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 line Zj/2D8 and its detection of nicotinamide-N-methylase antigen application

(1) Technical field

The invention relates to a protein immunological detection method, in particular to a monoclonal antibody capable of resisting nicotinamide-N-methylase protein, a cell line secreting the monoclonal antibody, a preparation method and the use of the monoclonal antibody .

(2) Background technology

Malignant tumors are a class of diseases that seriously endanger human health. According to the latest data from the World Health Organization, there are more than 10 million new cases of cancer worldwide each year, and more than 7 million deaths, accounting for 12% of the total death toll. By 2020, there will be 15 million new cancer patients every year. Not only that, the number of cancer deaths is also rising rapidly around the world, and this number may increase to 13.2 million in 2030.

Early diagnosis of tumors is the key to cancer prevention and treatment. The risk of death and recurrence of tumors is closely related to the stage at the time of initial diagnosis, and the sensitivity of chemotherapy and radiotherapy depends to a certain extent on the enzymes or protein molecules in cancer cells. Biomarkers are important signal indicators that represent the biological state of cells under specific conditions. In order to reduce the mortality of tumors or improve the effect of treatment, many studies are devoted to the discovery and screening of key biological standards of tumors. Therefore, seeking tumor markers for early diagnosis, staging or/and indication of chemotherapy, radiotherapy sensitivity, prognosis and potential therapeutic targets has always been a research hotspot in oncology.

Nicotinamide-N-methylase (NNMT, EC2.1.1.1) is normally expressed in human liver tissue, and other tissues such as intestine, lung, kidney, placenta, heart, skeletal muscle, brain, and pancreas have low or no expression Express. The activity of NNMT in the liver of different people varies greatly. The activity depends on the concentration of NNMT in the cytoplasm. The concentration of NNMT is related to the mRNA level of NNMT and has nothing to do with the polymorphism of the gene. It is generally believed that the mechanism of NNMT overexpression is Due to abnormal post-translational regulation of the gene, it may be activated by hepatocyte nuclear factor-1 (HNF-1), TGF-β1, STAT3, etc.

The main function of NNMT is to use S-adenosyl-L-methionine (SAM) as a donor to catalyze the methylation of nicotinamide and pyridines to form pyridinium salts and participate in the biotransformation of drugs in normal liver cells. , An enzyme that metabolizes xenobiotics. NNMT is the key enzyme to maintain the balance of nicotinamide, so it can be speculated that the increase of NNMT can affect the function of nicotinamide participating in cells. Niacinamide is an integral part of NAD molecule, which can produce NAD through the remedial synthesis pathway, and NAD is involved in many basic possibilities of cells such as metabolism, response and damage resistance, and cell lifespan. Parkinson's syndrome is considered to be related to the abnormal nicotinamide metabolism caused by the increase of NNMT. The excessive increase of this enzyme activity in brain tissue leads to Parkinson's syndrome through two pathways. One is to generate the neurotoxicant N-methylpyridine (such as N- Methyl-4-phenylpyridine (MPP+)) activation leads to damage to mitochondrial complex 1, and the second is to reduce nicotinamide leading to energy metabolism disorders. Niacinamide can also inhibit poly ADP polymerase (PARP), and PRAP maintains genome integrity through multicellular regulation such as base excision repair, necrosis, and apoptosis. Therefore, it is reasonable to speculate that NNMT may be involved in a variety of biological functions of tumor cells, related to the occurrence and development of tumors, or may affect the effects of chemotherapy and radiotherapy. If the role of NNMT in tumor cells can be clarified, and the mechanism of action of NNMT in tumors can be clarified or the modules or pathways of its downstream related molecules can be analyzed, which will further clarify the value of NNMT in cancer prevention and treatment.

In recent years, it was found that NNMT was abnormally expressed in various cancer tissues when protein-assisted learning and gene chip technology were used to compare the differences between different cancer tissues and adjacent tissues. Compared with normal tissues and paracancerous tissues, NNMT was overexpressed in tumor tissues such as colorectal cancer, lung cancer, bladder cancer, gastric cancer, papillary thyroid carcinoma, clear cell renal cell carcinoma, oral squamous cell carcinoma, breast cancer and pancreas, And it is elevated in the serum of patients with various tumors. These studies suggest that NNMT is closely related to tumorigenesis. It is speculated that NNMT may participate in various biological functions of tumor cells, be related to the occurrence and development of tumors, or affect the effects of chemotherapy and radiotherapy.

