CN104894073B - Hybridoma cell strain Zj/2D8 and its application for detecting niacinamide N methylase antigens - Google Patents

Abstract

A kind of application the invention discloses hybridoma cell strain Zj/2D8 and in niacinamide N methylase antigens are detected, China typical culture collection center is preserved in, preservation date is on October 22nd, 2013, and deposit number is CCTCC NO：C2013149, preservation address are Wuhan, China, Wuhan University, postcode 430072；Hybridoma cell strain Zj/2D8 provided by the invention being capable of efficient secretion NNMT monoclonal antibody proteins, there is very high antibody titer to NNMT antigens, preferable affinity and higher purity, it can be applied to clinical detection, by carrying out SABC to the pathological section of different tissues, the expression of NNMT albumen in the tissue is detected.

Description

Hybridoma cell strain Zj/2D8 and its detection NNMT antigen Using

(1) technical field

The present invention relates to a kind of protein immunization detection method, and in particular to one kind can resist niacinamide-N- to methylate The monoclonal antibody of zymoprotein, the cell line for secreting the monoclonal antibody, the purposes of preparation method and the monoclonal antibody.

(2) background technology

Malignant tumour is a kind of disease for seriously endangering human health.Shown according to World Health Organization's latest data, every year Global cancer new cases more than 1,000 ten thousand, more than dead 700 ten thousand, account for the 12% of total death toll., every year will be newly-increased to before the year two thousand twenty 15000000 cancer patients, moreover, the death toll of cancer is also in global rapid rising, 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, when the death and the danger of recurrence and first visit of tumour by stages It is closely related, and the enzyme or protein molecular that chemotherapy, the sensitiveness of radiotherapy are depended in cancer cell to a certain extent.Biomarker It is that cell is under specified conditions the signal of interest indication molecule for representing the cell biology state.In order to reduce the death of tumour Rate or the effect for improving treatment, it is many to study the discovery and screening for being directed to tumour key organism reference material.Therefore, tumour is sought Early diagnosis, by stages or/and instruction chemotherapy, the sensitiveness of radiotherapy, prognosis and potential therapy target tumor markers always It is the study hotspot of oncology.

NNMT (NNMT, EC2.1.1.1) normal expression is in human liver organization, and its hetero-organization is such as Intestines, lung, kidney, placenta, heart, skeletal muscle, brain, pancreas are expressed very low or not expressed.NNMT is living in the liver of different people Sex differernce is larger, and active height depends on the concentration of NNMT in endochylema, and NNMT concentration is relevant with NNMT mRNA level in-site, with Gene polynorphisms are unrelated, it is generally recognized that the mechanism that NNMT is overexpressed is due to regulate and control caused by abnormal, Ke Nengshou after gene translation HNF -1 (HNF-1), TGF-β 1, STAT3 etc. are activated.

NNMT major function is for donor with S- adenosine-L methionine (SAM), is catalyzed niacinamide and pyridines material Methylate, formed pyridiniujm, in normal liver cell participate in medicine bioconversion, be catalysis medicine, xenobiotic generation A kind of enzyme thanked.NNMT is the key enzyme for maintaining niacinamide balance, it is possible to speculates that increasing for NNMT can influence Buddhist nun gram acyl Amine participates in the function of cell.Niacinamide is the part of NAD molecules, can produce NAD, and NAD by salvage route Participating in many of cell substantially may be such as ability metabolism, corresponding and damage resistance, the life-span of cell etc..Parkinsonism quilt Think relevant with niacinamide metabolic disorder caused by NNMT increases, the enzymatic activity excessively increases by two ways in brain tissue Footpath causes parkinsonism, first, generation neurotoxic substance N- picolines (such as phenylpyridine of N- methyl -4 (MPP+)) activation is led Cause Mitochondria complex 1 impaired, two, which are reduction of niacinamide, causes energy metabolism impairment.Niacinamide can also suppress poly ADP polymerases (PARP), and PRAP is adjusted by many cells such as base excision repair, necrosis, apoptosis to keep the complete of genome Whole property.Therefore have reason to speculate a variety of biological functions that NNMT may participate in tumour cell, it is relevant with the occurrence and development of tumour, Or can influence, radiotherapeutic effect.If effects of the clear and definite NNMT in tumour cell, and illustrate effect machines of the NNMT in tumour System or the analysis module that correlation molecule forms downstream or path, must will be further appreciated NNMT as the valency in treatment and prevention of tumour Value.

In recent years, with albumen give financial aid to students more different cancerous tissues and cancer beside organism's difference with biochip technology when find NNMT The unconventionality expression in multiple cancerous tissues.Compared with normal structure and cancer beside organism, NNMT is in colorectal cancer, lung cancer, carcinoma of urinary bladder, stomach The tumor groups such as cancer, thyroid papillary carcinoma, clear cell carcinoma of kidney, OSCC, breast cancer and pancreas are woven with overexpression, And raised in the serum of kinds of tumors patient.These researchs prompting NNMT occurs closely related with tumour, thus it is speculated that NNMT may A variety of biological functions of tumour cell are participated in, it is relevant with the occurrence and development of tumour, or can influence, radiotherapeutic effect.

