Hybridoma cell line of secreting meloxicam monoclonal antibodies and application thereof

Abstract

A hybridoma cell line of secreting meloxicam monoclonal antibodies with a preservation number of hybridoma cell line of CGMCC No. 14700 belongs to the field of food safety immunological detection. The. BALB/c mice are immunized through one time immunization with complete freund's adjuvant, three times of booster immunization with incomplete freund's adjuvant and one time of rush immunization with meroxicam complete antigen without adjuvant; the spleen cells from BALB/C mice immunized with high potency and low value of IC50 are fused with murine myeloma cells; and then the hybridoma cell line is obtained through indirect competitive ELISA screening and three sub-clones. The monoclonal antibody secreted by this cell line has good specificity and detection sensitivity to meloxicam (value of IC50 is 0.1 ng/ml), being suitable for detection of meroxicam in food.

Claims

1. A hybridoma cell line of secreting meloxicam monoclonal antibodies deposited with the general microbiological center of China General Microbiological Culture Collection Center (No. 3, Yard 1, West Beichen Road, Chaoyang District, Beijing, China) under Accession Number CGMCC No. 14700 on Dec. 5, 2017.

2. A meroxicam monoclonal antibody obtained from the hybrid tumor cell line of claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the synthetic process of meroxicam haptens.

(2) FIG. 2 shows the standard curve of inhibition of meloxicam by meroxicam monoclonal antibody.

DESCRIPTION OF PREFERRED EMBODIMENTS

(3) The detailed implementation of the invention is further described as follows. The following embodiments are used to illustrate the invention, but not to limit the scope of the invention.

(4) The media involved in the following embodiments are as follows:

(5) RPMI-1640 medium: Fetal bovine serum 10%, 1-arginine 290 mg/L, calcium nitrate 100 mg/L, 1-asparagine 50 mg/L, anhydrous sulfate magnesium 488 mg/L, L-asparagine 20 mg/L, potassium chloride 400 mg/L, L-glutamic acid 20 mg/L, sodium chloride 6000 mg/L, glycine 10 mg/L, glucose 2000 mg/L, L-histidine 15 mg/L, reduced glutathione 1 mg/L, L-hydroxyproline 20 mg/L, phenol red 5 mg/L, L-isoleucine 50 mg/L, L-glutamine 50 mg/L, biotin 50 mg/L, L-lysine hydrochloride 40 mg/L, D-pantothenic acid 15 mg/L, L-threonine 20 mg/L, pyridrol hydrochloride 1 mg/L, L-tryptophan 5 mg/L, riboflavin 0.2 mg/L, L-tyrosine 23.19 mg/L, thiamine hydrochloride 1 mg/L, L-valine 20 mg/L, vitamin B12 0.005 mg/L, para aminobenzoic acid 1 mg/L.

(6) The reagents involved in the following embodiments are as follows:

(7) Carbonate buffer solution (CBS): Weigh Na2CO3 1.59 g and NaHCO.sub.31.59 g, then mixture with steaming water, plus double steamed water to 800 mL, adjust pH value to 9.6, Add double steaming water for constant volume (1000 mL), state for 4 V;

(8) Phosphate Buffer solution (PBS): 8.00 g NaCl, 0.2 g KCl, 0.2 g KH2PO4, 2.9 g Na2HPO4.12 H2O, dissolved in 800 mL pure water, pH was adjusted to 7.27.4 with NaOH or HCl, then the capacity was constant to 1000 mL;

(9) PBST: Add 0.5 ml Tween-20 to 1000 mL PBS solution (0.01 mol/L, pH 7.4);

(10) Antibody Diluent: Washing buffer containing 0.1% gelatin;

(11) TMB Substrate: Mixture of solution A and solution B with 1:5;

(12) Solution A: Na2HPO4.12H2O 18.43 g, citric acid 9.33 g, constant to 1000 mL with pure water. Solution B: 60 mg TMB dissolved in 100 mL ethylene glycol.

(13) The test methods involved in the following embodiments are as follows:

(14) Test method for the yield of meroxicam hapten: the ratio of the quality of the final product after purification to that of the raw material.

(15) Melo standard product inhibition test method:

(16) Indirect competitive enzyme-linked immunosorbent assays (ic-ELISA): The synthetic coating antigen were used to sheathe. Mouse serum was diluted with antibody dilutions of 1000, 3000, 9000 and 27000 times, and the standard product solution was prepared. The above four rows of the enzyme label plate were added with 50 mm L/hole in PBS, and the next four rows with 50 mm L/hole in the standard solution, and then the mice serum of different concentrations were added with 50 mm L/hole at 37 C. for 30 min incubation, adding enzyme mark after two resistance. After color development and termination, OD450 nm are measured. The inhibition rate is calculated according to the absorbance value, inhibition rate=the OD value of the hole with the inhibitor/OD value of the control hole without the inhibitor.

Example 1

Synthesis of Meroxicam Hapten

(17) The synthesis path is shown in FIG. 1.

(18) 1 g of compound 1 was dissolved in 50 ml of methanol solution, and stirred at 157 C. for the night after adding 157 mg MeONa, and then the compound 1 was gotten. 1.20 g of compound 2 was dissolved 30 ml DMF, and added 1.20 mg Ethyl 4-bromobutyrate stir at 80 C. for the night, and then the mixture 2 was gotten. The step 4 is to concentrate the obtained mixture 2 and then purify it with a silica gel column to obtain compound 3. The 800 mg compound 3 was dissolved into 3 mL tetrahydrofuran and 1 mL water, adding 180 mg 1-hydrated lithium hydroxide, adjusting pH to 4-6, and stirring at room temperature overnight to get mixture 4. The aqueous solution layer with ethylamine from mixture 4 was extracted, and the organic layer was combined, washing with salt water, drying with anhydrous sodium sulfate and being concentrated to obtain meloxicam hapten.