(3) Contents of the invention

The present invention uses nicotinamide-N-methylase protein as an immunogen to immunize mice to prepare a cell line capable of stably secreting anti-NNMT monoclonal antibody, and then obtains a cell line capable of secreting NNMT through indirect ELISA method screening and further purification of NNMT protein The cell line of the antibody and the monoclonal antibody extracted by the cell line, so the purpose of the present invention is to provide a hybridoma cell line that efficiently secretes anti-NNMT (nicotinamide-N-methylase) antibody, and use the cell line to extract The NNMT antibody uses the NNMT antibody to detect the NNMT antigen in the tissue to be tested, and then determines the content of the NNMT antigen in the tissue to be tested, which is helpful for clinical diagnosis.

The technical scheme adopted in the present invention is:

The present invention provides a hybridoma cell line Zj/2D8, which is preserved in the China Center for Type Culture Collection. The preservation date is October 22, 2013, the preservation number is CCTCC NO: C2013149, and the preservation address is Wuhan, China, Wuhan University, zip code :430072.

The present invention also relates to an application of the hybridoma cell line Zj/2D8 in the detection of nicotinamide-N-methylase (NNMT) antigen, specifically the application is to extract the hybridoma cell line Zj/2D8 NNMT antibody, and then according to the principle of antigen-antibody specific binding, use NNMT antibody to detect the content of NNMT antigen in the sample to be tested.

The extraction method of the NNMT antibody of the present invention is as follows: (1) the hybridoma cell line Zj/2D8 is used to contain 10% fetal bovine serum 1640 culture medium, at 37 ℃, containing 5% CO ₂ Expanded culture in the cell incubator , collect cells, prepare 0.4×10 ⁶ /ml cell solution with PBS (pH value 7.4), and then intraperitoneally inject the cell solution into paraffin-sensitized BALB/c mice (not limited to BALB/c mice, in the art Known mouse models can be used), each injected with 1ml, and the ascites was harvested 9 days after inoculation, centrifuged at 1200 rpm, the precipitate was removed, and the supernatant was collected;

(2) Add 2 times of volume of pH 5.0, 0.06mol/L sodium acetate buffer in the supernatant collected in step (1), then adjust the pH to 4.8 with 1mol/L HCl to make the diluted supernatant; then According to the proportion of adding 11 μl octanoic acid per ml of diluted supernatant, add octanoic acid dropwise to the diluted supernatant under stirring at room temperature, complete the addition within 30 minutes, let stand at 4°C for 2 hours, take it out, centrifuge at 15000g for 30 minutes, discard Precipitation; after the supernatant is filtered through a 125 μm nylon sieve, add 0.01mol/LPBS of 1/10 volume to the filtrate, and adjust the pH to 7.2 with 1mol/L NaOH; add ammonium sulfate to 45% saturation at 4°C (the 45% Saturation refers to the mass concentration), stirred for 30 minutes, stood still for 1 hour, centrifuged at 10000g for 30 minutes, discarded the supernatant, and precipitated with PBS containing 137mmol/L NaCl+2.6mol/LKCl+0.2mmol/L EDTA as the dialysate Dialyze at 4°C overnight until the retentate is detected to be free of SO ₄ ions with 10g/L BaCl ₂ aqueous solution, take out the retentate, and centrifuge at 10,000g for 30 minutes to remove insoluble sediment, and the supernatant is the NNMT antibody.

The monoclonal NNMT antibody produced by the hybridoma cell Zj/2D8 provided by the present invention has a high level of antibody titer for the NNMT antigen, so the monoclonal NNMT antibody, its active fragment or its conservative mutant, and the labeled active fragment or Its conservative mutants, or any combination thereof, can be used to prepare reagents, medicaments or kits for detecting NNMT antigens. The preparation of the reagent, medicament or kit for detecting NNMT antigen in the present invention preferably uses the double-antibody sandwich method to detect NNMT antigen.

The marked active fragments or conservative mutants thereof in the present invention refer to biological markers or chemical markers known in the art. For example enzymatic labels, fluorescent labels (eg fluorescein labels) or chemiluminescent labels. Preferably, the enzyme marker is a horseradish peroxide, pyruvate kinase or glucose oxidase marker.

The monoclonal antibody described in the present invention is preferably a humanized monoclonal antibody (humanized antibody mainly refers to a mouse monoclonal antibody transformed by gene cloning and DNA recombination technology, and re-expressed antibody, most of its amino acid sequence is replaced by human sequence , basically retain the affinity and specificity of the parental mouse monoclonal antibody, and reduce its heterogeneity).

Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

(1) The monoclonal hybridoma cell line Zj/2D8 provided by the present invention can be efficiently secreted (meaning that the antibody secreted by the cells in the supernatant after being diluted 1000 times in 1.3 of Example 1 is also positive) ) NNMT monoclonal antibody protein.

(2) The NNMT monoclonal antibody extracted from the monoclonal hybridoma cell line Zj/2D8 of the present invention has very high (1:1,000,000) antibody titer, better affinity and higher purity to the NNMT antigen, suggesting that the monoclonal antibody The cloned antibody can be applied to the scientific research field of detecting NNMT antigen.