(3) content of the invention

The present invention is prepared for energy stably excreting as immunogen immune mouse using NNMT albumen and resisted The cell line of NNMT monoclonal antibodies, is then screened and is further purified by the indirect elisa method of NNMT albumen and obtain energy The monoclonal antibody secreted the cell line of NNMT antibody and extracted by affiliated cell line, therefore it is an object of the present invention to provide a plant height Effect secretes the hybridoma cell strain of anti-NNMT (NNMT) antibody, and NNMT antibody is extracted using the cell line, The NNMT antigens in test serum are detected using NNMT antibody, and then determine the content of NNMT antigens in test serum, Contribute to clinical diagnosis.

The technical solution adopted by the present invention is：

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

The invention further relates to a kind of hybridoma cell strain Zj/2D8 in detection NNMT (NNMT) Application in antigen, specific described application are to extract the NNMT antibody in hybridoma cell strain Zj/2D8, are resisted further according to antigen Body specifically binds principle, utilizes NNMT antigenic contents in NNMT antibody test testing samples.

The extracting method of NNMT antibody of the present invention is：(1) hybridoma cell strain Zj/2D8 is used and contains 10% tire ox blood Clear 1640 culture medium, at 37 DEG C, containing 5%CO₂Cell culture incubator in expand culture, collect cell, (pH value is with PBS 7.4) it is configured to 0.4 × 10⁶/ ml cell liquid, then the BALB/c mouse of cell liquid intraperitoneal injection paraffin sensitization (is not limited to BALB/c mouse, mouse model well known in the art), every injection 1ml, 9 days harvest ascites after inoculation, 1200 revs/min Zhongli's heart, precipitation is removed, collect supernatant；

(2) pH 5.0, the 0.06mol/L sodium-acetate buffers of 2 times of volumes are added in the supernatant collected to step (1), PH to 4.8 is adjusted with 1mol/L HCl again, dilution supernatant is made；Then by the ratio for adding 11 μ l octanoic acids in every milliliter of dilution supernatant Example, it is stirred at room temperature in the lower supernatant to dilution and octanoic acid is added dropwise, added in 30 minutes, 4 DEG C stand 2 hours, take out, 15000g is centrifuged 30 minutes, abandons precipitation；For supernatant after 125 μm of nylon mesh filter, filtrate adds the 0.01mol/L of 1/10 volume PBS, pH to 7.2 is adjusted with 1mol/L NaOH；Ammonium sulfate is added at 4 DEG C, and to 45% saturation degree, (45% saturation degree refers to matter Measure concentration), stir 30 minutes, stand 1 hour, 10000g is centrifuged 30 minutes, is abandoned supernatant, is precipitated with NaCl+ containing 137mmol/L 2.6mol/LKCl+0.2mmol/L EDTA PBS is that dialyzate is dialysed, and 4 DEG C overnight, until using 10g/L BaCl₂It is water-soluble Liquid detects trapped fluid without SO₄Ion, trapped fluid is taken out, 10000g is centrifuged 30 minutes, removes insoluble sediment, supernatant is NNMT Antibody.

Monoclonal NNMT antibody caused by hybridoma Zj/2D8 provided by the invention has height for NNMT antigens Horizontal antibody titer, therefore monoclonal NNMT antibody, its active fragment or the active tablet of its conservative mutation body and mark Section or its conservative mutation body, or its any combination are used equally for preparing reagent, medicament or the kit of detection NNMT antigens.This Reagent, medicament or the kit for preparing detection NNMT antigens is invented preferably using double-antibody method detection NNMT antigens.

Active fragment or its conservative mutation body for mark of the present invention, refer to biomarker well known in the art Or chemical labeling.Such as enzyme mark, fluorescence labeling (such as fluorescein mark) or chemiluminescent labeling.Preferably, the enzyme mark It is designated as horseradish peroxidase, pyruvate kinase or glucose oxidase mark.

Monoclonal antibody of the present invention is preferably that (humanized antibody refers mainly to mouse source Dan Ke for the monoclonal antibody of humanization Grand antibody is transformed with gene cloning and DNA recombinant techniques, the antibody expressed again, its Most amino-acids sequence behaviour source sequence Substitution, the basic affinity and specificity for retaining parent's mouse monoclonal antibody, reduces its heterologous again).

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 (refer in embodiment 1 The antibody that is secreted into supernatant of detection cell is also the positive after nutrient solution supernatant dilutes 1000 times in 1.3) NNMT monoclonals resist Body protein.

