 10 minutes.

(6) antigen retrieval is carried out with citrate buffer.

(7) often open section and drip the NNMT antibody of embodiment 1 preparation as primary antibodie (1 × PBS 1:16000 dilutes), 37 DEG C, 60 minutes or 4 DEG C of refrigerator overnight.

(8) 1 × PBS rinse, each 5 minutes, totally 3 times.

(9) two anti-(1:5000) of the rabbit anti-mouse that horseradish peroxidase (HRP) marks are dripped, 37 DEG C, 40 minutes.

(10) 1 × PBS rinse, each 5 minutes, totally 3 times.

(11) 100 μ l freshly prepared 3,3-diaminobenzidines (DAB) chromophoric solution (GENMEM company of the U.S.) are added, 10 minutes.

(12) tap water, Hematorylin is redyed, neutral gum sealing.

Similarity condition with HCE8693 cell in contrast.

2, the immunoblotting analysis of colorectal cancer cell lines

Wherein HCE8693 and the HT-29 cell display NNMT positive (Fig. 7), after HT-29 cell slow virus interference NNMT expresses, the NNMT positive weakens (Fig. 7).

3, the immunohistochemical methods of HT-29 Transplanted cells nude mice after HT-29 cell and NNMT interference

The nude mice of HT-29 Transplanted cells is obviously eager to excel (Fig. 8) than the nude mice positive of the HT-29 Transplanted cells after NNMT interference.

Embodiment 3: monoclonal antibody is applied to clinical serum Samples detection (employing double antibody sandwich method)

In embodiment 1, the NNMT monoclonal antibody of 1.5 final purifying can be applied to the test experience of NNMT antigen in clinical serum sample.We select Zj/2D8, and the monoclonal antibody that 1E7 with 2F8 is separated carries out NNMT antigen detection assay to the different concns sample diluted with serum dilution (the NNMT antigen standard (ENZ-418) purchased from Prospec company).First by monoclonal antibody bag by solid phase carrier, form insolubilized antibody, add serum specimen to be checked, form solid phase antigen antibody complex, add the NNMT antigen of monoclonal antibody linked with peroxidase in mixture to be again combined, form insolubilized antibody-determined antigen-enzyme labelled antibody sandwich complex, finally use substrate chromogenic reagent.Concrete grammar is as follows:

(1) bag quilt: be buffered liquid with bag and NNMT antibody (monoclonal antibody that Zj/2D8,1E7 and 2F8 are separated being tested respectively) is diluted to 10 μ g/mL, add 0.1ml in the reacting hole of each polystyrene board, 4 DEG C are spent the night.Next day, discard solution in hole, wash 3 times with lavation buffer solution, each 3 minutes.

(2) application of sample: add serum sample 0.1ml to be checked and wrapped in the reacting hole of quilt in above-mentioned, places 37 DEG C and hatches 1 hour.Then wash 3 times.

(3) add enzyme labelled antibody: in each reacting hole, add the NNMT antibody 0.1ml of the horseradish peroxidase-labeled of diluted fresh.Hatch 0.5 ~ 1 hour for 37 DEG C, wash 3 times.

(4) substrate solution colour developing is added: in each reacting hole, add tmb substrate solution 0.1ml, 37 DEG C, 15 minutes.

(5) termination reaction: add stop buffer 0.05ml in each reacting hole.

(6) OD value is measured with Elisa detector in 450nm place.

According to foreign literature report (Markus Roe β ler, Wolfgang Rollinger, Stefan Palme, et al.Identification of Nicotinamide N-Methyltransferase as a Novel Serum Tumor Marker for Colorectal Cancer, Clinical Cancer Research, 2005), use the NNMT antigenic content in Elisa method detection normal human serum, the lowest content is positioned at 169pg/ml, and Partial tumors patients serum concentration raises.So NNMT antigen standard is diluted to 0pg/ml, 50pg/ml, 100pg/ml, 200pg/ml, 500pg/ml, 1ng/ml, 5ng/ml by us, the concentration of 10ng/ml and 100ng/ml, detected result is as table 4.

Table 4 Zj/2D8,1E7 and 2F8 monoclonal antibody detects the OD value of different N NMT antigen concentration

From result, we see, the monoclonal antibody that Zj/2D8 is separated can detect 50pg/ml, and when 1E7 and 2F8 detects 200pg/ml, resolving power is not strong.So the monoclonal antibody that Zj/2D8 is separated is applicable to the concentration making NNMT in classes of agents and test kit detection human serum, detects NNMT antigen.

Claims (4)

1. a strain of hybridoma strain Zj/2D8, is preserved in China typical culture collection center, and preservation date is on October 22nd, 2013, and deposit number is CCTCC NO:C2013149, and preservation address is Wuhan, China, Wuhan University, postcode 430072.

2. hybridoma cell strain Zj/2D8 described in a claim 1 is detecting the application in NNMT antigen.

3. apply as claimed in claim 2, it is characterized in that the NNMT antibody in hybridoma cell strain Zj/2D8 is extracted in described application, then use NNMT antigenic content in NNMT antibody test testing sample.

4. apply as claimed in claim 3, it is characterized in that the extracting method of described NNMT antibody is: hybridoma cell strain Zj/2D8 is used the RPMI-1640 containing 10% foetal calf serum by (1), at 37 DEG C, containing 5%CO ₂cell culture incubator in enlarged culturing, collecting cell, is mixed with 0.4 × 10 with PBS ⁶the enchylema of/ml, then by the BALB/c mouse of enchylema abdominal injection paraffin sensitization, often only inject 1ml, inoculate latter 9 days results ascites, 1200 revs/min are centrifugal, and removing precipitation, collects supernatant liquor;

(2) add pH 5.0, the 0.06mol/L sodium-acetate buffer of 2 times of volumes in the supernatant liquor collected to step (1), then adjust pH to 4.8 with 1mol/L HCl, make dilution supernatant liquor; Then add the sad ratio of 11 μ l in every milliliter of dilution supernatant liquor, it is sad dropwise to add in dilution supernatant liquor under stirring at room temperature, adds in 30 minutes, and 4 DEG C leave standstill 2 hours, and take out, centrifugal 30 minutes of 15000g, abandons precipitation; Supernatant is after 125 μm of nylon mesh are filtered, and filtrate adds the 0.01mol/L PBS of 1/10 volume, adjusts pH to 7.2 with 1mol/L NaOH; At 4 DEG C, add ammonium sulfate to 45% saturation ratio, stir 30 minutes, leave standstill 1 hour, centrifugal 30 minutes of 10000g, abandons supernatant, precipitates with the PBS containing 137mmol/L NaCl+2.6mol/L KCl+0.2mmol/L EDTA for dialyzate is dialysed, 4 DEG C are spent the night, until use 10g/L BaCl ₂aqueous assay trapped fluid is without SO ₄ion, take out trapped fluid, centrifugal 30 minutes of 10000g, remove insoluble sediment, supernatant is NNMT antibody.