(2) The NNMT monoclonal antibody extracted from the monoclonal hybridoma cell line Zj/2D8 of the present invention can be applied to clinical detection, and the expression of NNMT protein in the tissue is detected by performing immunohistochemistry on pathological sections of different tissues.

(4) Description of drawings

Figure 1 is the SDS-PAGE electrophoresis of Zj/2D8, 1E7 and 2F8 pure antibodies, M: Marker, 1: Zj/2D8 pure antibodies, 2: 1E7 pure antibodies, 3: 2F8 pure antibodies.

Figure 2 shows the subclasses and light chain types of antibodies secreted by Zj/2D8, 1E7 and 2F8 cell lines. A is Zj/2D8, B is 1E7, and C is 2F8C.

Fig. 3 is a gel scanning picture of Zj/2D8 pure antibody.

Fig. 4 is the affinity response curves of pure antibodies of Zj/2D8, 2F8 and 1E7 cell lines.

Figure 5 is the immunoblotting of Zj/2D8 cell line monoclonal antibody, 1: GST protein, 2: NNMT-GST fusion protein, 3: NNMT protein.

Figure 6 is the western blot of 10 synthetic short peptides.

Figure 7 is the immunoblotting of NNMT lentivirus interference of colorectal cancer cell line and positive cell line HT-29.

Figure 8 shows the immunohistochemistry of HT-29 cells (A) and HT-29 cells (B) after NNMT interference in nude mice (×100).

(5) Specific implementation methods

The present invention is further described below in conjunction with specific embodiment, but protection scope of the present invention is not limited thereto:

The percentage concentration of the present invention, if not specified, is all mass concentration, and the solvent is all water.

Example 1: Obtaining of hybridoma cell lines, screening, preparation and identification of monoclonal antibodies

1. Preparation of monoclonal antibodies

Prepare the anti-NNMT monoclonal antibody hybridoma cell line Zj/2D8 of the present invention according to conventional methods known in the art, and mainly refer to the principles and methods described in US Patent No. 5,585,089 to prepare a humanized form of the anti-NNMT monoclonal antibody hybrid Tumor cell line Zj/2D8 (commonly known as Kohler and MIlrtein, Nature 256:495-96, 1975), the following reagents will be mentioned in the method:

(1) Coating solution: 1.5g of Na ₂ CO ₃ , 2.9g of NaHCO ₃ , and 0.2g of NaN ₃ were dissolved in 1L of water, and the pH was adjusted to 9.6.

(2) Blocking solution: 1% bovine serum albumin.

(3) Washing solution: 0.5ml Tween-20 was dissolved in 1000ml 1×PBS.

(4) TMB chromogenic solution (including substrate chromogenic solution A and B solution), substrate chromogenic solution A: sodium acetate 13.6g, citric acid 1.6g, 30% hydrogen peroxide 0.3ml, distilled water added to 500ml; Chromogenic solution B: 0.2 g disodium edetate, 0.95 g citric acid, 50 ml glycerin, 0.15 g tetramethylbenzidine (TMB) was dissolved in 3 ml DMSO, and distilled water was added to 500 ml. When using, take 50 μl each and mix them.

(5) Termination solution: 2.0 mol/L H ₂ SO ₄ aqueous solution.

1.1. Preparation of immunogen

The immunogen is GST-NNMT fusion protein and NNMT protein, and the NNMT protein is prepared by genetic engineering technology: according to the NCBI nucleotide database, it is coded as gi:12652950 human NNMT cDNA sequence, and the PCR primers of the NNMT gene are synthesized,