(2) present invention has from the NNMT monoclonal antibodies of monoclonal hybridoma strain Zj/2D8 extractions to NNMT antigens Very high (1：1000000) antibody titer, preferable affinity and higher purity, prompt the monoclonal antibody to can be applied to examine Survey the scientific research field of NNMT antigens.

(2) the NNMT monoclonal antibodies from monoclonal hybridoma strain Zj/2D8 extractions of the invention can be applied to clinic Detection, by carrying out SABC to the pathological section of different tissues, detect the expression of NNMT albumen in the tissue.

(4) illustrate

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

Fig. 2 be Zj/2D8,1E7 and 2F8 cell line secretory antibody subclass, light chain type figure, A Zj/2D8, B 1E7, C For 2F8C.

Fig. 3 is Zj/2D8 sterling antigen gel scanning figures.

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

Fig. 5 be Zj/2D8 cell line monoclonal antibodies Western blotting, 1：GST albumen, 2：NNMT-GST fusion proteins, 3：NNMT eggs In vain.

Fig. 6 is the Western blotting of 10 synthesis small peptides.

Fig. 7 is the Western blotting after colorectal cancer cell lines and the interference of positive cell strain HT-29 cell NNMT slow virus.

Fig. 8 is the SABC (× 100) of HT-29 cells (B) after transplanted tumor in nude mice HT-29 cells (A) and NNMT interference.

(5) embodiment

With reference to 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, all it is mass concentration if being indicated without special, solvent is all water.

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

1st, the preparation of monoclonal antibody

The anti-NNMT of present invention monoclonal antibody hybridoma cell strain Zj/ is prepared in accordance with conventional methods known in the art 2D8, the monoclonal that the anti-NNMT of humanization form is prepared referring especially to the principle and method described in United States Patent (USP) 5585089 resist Body hybridoma cell strain Zj/2D8 (common Kohler and MIlrtein, Nature 256:495-96,1975), will in method Mention following reagent：

(1) coating buffer：Na₂CO₃1.5g, NaHCO₃2.9g, NaN₃0.2g, it is dissolved in 1L water, adjusts pH to 9.6.

(2) confining liquid：1% bSA.

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

(4) TMB nitrite ions (including 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 add to 500ml；Substrate colour developing B liquid：Disodium ethylene diamine tetraacetate 0.2g, citric acid 0.95g, glycerine 50ml, 0.15g tetramethyl benzidines (TMB) are taken to be dissolved in 3ml DMSO, distilled water adds to 500ml.During use 50 μ l are respectively taken to mix.

(5) terminate liquid：2.0mol/L H₂SO₄The aqueous solution.

1.1st, the preparation of immunogene

Immunogene is GST-NNMT fusion proteins and NNMT albumen, and NNMT albumen is prepared using technique for gene engineering：According to NCBI RiboaptDBs are encoded to gi:12652950 people's NNMT cDNA sequences, the PCR primer of NNMT genes is synthesized,

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

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

At P1 5' ends and P2 3' ends design BamHI and XhoI sites.Using liver c DNA as template (sequence with it is above-mentioned NCBI RiboaptDBs are encoded to gi:12652950 people's NNMT cDNA sequences are consistent), carried out under primer P1, P2 effect PCR expands NNMT fragments, and response parameter is 94 DEG C of pre-degeneration 2min, 94 DEG C of 20s, 60 DEG C of 30s, 72 DEG C of 45s.Circulate it 20 times Afterwards, 0.8 μ l Taq enzymes, 72 DEG C of extension 10min, 4 DEG C of preservations are added immediately.For PCR primer after glue reclaim, T-A is cloned into PGEM- Teasy carriers, pGEM-T-Easy-NNMT plasmids are obtained, plasmid uses BamHI and XhoI digestions after sequence verification, reclaims DNA pieces Section, p GEX-4T-2 carriers are connected to, build pGEX-4T-2/NNMT plasmids, be transformed into Escherichia coli BL-21-STAR (DE3) In to express GST-NNMT fusion proteins, obtain recombination bacillus coli BL-21-STAR (DE3)-GST-NNMT, 37 DEG C of culture 2h Afterwards, 3h is induced at 30 DEG C with final concentration 0.1mM IPTG, 4 DEG C, 5000rpm is centrifuged 10 minutes, collects thalline, is used The D ultrasonic cell disruptors of scientz- II, at 100 watts, ultrasound 10 seconds, stop 10 seconds, ultrasonication under conditions of 60 circulations Thalline, after 13000rpm is centrifuged 3 minutes, supernatant is left and taken, by supernatant through Glutathione Sepharose 4B (purchased from U.S. Pharmacia companies of state) affinity column purifying GST-NNMT albumen.Then it is (every that fibrin ferment is added into GST-NNMT albumen 1mg albumen adds 10U fibrin ferments) make its abundant digestion, again pass by Glutathione Sepharose 4B and (be purchased from the U.S. Pharmacia companies) affinity column, room temperature jog 30 minutes, GST albumen is fully combined with affinity column, collect stream Go out liquid, 500g is centrifuged 5 minutes, is collected supernatant, is only contained NNMT albumen in supernatant, obtain NNMT albumen after purification.With adjacent benzene The red method measure protein concentration of three acid, 10%SDS-PAGE electrophoresis and Western-blot identify NNMT albumen, only contained in supernatant There is NNMT albumen.

1.2nd, animal immune：Immune programme for children is：Initial immunity takes 8 week old BALB/C female mices, by NNMT albumen and not After family name's Freund's complete adjuvant mixes in equal volume, in the subcutaneous multi-point injection of mouse back, dosage is every μ g of mouse 140.After 30 days, again After being mixed in equal volume with NNMT albumen and incomplete Freund's adjuvant, in the subcutaneous multi-point injection of mouse back, dosage is every mouse 140μg.It is immune once with the isometric homomixture of incomplete Freund's adjuvant with NNMT albumen again every 10 days afterwards, altogether three times, make For booster immunization.First three day of cell fusion is finally carried out, NNMT albumen mixes pneumoretroperitoneum injection, dosage in equal volume with physiological saline For every μ g of mouse 80.Incomplete Freund's adjuvant composition mixes for atoleine with lanolin, and volume components ratio is 2：1, Freund's complete adjuvant composition is in incomplete Freund's adjuvant plus BCG vaccine (concentration 5mg/ml).

1.3rd, cell fusion：Immune BALB/C mouse spleen cell is taken to be pressed with SP2/0 myeloma (being purchased from Chinese Academy of Sciences's cell bank) Ratio (5：1) merge, 1200rpm is centrifuged 5 minutes, abandons supernatant.At 37 DEG C, 50%PEG is added to cell precipitation in 1 minute (0.8ml every 10⁸Splenocyte), fusion 90 seconds are stood, 1200rpm is centrifuged 5 minutes, abandons supernatant, adds 20ml HAT selection cultures Liquid (contains 1640 culture medium 400ml, hyclone 100ml, 50 × HAT 10ml in per 500ml HAT selection nutrient solutions (sigma) 96 porocyte culture plates), are spread, using HAT select nutrient solution culture hybridoma (normal cell culture, 37 DEG C, 5%CO₂Cell culture incubator in), select to survive is hybridoma.BALB/C mice splenocyte with SP2/0 myeloma cell completes cell fusion, cell confluency 96.7%.7th day using indirect elisa method detection cell Supernatant, to screen positive cell.Indirect elisa method is as follows：NNMT albumen is diluted to 0.025 μ g/ml with coating buffer, per hole 100 μ l coated elisa plates, GST-NNMT albumen are diluted to 0.04 μ g/ml with coating buffer, and per the μ l coated elisa plates of hole 100,4 DEG C put Put overnight.Next day, cleaning solution wash 3 times, add the μ l/ holes of confining liquid about 350,37 DEG C, 1 hour, are patted dry after cleaning solution washing.Per hole The μ l of hybridoma supematant 100 are added, if positive serum controls (serum of immunized mice, how anti-), negative serum control (are not immunized The serum of mouse), while blank control wells (1 × PBS) are set, after 37 DEG C are reacted 2 hours, cleaning solution washing ELISA Plate, add 100 μ l rabbit anti-mouses IG-HRP (Cell signaling Technology) (use PBS1：4000 dilutions), 37 DEG C are reacted 1 hour After wash ELISA Plate, add TMB nitrite ions, 37 DEG C are developed the color 15 minutes, add terminate liquid terminating reaction, spectrophotometer (BIO RAD, Model 680, Japan) on read OD₄₅₀Absorbance.The primary dcreening operation positive rate of fused cell is 62.4%.About one Select anti-NNMT titres are high (to reach 1 in hundred primary dcreening operation positives：1000) and anti-GST-NNMT titres are very low (less than 1：10) miscellaneous Hand over tumor cell strain 21, further cloning (the cell picking monoclonal in a positive hole is expanded to culture into 96 orifice plates, Above-mentioned identification is carried out again, until what the cell in this hole picked out is all the positive, as monoclonal antibody strain) until positive rate reaches To 100%, so as to screen monoclonal antibody hybridoma cell strain Zj/2D8, hybridoma cell strain 1E7 and hybridoma cell strain 2F8, hybridoma cell strain 1E7 are preserved in China typical culture collection center, and preservation date August in 2011 31 days, preservation is compiled Number CCTCC NO:C201167, preservation address are Wuhan, China Wuhan University, postcode 430072；Hybridoma cell strain 2F8 preservations In China typical culture collection center, preservation date on January 15th, 2015, deposit number CCTCC NO:C201506, preservation Address is Wuhan, China Wuhan University, postcode 430072.