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

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

BamHI and XhoI sites were designed at the 5' end of P1 and the 3' end of P2. Using liver cDNA as a template (sequence consistent with the NCBI nucleotide database coded as gi:12652950 human NNMT cDNA sequence), the NNMT fragment was amplified by PCR under the action of primers P1 and P2, and the reaction parameters were pre-denaturation at 94°C for 2 minutes. 94°C for 20s, 60°C for 30s, 72°C for 45s. After 20 cycles, 0.8 μl Taq enzyme was added immediately, extended for 10 min at 72°C, and stored at 4°C. After the PCR product was recovered by gel, T-A was cloned into the PGEM-Teasy vector to obtain the pGEM-T-Easy-NNMT plasmid. After the plasmid was sequenced and verified, it was digested with BamHI and XhoI, and the DNA fragment was recovered and connected to the pGEX-4T-2 vector , construct the pGEX-4T-2/NNMT plasmid, transform it into Escherichia coli BL-21-STAR (DE3) to express the GST-NNMT fusion protein, and obtain recombinant Escherichia coli BL-21-STAR (DE3)-GST-NNMT, 37 After incubating at ℃ for 2 hours, induce with IPTG with a final concentration of 0.1 mM at 30 ℃ for 3 hours, centrifuge at 5000 rpm for 10 minutes at 4 ℃, collect the bacteria, use a scientz-ⅡD ultrasonic cell pulverizer, at 100 watts, ultrasonic for 10 seconds, stop for 10 seconds , under the condition of 60 cycles, sonicate the bacteria, centrifuge at 13000rpm for 3 minutes, take the supernatant, and purify the supernatant GST-NNMT protein through Glutathione Sepharose 4B (purchased from Pharmacia, USA) affinity chromatography column. Then add thrombin (10 U thrombin per 1 mg protein) to the GST-NNMT protein to make it fully digested, and then pass through the affinity chromatography column of Glutathione Sepharose 4B (purchased from Pharmacia, USA) again, and shake gently at room temperature for 30 minutes, so that The GST protein is fully combined with the affinity chromatography column, the effluent is collected, centrifuged at 500g for 5 minutes, and the supernatant is collected, which only contains NNMT protein, and the purified NNMT protein is obtained. The protein concentration was determined by the phthalic acid red method, and the NNMT protein was identified by 10% SDS-PAGE electrophoresis and Western-blot. The supernatant contained only NNMT protein.

1.2. Animal immunization: The immunization procedure is as follows: take 8-week-old BALB/C female mice for the first immunization, mix the NNMT protein and Freund's complete adjuvant in equal volumes, and inject subcutaneously at multiple points on the back of the mice. 140 μg for mice. After 30 days, the NNMT protein and Freund's incomplete adjuvant were mixed again in equal volumes, and injected subcutaneously at multiple points on the back of the mouse, with a dose of 140 μg per mouse. Afterwards, every 10 days, they were immunized once with equal volume mixture of NNMT protein and Freund's incomplete adjuvant, for a total of three times, as a booster immunization. Three days before the final cell fusion, the NNMT protein was mixed with normal saline in equal volumes and injected intraperitoneally at a dose of 80 μg per mouse. Freund's incomplete adjuvant is composed of liquid paraffin and lanolin mixed with a volume ratio of 2:1, and Freund's complete adjuvant is composed of Freund's incomplete adjuvant plus BCG (concentration 5mg/ml).

1.3. Cell fusion: Splenocytes from immune BALB/C mice were fused with SP2/0 myeloma (purchased from the Cell Bank of Chinese Academy of Sciences) in proportion (5:1), centrifuged at 1200 rpm for 5 minutes, and the supernatant was discarded. At 37°C, add 50% PEG (0.8ml per 108 splenocytes) to the cell pellet within ¹ minute, let it stand for fusion for 90 seconds, centrifuge at 1200rpm for 5 minutes, discard the supernatant, and add 20ml of HAT selection medium (per 500ml of HAT selection culture Contain 400ml of 1640 culture medium in the liquid, 100ml of fetal bovine serum, 50×HAT 10ml (sigma)), spread 96 hole cell culture plate, adopt HAT selection medium to cultivate hybridoma cells (normal cell culture, 37 ℃, 5%CO ₂ in the cell culture incubator), the hybridoma cells were selected to survive. BALB/C mouse spleen cells and SP2/0 myeloma cells completed cell fusion, and the cell fusion rate was 96.7%. On the seventh day, the cell supernatant was detected by indirect ELISA method to screen positive cells. The indirect ELISA method is as follows: Dilute NNMT protein to 0.025 μg/ml with coating solution, coat 100 μl per well with enzyme label plate, dilute GST-NNMT protein with coating solution to 0.04 μg/ml, coat with 100 μl per well with enzyme label plate at 4°C overnight. The next day, wash 3 times with washing solution, add blocking solution about 350 μl/well, 37°C for 1 hour, wash with washing solution and pat dry. Add 100 μl of hybridoma supernatant to each well, set positive serum control (serum of immunized mice, polyclonal antibodies), negative serum control (serum of non-immunized mice), and set blank control wells (1×PBS), at 37°C After reacting for 2 hours, wash the microtiter plate with washing solution, add 100 μl rabbit anti-mouse IG-HRP (Cell signaling Technology) (diluted with PBS1:4000), wash the microtiter plate after reacting at 37°C for 1 hour, and add TMB chromogenic solution , develop color at 37°C for 15 minutes, add stop solution to terminate the reaction, and read the absorbance at OD ₄₅₀ on a spectrophotometer (BIORAD, Model 680, Japan). The positive rate of primary screening of fusion cells was 62.4%. 21 cases of hybridoma cell lines with high anti-NNMT titer (up to 1:1000) and low anti-GST-NNMT titer (less than 1:10) were selected from about one hundred cases of positive initial screening, and further cloned (incorporated Cells in a positive well are selected for monoclonal expansion in a 96-well plate, and then the above identification is carried out until the cells in this well are all positive, which is a monoclonal antibody strain) until the positive rate reaches 100%, so that The monoclonal antibody hybridoma cell line Zj/2D8, hybridoma cell line 1E7 and hybridoma cell line 2F8 were screened, and the hybridoma cell line 1E7 was preserved in the China Center for Type Culture Collection, with the date of preservation on August 31, 2011, and the preservation number CCTCC NO:C201167, the deposit address is Wuhan University, Wuhan, China, postcode 430072; the hybridoma cell line 2F8 was deposited in the China Center for Type Culture Collection, the deposit date was January 15, 2015, the deposit number CCTCC NO:C201506, the deposit address is China Wuhan University, Wuhan, postcode 430072.