As a result, the OD values of positive serum controls are 2.192, and the OD values of negative serum control are 0.101, and blank well OD values are 0.097, by screening, the OD values corresponding to aim cell strain obtained are as shown in table 1.

The cell strain of monoclonal antibody of table 1 screens cell conditioned medium OD values

Thus the 3 strain of hybridoma strains filtered out can secrete the antibody of anti-NNMT albumen and OD values are more than 1.4.And Anti- GST-NNMT OD values are very low, almost consistent with the OD values of blank well.Wherein hybridoma cell strain Zj/2D8 anti-NNMT Albumen OD values are apparently higher than hybridoma cell strain 1E7 and hybridoma cell strain 2F8, and anti-GST-NNMT OD values are lower.

1.4th, prepared by ascites：By hybridoma cell strain Zj/2D8 expand culture (normal cell culture, 37 DEG C, 5%CO₂ Cell culture incubator in), with PBS be configured to 0.4 × 10 after collecting cell⁶/ ml cell liquid, paraffin is injected intraperitoneally by cell liquid The BALB/c mouse of sensitization (paraffin sensitization refer to be injected intraperitoneally 1ml paraffin/only), every injection 1ml, 9 days harvest abdomens after inoculation Water, 1200 revs/min of centrifugations, removes precipitation, collects supernatant, as upper strata monoclonal antibody ascites.

1.5th, antibody purification：2 parts of sodium-acetate buffers (0.06mol/L, pH are added in 1 part of supernatant (prepared by step 1.4) 5.0) pH to 4.8, is adjusted with 1mol/L HCl, dilution supernatant is made.Add the ratio of 11 μ l octanoic acids in every milliliter of dilution supernatant, Be stirred at room temperature it is lower to dilution supernatant in octanoic acid is added dropwise, added in 30 minutes, 4 DEG C stand 2 hours, take out 15000g from The heart 30 minutes, abandons precipitation；For supernatant after nylon mesh filters (125 μm), filtrate adds the 0.01mol/L PBS of 1/10 volume, uses 1mol/L NaOH adjust pH to 7.2；Ammonium sulfate is added at 4 DEG C to 45% saturation degree, stirs 30 minutes, stands 1 hour； 10000g is centrifuged 30 minutes, abandons supernatant；Precipitation is with the KCl+0.2mmol/L of NaCl+2.6mol/L containing 137mmol/L EDTA's PBS is dialyzate (molecular cut off of bag filter is 14000), is dialysed with the dialyzate of 50-100 times of precipitation volume, 4 DEG C Overnight, dialyzate is changed therebetween more than 3 times, until the BaCl with 10g/L₂Aqueous assay trapped fluid is without SO₄Ion.Take out retention Liquid, 10000g are centrifuged 30 minutes, remove insoluble sediment, the NNMT monoclonal antibodies that supernatant as purifies, uv-spectrophotometric After method measure protein content, 0.02%NaN is added₃Packing saves backup.Ascites crude product is through 4% octanoic acid extraction and 50% sulfuric acid Ammonium salt is analysed, and main foreign protein, such as albumin almost all is removed.SDS-PAGE electrophoresis showeds (Fig. 1) are after purification except anti- Miscellaneous band outside the heavy chain (50KD) and light chain (25KD) band of body is less, shows that purification effect is preferable.

1.6th, the identification of antibody

1.6.1, the subclass and light chain type of the antibody of hybridoma secretion, uses sigma-aldrich companies IsoQuick Kits for Mouse Monoclonal Isotyping are detected：(antibody prepared by this experiment mentioned hereinafter Either Zj/2D8 all refer to 1., 5 purifying prepare gained) 1.5 purifying prepare gained Zj/2D8 secretion NNMT antibody, be IgG2a κ classes (A in Fig. 2), (B in Fig. 2) identical with 1E7 hybridoma cell strain types, but be IgG2b κ with the 2F8 antibody secreted Class difference (C in Fig. 2).

1.6.2, the purity and molecular weight of sterling antibody：1.5 purifying prepare gained NNMT antibody through SDS-PAGE electrophoresis and After gel image scanning analysis, antibody purity is 91.4% (Fig. 3).Light chain molecule amount is about 25KD, and heavy chain molecule amount is about 50KD, with The characteristic of antibody molecule is consistent.

1.6.3, sterling antibody titer and affinity analysis：1.5 purifying prepare gained NNMT antibody, 1E7,2F8 cell line Sterling antibody is after indirect ELISA is analyzed, and antibody titer is more than 100000, but the NNMT antibody of ZJ/2D8 separation will height In the antibody that 1E7 and 2F8 (such as table 2 and Fig. 4) is separated.Specific method is as follows：NNMT albumen is diluted to 0.025 μ with coating buffer Bag ELISA Plate after g/ml, 4 DEG C overnight.Add confining liquid in ELISA Plate, after 37 DEG C are closed 1 hour, cleaning solution washing.Washing pats dry Afterwards, the NNMT antibody of Zj/2D8 separation, the antibody of 1E7 separation, the antibody (10mg/ml) of 2F8 separation are added respectively, by 1:100~ 1:10⁷10 times of ratios (table 2) and 2 times of ratio (Fig. 4) amount of dilution enter hole, after 37 DEG C of washings 2 hours, add the anti-rabbit of 100 μ l bis- Anti- mouse HRP-IgG is washed after 1 hour.Last TMB nitrite ions develop the color 15 minutes, add terminate liquid terminating reaction, determine wavelength 450nm OD values (table 2).Show it is purified after, the potency of antibody has increased, and maintains the immunocompetence of antibody.

The monoclonal antibody of table 2 Zj/2D8,1E7 and 2F8 extraction is to NNMT albumen indirect method antibody titer results

As a result using the logarithm of antibody concentration as abscissa, OD values are ordinate, draw the measure curve of monoclonal antibody affinity (such as Fig. 4).Antibody concentration (unit μ g/ml) when Zj/2D8 cell lines reach maximum combined 50% is 0.11 μ g/ml, than 1E7's 0.34 μ g/ml and 2F8 0.22 μ g/ml will be low, show antibody secreted by Zj/2D8 cell lines relative affinity ratio 1E7 and 2F8 strains will be high, and its affinity can be satisfied with all kinds of detection reagents of production and kit.

1.7th, antibody specificity identification (using Western-blot)

1.7.1, method

A. polyacrylamide gel electrophoresis (SDS-PAGE)

(1) separation gel of polyacrylamide 15% is prepared by table.Final step adds TEMED, carefully will separation after mixing Glue is injected in ready glass gels mould, to be the space that concentration glue stays 1.5-2cm above.Covered in top layer plus water, make boundary Face is smooth, and prevents the oxygen in air from playing inhibitory action to gel polymerisation, places at room temperature about 30 minutes, is allowed to polymerize.It is poly- After the completion of conjunction, the water that inclines on separation gel, the remaining liquid on gel top is blotted with blotting paper as far as possible.

(2) 5% concentration glue is prepared by table, glass gels mould is added after mixing, plugs comb, is placed 30 minutes at room temperature. Gel electrophoresis plate is put into electrophoresis tank, is immersed in 1 × Tris glycine running buffers, carefully removes comb.

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

(4) loading：Each swimming lane is loaded 10 μ l with micro sample adding appliance respectively.

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

B. transferring film and development

(1) take 4 with running gel filter paper of the same size and polyvinylidene fluoride filter membrane (PVDF, Polyvinylidene-Fluoride), with methanol soak 15 minutes it is transparent to filter membrane.

(2) two layers of filter paper is placed on fixed transfer device, the good polyacrylamide gel of own electrophoresis is placed on filter paper On, then PVDF filter membranes are placed on polyacrylamide gel, cover two layers of filter paper on PVDF filter membranes again, each step will use glass Glass rod is rushed bubble, then fixing device.

(3) whole device is placed in electrophoresis tank, pays attention to making gel layer be located at negative pole, filter membranous layer is located at positive pole, shifted The transfering buffering liquid of precooling is added in electrophoresis tank.

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

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

(6) primary antibody is added：Primary antibody (anti-NNMT monoclonal antibodies) is diluted with confining liquid, with primary antibody 2ml (volume ratios 1 after dilution： 12000), film is put into wherein, 4 DEG C rotate overnight.

(7) film is embathed 3 times with confining liquid room temperature, 10 minutes every time.

(8) secondary antibody is added：By secondary antibody rabbit anti-mouse (IgG-HRP) (Cell signaling Technology), confining liquid is used 1:5000 dilutions, with secondary antibody 2ml after dilution, film are put into wherein, rocked at room temperature 2 hours.

(9) band is embathed 3 times with dip lotion room temperature, 10 minutes every time.

(10) plus after ECL developer solutions develop.

As a result monoclonal antibody is identified through Western-blot, on PVDF (Polyvinylidene-Fluoride) film, Only there is an obvious spy in the corresponding molecular weight region of NNMT-GST fusion proteins (55KD) and NNMT albumen (29KD) swimming lane Different in nature band, and do not occur respective strap (as shown in Figure 5) in GST albumen (26KD) swimming lane.

1.8th, antibody antigen Epitope Identification

1.8.1, method

The synthesis of a.NNMT small peptides：According to NNMT protein amino acid sequences (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, the design of commission Zhongtai Bio-Chem. Co., Ltd., Hangzhou And 10 small peptides (N1-N10) (table 3) of chemical synthesis.