As a result, the OD value of the positive serum control was 2.192, the OD value of the negative serum control was 0.101, and the OD value of the blank well was 0.097. After screening, the OD values corresponding to the obtained target cell lines are shown in Table 1.

Table 1 OD value of cell supernatant screened by monoclonal antibody cell lines

All the three hybridoma cell lines screened out can secrete antibodies against NNMT protein and the OD value is greater than 1.4. Moreover, the OD values of anti-GST-NNMT were all very low, almost consistent with the OD values of blank wells. Among them, the OD value of anti-NNMT protein of hybridoma cell line Zj/2D8 was significantly higher than that of hybridoma cell line 1E7 and hybridoma cell line 2F8, and the OD value of anti-GST-NNMT was lower.

1.4. Preparation of ascites: expand the hybridoma cell line Zj/2D8 (normal cell culture, 37°C, 5% CO ₂ cell culture incubator), collect the cells and prepare 0.4×10 ⁶ /ml cells with PBS BALB/c mice were injected intraperitoneally with paraffin sensitization (paraffin sensitization refers to the intraperitoneal injection of 1ml paraffin per mouse) into the cell fluid, and each mouse was injected with 1ml, and the ascites was harvested 9 days after inoculation, and centrifuged at 1200 rpm to remove the precipitate , collect the supernatant, which is the upper layer of monoclonal antibody ascites.

1.5. Antibody purification: Add 2 parts of sodium acetate buffer (0.06mol/L, pH5.0) to 1 part of supernatant (prepared in step 1.4), adjust the pH to 4.8 with 1 mol/L HCl to prepare diluted supernatant . According to the ratio of adding 11 μl octanoic acid per milliliter of the diluted supernatant, add octanoic acid dropwise to the diluted supernatant under stirring at room temperature, complete the addition within 30 minutes, let stand at 4°C for 2 hours, take out 15000g and centrifuge for 30 minutes, and discard the precipitate; After the supernatant was filtered through a nylon mesh (125 μm), add 1/10 volume of 0.01mol/L PBS to the filtrate, adjust the pH to 7.2 with 1mol/L NaOH; add ammonium sulfate to 45% saturation at 4°C, and stir for 30 minutes , let stand for 1 hour; centrifuge at 10000g for 30 minutes, discard the supernatant; precipitate with PBS containing 137mmol/L NaCl+2.6mol/L KCl+0.2mmol/L EDTA as the dialysate (the molecular weight cut-off of the dialysis bag is 14000), use Dialyze 50-100 times the precipitation volume of the dialysate at 4°C overnight, during which the dialysate was changed more than 3 times until the retentate was detected to be free of SO ₄ ions with 10 g/L BaCl ₂ aqueous solution. The retentate was taken out and centrifuged at 10,000g for ₃₀ minutes to remove insoluble sediment. The supernatant was the purified NNMT monoclonal antibody. After the protein content was determined by UV spectrophotometry, 0.02% NaN was added and stored in aliquots for future use. The crude ascites was extracted with 4% octanoic acid and salted out with 50% ammonium sulfate, and the main impurities, such as albumin, were almost completely removed. SDS-PAGE electrophoresis showed (Fig. 1) that after purification, there were few bands except the heavy chain (50KD) and light chain (25KD) bands of the antibody, indicating that the purification effect was better.

1.6. Antibody Identification

1.6.1. The subtypes and light chain types of antibodies secreted by hybridoma cells were detected using IsoQuick Kits for Mouse Monoclonal Isotyping from sigma-aldrich: (The antibodies prepared in this experiment mentioned later or Zj/2D8 are both Refer to 1., 5 purified and prepared) 1.5 Purified and prepared NNMT antibody secreted by Zj/2D8, which is IgG2aκ class (A in Figure 2), the same type as the 1E7 hybridoma cell line (B in Figure 2), but secreted with 2F8 The antibodies were different for the IgG2bκ class (Fig. 2, C).

1.6.2. Purity and molecular weight of pure antibody: 1.5 Purification The prepared NNMT antibody was analyzed by SDS-PAGE electrophoresis and gel scanning, and the purity of the antibody was 91.4% (Fig. 3). The molecular weight of the light chain is about 25KD, and the molecular weight of the heavy chain is about 50KD, which is consistent with the characteristics of antibody molecules.

1.6.3. Antibody titer and affinity analysis of pure product: 1.5 Purification and preparation of NNMT antibody, 1E7, 2F8 cell line pure antibody after indirect ELISA analysis, the antibody titer is above 100,000, but NNMT isolated from ZJ/2D8 The antibodies were higher than those isolated from 1E7 and 2F8 (as shown in Table 2 and Figure 4). The specific method is as follows: the NNMT protein was diluted to 0.025 μg/ml with the coating solution, then coated with a microtiter plate, and left overnight at 4°C. Add blocking solution to the ELISA plate, block for 1 hour at 37°C, and wash with washing solution. After washing and patting dry, add NNMT antibody isolated from Zj/2D8, antibody isolated from 1E7, antibody isolated from 2F8 (10mg/ml) respectively, at a ratio of ¹⁰ times (Table 2) and 2 times of 1:100～1:107 The ratio (Figure 4) was diluted into the wells, washed at 37°C for 2 hours, and washed after adding 100 μl of secondary antibody rabbit anti-mouse HRP-IgG for 1 hour. Finally, the TMB chromogenic solution developed the color for 15 minutes, and the stop solution was added to terminate the reaction, and the OD value at a wavelength of 450 nm was measured (Table 2). It shows that after purification, the titer of the antibody is increased and the immunological activity of the antibody is maintained.

Table 2 Antibody titer results of Zj/2D8, 1E7 and 2F8 extracted monoclonal antibodies against NNMT protein indirect method

As a result, the logarithm of the antibody concentration was taken as the abscissa, and the OD value was used as the ordinate, and the assay curve of the monoclonal antibody affinity was drawn (as shown in FIG. 4 ). The antibody concentration (unit μg/ml) of the Zj/2D8 cell line when it reaches 50% of the maximum binding is 0.11 μg/ml, which is lower than 0.34 μg/ml of 1E7 and 0.22 μg/ml of 2F8, indicating that the Zj/2D8 cell line The relative affinity of the secreted antibody is higher than that of 1E7 and 2F8 strains, and its affinity is sufficient for the production of various detection reagents and kits.

1.7. Identification of antibody specificity (using Western-blot)

1.7.1. Method

a. Polyacrylamide gel electrophoresis (SDS-PAGE)

(1) Prepare polyacrylamide 15% separating gel according to the table. In the last step, add TEMED, mix well, and carefully pour the separation gel into the prepared glass gel mold, leaving a gap of 1.5-2cm for the stacking gel. Cover the top layer with water to make the interface smooth and prevent the oxygen in the air from inhibiting the polymerization of the gel. Place it at room temperature for about 30 minutes to allow it to polymerize. After the polymerization is completed, pour off the water on the separation gel, and try to absorb the remaining liquid on the top of the gel with absorbent paper.

(2) Prepare a 5% stacking gel according to the table, mix well, add to a glass gel mold, insert a comb, and place at room temperature for 30 minutes. Put the gel electrophoresis plate into the electrophoresis tank, submerge it in 1×Tris glycine electrophoresis buffer, and carefully remove the comb.

(3) Sample preparation: Take 8 μl of loading buffer, 2 μl of 1M DTT, and 10 μl of protein sample, put them in a 0.5ml centrifuge tube, put them in a boiling water bath for 10 minutes, and place them on ice immediately.

(4) Sample loading: add 10 μl of sample to each swimming lane with a micro-sampler.

(5) Turn on the power, run 35mA electrophoresis for 10-15 minutes, so that the protein can quickly swim to the upper layer of the separation gel and concentrate into a narrow band; change the voltage to 25mA, and continue electrophoresis for about 1.5 hours until the tracer dye is close to the bottom of the gel .

b. Film transfer and development

(1) Take 4 pieces of filter paper with the same size as the electrophoresis gel and a fluorinated polyvinylidene filter membrane (PVDF, Polyvinylidene-Fluoride), soak in methanol for 15 minutes until the filter membrane becomes transparent.

(2) Put two layers of filter paper on a fixed transfer device, put the electrophoresed polyacrylamide gel on the filter paper, then put the PVDF filter membrane on the polyacrylamide gel, and cover the PVDF filter membrane Two layers of filter paper, each step with a glass rod to drive out air bubbles, and then fix the device.

(3) Put the whole device in the electrophoresis tank, make sure that the gel layer is located at the negative pole, and the filter membrane layer is located at the positive pole, and add pre-cooled transfer buffer into the transfer electrophoresis tank.