B.Western-blot and Elisa methods identification antibody identification epitope：Antigen table is carried out using Elisa methods Position identification, using 10 small peptides of synthesis as envelope antigen, using as negative control, according to indirect Elisa methods (such as 1.6.3) Carry out, as a result show that the 3rd article of small peptide of Zj/2D8 groups is positive, the epitope for illustrating Zj/2D8 identifications is N3 (SGPTIYQLLSACESFKEIVVTD), and 1E7 and 2F8 without response small peptide it is corresponding.Utilize Western-blot simultaneously Verified, after these small peptides are carried out into SDS-PAGE electrophoresis, be transferred on NC films, be used as primary antibody, goat-anti by the use of 3 plants of monoclonal antibodies respectively Mouse HRP-IgG is developed the color through DAB as secondary antibody, as a result consistent with Elisa experimental results (Fig. 6).Because Zj/2D8 has clearly anti- Original identification epitope, and the also no clear and definite antigen recognizing epitope of 1E7 and 2F8, so Zj/2D8 is more suitable for producing all kinds of detections Reagent and kit, for all kinds of detection and research.

3 10 synthesis short peptide sequence information of table

Title	Original 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

1.10th, the measure of antibody-secreting stability

3 strain of hybridoma of preparation are recovered freezing 6 months and 12 months, growth conditions are good, with indirect The identification of Elisa methods, OD_450nmIt is worth for 1.3~2.0, shows that the ability of secretory antibody is stable.

Embodiment 2：Monoclonal antibody is applied to SABC detection clinical samples (using two step indirect methods)

1st, the anti-NNMT monoclonal antibodies of this experiment production can apply to the immunohistochemical experiment of clinical pathology sample.I From hybridoma cell strain Zj/2D8 separation NNMT monoclonal antibodies (1.5 prepare gained after purification in embodiment 1) to not Immunohistochemical experiment is carried out with tissue.First by primary antibody (specific antibody -- NNMT monoclonal antibodies) and corresponding tissue antigen knot Close, form antigen antibody complex, then combined with secondary antibody (antibody that enzyme marks) with the specific antibody in compound, formed anti- Antigen-antibody-hrp-antibody complex, finally with substrate chromogenic reagent.Specific method is as follows：

(1) HT-29 cells (being purchased from Chinese Academy of Sciences's cell bank) the transplanting nude mice after paraffin NNMT interference, the tissue of transplantable tumor are cut Piece dewaxing to water (general term, exactly slough paraffin with dimethylbenzene-absolute alcohol, then by the alcohol of absolute alcohol 70%, 50% alcohol is transitioned into water successively).

(2) 1 × PBS are rinsed, 5 minutes every time, totally 3 times.

(3) 3% hydrogen peroxide methanol solutions act on 20 minutes, block endogenous peroxidase activity.

(4) 1 × PBS are rinsed, 5 minutes every time, totally 3 times.

(5) confining liquid is closed 10 minutes.

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

(7) the NNMT antibody of the preparation of embodiment 1 is added dropwise as primary antibody (1 × PBS 1 in every section：16000 dilutions), 37 DEG C, 60 minutes or 4 DEG C of refrigerator overnights.

(8) 1 × PBS are rinsed, 5 minutes every time, totally 3 times.

(9) secondary antibody (1 of the rabbit anti-mouse of horseradish peroxidase (HRP) mark is added dropwise:5000), 37 DEG C, 40 minutes.

(10) 1 × PBS are rinsed, 5 minutes every time, totally 3 times.

(11) 3,3- diaminobenzidines (DAB) chromophoric solution (GENMEM companies of the U.S.) of 100 μ l Fresh is added, 10 minutes.

(12) running water is rinsed, and haematoxylin is redyed, neutral gum sealing.

Similarity condition is used as control using HCE8693 cells.

2nd, the immunoblotting analysis of colorectal cancer cell lines

Wherein HCE8693 and HT-29 cells show that NNMT is positive (Fig. 7), after the interference NNMT expression of HT-29 cells slow virus NNMT is positive to weaken (Fig. 7).

3rd, the SABC of the HT-29 cell transplantation nude mices after HT-29 cells and NNMT are disturbed

The nude mice of HT-29 cell transplantations is substantially than the nude mice positive (figure eager to excel in whatever one does of the HT-29 cell transplantations after NNMT interference 8)。

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

The 1.5 NNMT monoclonal antibodies finally purified can apply to NNMT antigens in clinical serum sample in embodiment 1 Test experience.We select the monoclonal antibody that Zj/2D8,1E7 and 2F8 are separated to (being purchased from Prospec with serum dilution The NNMT antigen standards (ENZ-418) of company) dilution various concentrations sample carry out NNMT antigen detection assays.First by Dan Ke Grand antibody is coated with solid phase carrier, forms insolubilized antibody, adds serum specimen to be checked, forms solid phase antigen antibody complex, then add Monoclonal antibody linked with peroxidase and the NNMT antigen bindings in compound, it is sandwich compound to form insolubilized antibody-determined antigen-enzyme labelled antibody Thing, finally with substrate chromogenic reagent.Specific method is as follows：

(1) it is coated with：With coating buffer solution by NNMT antibody (respectively by the monoclonal antibody of Zj/2D8,1E7 and 2F8 separation Tested) 10 μ g/mL are diluted to, 0.1ml is added in the reacting hole of each XPS, 4 DEG C are overnight.Next day, discard Solution in hole, washed 3 times, every time 3 minutes with lavation buffer solution.