(4) Turn on the power supply and transfer at 250mA for 2 hours.

(5) Blocking: rotate in blocking solution for 60 minutes at room temperature.

(6) Add primary antibody: the primary antibody (anti-NNMT monoclonal antibody) is diluted with blocking solution, and 2ml of the diluted primary antibody (volume ratio 1:12000) is added, and the membrane is placed in it, and rotated overnight at 4°C.

(7) Rinse the membrane with blocking solution at room temperature for 3 times, 10 minutes each time.

(8) Add secondary antibody: Dilute the secondary antibody rabbit anti-mouse (IgG-HRP) (Cell signaling Technology) with blocking solution 1:5000, mix the diluted secondary antibody 2ml, put the membrane in it, and rotate at room temperature for 2 hours .

(9) Rinse the strip with the soaking solution at room temperature for 3 times, 10 minutes each time.

(10) After adding ECL developer, develop.

Results The monoclonal antibody was identified by Western-blot. On the PVDF (Polyvinylidene-Fluoride) membrane, only NNMT-GST fusion protein (55KD) and NNMT protein (29KD) had an obvious specific band in the corresponding molecular weight region of the swimming lane, while in No corresponding band appeared in the GST protein (26KD) lane (as shown in Figure 5).

1.8. Antibody epitope identification

1.8.1. Method

a.NNMT短肽的合成：根据NNMT蛋白氨基酸序列(mesgftskdt ylshfnprdylekyykfgsr hsaesqilkh llknlfkifc ldgvkgdlli digsgptiyq llsacesfke ivvtdysdqnlqelekwlkk epeafdwspv vtyvcdlegn rvkgpekeek lrqavkqvlk cdvtqsqplg avplppadcvlstlcldaac pdlptycral rnlgsllkpg gflvimdalk ssyymigeqk fsslplgrea veaavkeagytiewfevisq sysstmanne glfslvarkl srpl)及结构分析，委托杭州中肽生化有限公司设计And chemically synthesized 10 short peptides (N1-N10) (Table 3).

b. Western-blot and Elisa method to identify the antigenic epitope recognized by the antibody: use the Elisa method to identify the antigenic epitope, and use the synthesized 10 short peptides as the coating antigen as a negative control, follow the indirect Elisa method (such as 1.6.3 ), the results showed that the third short peptide of the Zj/2D8 group was positive, indicating that the antigenic epitope recognized by Zj/2D8 was N3 (SGPTIYQLLSACESFKEIVVTD), and neither 1E7 nor 2F8 had corresponding short peptides corresponding to it. At the same time, Western-blot was used for verification. After these short peptides were subjected to SDS-PAGE electrophoresis, they were transferred to NC membranes. Three monoclonal antibodies were used as primary antibodies, and goat anti-mouse HRP-IgG was used as secondary antibody, and the color was developed by DAB. , the results are consistent with the results of the Elisa experiment (Fig. 6). Because Zj/2D8 has a clear antigen recognition epitope, while 1E7 and 2F8 have no clear antigen recognition epitope, Zj/2D8 is more suitable for the production of various detection reagents and kits for various detection and research.

Table 3 Sequence information of 10 synthetic short peptides

name starting point sequence end 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. Determination of antibody secretion stability

The 3 strains of hybridoma cells were recovered after cryopreservation for 6 months and 12 months, and the growth status was good. The indirect Elisa method was used to identify the OD _450nm value of 1.3-2.0, indicating that the ability to secrete antibodies was stable.

Example 2: Application of monoclonal antibody to immunohistochemical detection of clinical specimens (two-step indirect method)

1. The anti-NNMT monoclonal antibody produced in this experiment can be applied to immunohistochemical experiments of clinical pathological specimens. We selected the NNMT monoclonal antibody isolated from the hybridoma cell line Zj/2D8 (prepared after purification at 1.5 in Example 1) to perform immunohistochemical experiments on different tissues. First, the primary antibody (specific antibody - NNMT monoclonal antibody) is combined with the corresponding tissue antigen to form an antigen-antibody complex, and then the secondary antibody (enzyme-labeled antibody) is combined with the specific antibody in the complex to form an antigen- The antibody-enzyme-labeled antibody complex is finally developed with a substrate chromogenic reagent. The specific method is as follows:

(1) HT-29 cells (purchased from the Chinese Academy of Sciences Cell Bank) after paraffin NNMT interference were transplanted into nude mice, and the tissue sections of transplanted tumors were dewaxed to water (conventional terms, that is, deparaffinized with xylene-absolute alcohol, and then passed through Anhydrous alcohol 70% alcohol, 50% alcohol transitions to water).

(2) Wash with 1×PBS, 5 minutes each time, 3 times in total.

(3) 3% methanol solution of hydrogen peroxide acts for 20 minutes to block endogenous peroxidase activity.

(4) Rinse with 1×PBS, 5 minutes each time, 3 times in total.

(5) The blocking solution was blocked for 10 minutes.

(6) Antigen retrieval was carried out with citrate buffer.

(7) Add the NNMT antibody prepared in Example 1 as the primary antibody (diluted 1:16000 in 1×PBS) dropwise to each slice, and store at 37°C for 60 minutes or overnight in a refrigerator at 4°C.

(8) Rinse with 1×PBS, 5 minutes each time, 3 times in total.

(9) Add horseradish peroxidase (HRP)-labeled rabbit anti-mouse secondary antibody (1:5000) dropwise at 37°C for 40 minutes.

(10) Rinse with 1×PBS, 5 minutes each time, 3 times in total.

(11) Add 100 μl of freshly prepared 3,3-diaminobenzidine (DAB) chromogenic solution (GENMEM, USA) for 10 minutes.

(12) Rinse with tap water, counterstain with hematoxylin, and mount with neutral gum.

Under the same conditions, HCE8693 cells were used as a control.

2. Immunoblotting of colorectal cancer cell lines

Among them, HCE8693 and HT-29 cells were positive for NNMT (Figure 7), and the NNMT positiveness of HT-29 cells was weakened after the lentivirus interfered with the expression of NNMT (Figure 7).

3. Immunohistochemistry of HT-29 cells and HT-29 cells transplanted into nude mice after NNMT interference

The nude mice transplanted with HT-29 cells were significantly more positive than the nude mice transplanted with HT-29 cells after NNMT interference (Fig. 8).

Example 3: Application of monoclonal antibodies to detection of clinical serum samples (using double-antibody sandwich method)

The final purified NNMT monoclonal antibody in 1.5 of Example 1 can be applied to the detection experiment of NNMT antigen in clinical serum samples. We selected monoclonal antibodies isolated from Zj/2D8, 1E7 and 2F8 to conduct NNMT antigen detection experiments on samples with different concentrations diluted with serum diluent (NNMT antigen standard (ENZ-418) purchased from Prospec Company). First, the monoclonal antibody is coated with a solid-phase carrier to form a solid-phase antibody, and the serum sample to be tested is added to form a solid-phase antigen-antibody complex, and then the enzyme-labeled monoclonal antibody is combined with the NNMT antigen in the complex to form a solid-phase antibody -The antigen-to-be-tested-enzyme-labeled antibody sandwich complex is finally developed with a substrate chromogen. The specific method is as follows:

(1) Coating: Dilute the NNMT antibody (monoclonal antibodies isolated from Zj/2D8, 1E7 and 2F8) to 10 μg/mL with coating buffer, add to the reaction well of each polystyrene plate 0.1ml, overnight at 4°C. The next day, the solution in the well was discarded, and washed 3 times with washing buffer, 3 minutes each time.

(2) Adding samples: Add 0.1 ml of the serum sample to be tested to the above-mentioned coated reaction wells, and incubate at 37°C for 1 hour. Then wash 3 times.

(3) Add enzyme-labeled antibody: Add 0.1 ml of freshly diluted horseradish peroxidase-labeled NNMT antibody to each reaction well. Incubate at 37°C for 0.5-1 hour and wash 3 times.

(4) Color development by adding substrate solution: add 0.1 ml of TMB substrate solution to each reaction well, 37°C, 15 minutes.

(5) Stopping the reaction: Add 0.05 ml of stop solution to each reaction well.

(6) Measure the OD value at 450 nm with an Elisa detector.

According to foreign literature reports (Markus Roeβler, Wolfgang Rollinger, Stefan Palme, etal. Identification of Nicotinamide N-Methyltransferase as a Novel Serum TumorMarker for Colorectal Cancer, Clinical Cancer Research, 2005), the NNMT antigen content in normal human serum was detected by Elisa method , the lowest content was located at 169pg/ml, and the serum concentration of some tumor patients increased. Therefore, we diluted the NNMT antigen standard to concentrations of 0pg/ml, 50pg/ml, 100pg/ml, 200pg/ml, 500pg/ml, 1ng/ml, 5ng/ml, 10ng/ml and 100ng/ml. As in Table 4.

Table 4 OD values of Zj/2D8, 1E7 and 2F8 monoclonal antibodies detecting different NNMT antigen concentrations

From the results, we can see that the monoclonal antibody isolated from Zj/2D8 can detect 50pg/ml, while the resolution ability of 1E7 and 2F8 is not strong when it detects 200pg/ml. Therefore, the monoclonal antibody isolated from Zj/2D8 is suitable for making various reagents and kits to detect the concentration of NNMT in human serum and to detect the NNMT antigen.

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.