(2) it is loaded：Add blood serum sample 0.1ml to be checked in above-mentioned coated reacting hole, place 37 DEG C and be incubated 1 hour. It is washed out 3 times.

(3) enzyme-added labeling antibody：In each reacting hole, the NNMT antibody of the horseradish peroxidase-labeled of diluted fresh is added 0.1ml.37 DEG C are incubated 0.5~1 hour, wash 3 times.

(4) plus substrate solution develops the color：The addition tmb substrate solution 0.1ml in each reacting hole, 37 DEG C, 15 minutes.

(5) terminating reaction：Terminate liquid 0.05ml is added in each reacting hole.

(6) OD values are determined at 450nm with Elisa detectors.

(Markus Roe β ler, Wolfgang Rollinger, Stefan Palme, et are reported according to foreign literature al.Identification of Nicotinamide N-Methyltransferase as a Novel Serum Tumor Marker for Colorectal Cancer, Clinical Cancer Research, 2005), detected using Elisa methods NNMT antigenic contents in normal human serum, the lowest content are located at 169pg/ml, the rise of Partial tumors patients serum concentration.Institute So that NNMT antigen standards are diluted to 0pg/ml, 50pg/ml, 100pg/ml, 200pg/ml, 500pg/ml, 1ng/ by us Ml, 5ng/ml, 10ng/ml and 100ng/ml concentration, testing result such as table 4.

Table 4 Zj/2D8,1E7 and 2F8 monoclonal antibody detect the OD values of different NNMT antigen concentrations

We have seen that, the monoclonal antibody of Zj/2D8 separation can detect 50pg/ml, and 1E7 and 2F8 are detected from result Resolution capability is not strong during 200pg/ml.So the monoclonal antibody of Zj/2D8 separation is adapted to classes of agents and examination is made NNMT concentration, is detected to NNMT antigens in agent box detection human serum.

Claims (4)

1. a strain of hybridoma strain Zj/2D8, is preserved in China typical culture collection center, preservation date is 2013 10 The moon 22, deposit number is CCTCC NO：C2013149, preservation address are Wuhan, China, Wuhan University, postcode 430072.

2. hybridoma cell strain Zj/2D8 described in a kind of claim 1 is preparing detection NNMT antigen Application in kit.

3. application as claimed in claim 2, it is characterised in that described application is in extraction hybridoma cell strain Zj/2D8 NNMT antibody, then with niacinamide-N- in NNMT antibody test testing sample Methylase antigenic content.

4. application as claimed in claim 3, it is characterised in that the extracting method of the NNMT antibody For：(1) hybridoma cell strain Zj/2D8 is used to the 1640 culture medium for containing 10% hyclone, at 37 DEG C, containing 5%CO₂Cell Expand culture in incubator, collect cell, 0.4 × 10 is configured to PBS⁶/ ml cell liquid, then stone is injected intraperitoneally in cell liquid The BALB/c mouse of wax sensitization, every injection 1ml, 9 days harvest ascites after inoculation, 1200 revs/min of centrifugations, precipitation is removed, is received Collect supernatant；

(2) pH 5.0, the 0.06mol/L sodium-acetate buffers of 2 times of volumes are added in the supernatant collected to step (1), then used 1mol/L HCl adjust pH to 4.8, and dilution supernatant is made；Then the ratio that in supernatant plus 11 μ l are sad is diluted in every milliliter, It is stirred at room temperature in the lower supernatant to dilution and octanoic acid is added dropwise, added in 30 minutes, 4 DEG C stands 2 hours, take out, 15000g Centrifugation 30 minutes, abandons precipitation；For supernatant after 125 μm of nylon mesh filter, filtrate adds the 0.01mol/L PBS of 1/10 volume, uses 1mol/L NaOH adjust pH to 7.2；Ammonium sulfate is added at 4 DEG C to 45% saturation degree, stirs 30 minutes, stands 1 hour, 10000g is centrifuged 30 minutes, is abandoned supernatant, is precipitated with the KCl+0.2mmol/L of NaCl+2.6mol/L containing 137mmol/L EDTA's PBS is that dialyzate is dialysed, and 4 DEG C overnight, until using 10g/L BaCl₂Aqueous assay trapped fluid is without SO₄Ion, take out and cut Liquid is stayed, 10000g is centrifuged 30 minutes, removes insoluble sediment, supernatant is NNMT antibody.