(19) The products of compound 3 and haptens are calculated.

(20) Compound 3 production rate of 53.3%, the yield of the hapten was 66.4%.

Example 2

Complete Antigen Synthesis of Meloxicam

(21) 1.7 mg Melo, 2.2 mg 1-ethyl carbodiimide hydrochloride and 1.4 mg N-hydroxysuccinylimide was dissolved into 400 phenyl-anhydrous N, N-dimethylamine, stirring 6-8 h at room temperature to obtain A1 solution. 10 mg keyhole blood blue protein (KLH) (Melo to KLH Moore ratio of 1500:1) was diluted with an appropriate amount of boric acid buffer solution, and B1 solution was obtained. At room temperature, A1 solution was added to B1 solution one drop at a time. Complete antigen (Melo-KLH) is obtained by separating complete antigen and uncoupled small molecule hapten (Melo) by dialysis.

(22) 1.7 mg Melo, 2.2 mg 1-ethyl carbodiimide hydrochloride and 1.4 mg N-hydroxysuccinylimide was dissolved into 400 phenyl-anhydrous N, N-dimethylamine, stirring 6-8 h at room temperature to obtain A1 solution. 10 mg keyhole blood blue protein (KLH) (Melo to KLH Moore ratio of 3000:1) was diluted with an appropriate amount of boric acid buffer solution, and B1 solution was obtained. At room temperature, A1 solution was added to B1 solution one drop at a time. Complete antigen (Melo-KLH) is obtained by separating complete antigen and uncoupled small molecule hapten (Melo) by dialysis.

(23) 1.7 mg Melo, 2.2 mg 1-ethyl carbodiimide and 1.4 mg N-hydroxysuccinylimide was dissolved into 400 phenyl-anhydrous N, N-dimethylamine, stirring 6-8 h at room temperature to obtain A1 solution. 10 mg keyhole blood blue protein (KLH) (Melo to KLH Moore ratio of 4500:1) was diluted with an appropriate amount of boric acid buffer solution, and B1 solution was obtained. At room temperature, A1 solution was added to B1 solution one drop at a time. Complete antigen (Melo-KLH) is obtained by separating complete antigen and uncoupled small molecule hapten (Melo) by dialysis.

Example 3

Synthesis of the Meroxicam Coating Antigen

(24) 1.8 mg Melo 2.4 mg 1-ethylenediamine hydrochloride salt 1.45 mg N-hydroxysuccinylimide was dissolved into 300 phenyl-anhydrous N, N-dimethylamine, and stir it 6-8 h at room temperature to obtain A2 solution. 5 mg of chicken egg albumin (OVA) was dissolved in 2 mL boric acid buffer solution to obtain B2 solution. At room temperature, A2 solution was added to B2 solution, and mixed solution was obtained after overnight reaction at room temperature. Complete antigens, uncoupled small molecule haptens (Melo) and coating antigen (Melo-OVA) were separated by dialysis.

Example 4

Preparation of Hybrid Tumor Cell Lines that Secrete the Monoclonal Antibody of Meloxicam

(25) Animal Immunization

(26) Healthy Balb/C mice aged 6-8 weeks were selected for immunization. BALB/c mice were immunized by subcutaneous injection of three different molar ratios of Melo complete antigen and equivalent freund's adjuvant. For the first immunization, 100 ug of each mouse was injected with complete freund's adjuvant, after which the whole freund's adjuvant was used. Each mouse was injected with 50 ug between the first immunization and the second booster immunization for 28 days, and between multiple booster immunization for 21 days, and blood was collected for 7 days after the third immunization (5 ul Tail blood of mice+995 ul antibody diluent=antiserum). The serum titer and inhibition of mice were determined by ic-elisa, and the mice with high titer and good inhibition were selected for the sprint immune after the fourth immunization session by intraperitoneal injection with the dosage halved and without any adjuvant.

(27) The serum titer and inhibition rate of mice were measured by IC-ELISA. It was found that when the anti-serum dilution multiple was 3K and the concentration of coating antigen was 0.1 g/mL, the potency of the mice immunized was 71%, 52% and 51 after the addition of 50 ppb Melo standard with immunogen Melo-KLH 1500:1, Melo-KLH 3000:1 and Melo-KLH 4500:1 were 1.648, 1.333 and 1.613 respectively.

(28) Obviously, the titer and inhibition rate of immunized mice with immunogen melo-klh 1500:1 were the highest, so this mouse was selected for the next experiment.

(29) Cell Fusion

(30) After 3 days of shock immunity, cell fusion was performed by PEG (polyethylene glycol, with a molecular weight of 1500). The steps are as follows:

(31) After mice were killed by cervical dislocation, the their eyeball blood was picked and soaked immediately in 75% alcohol disinfection about 5 min. The spleen of the mice was taken out by aseptic operating, grinded moderately by the glue head of the syringe and gotten the splenocyte suspension through 200 mesh cell screen. And the splenocyte suspension was collected and centrifuged (1200 RPM, 8 min). And then washing spleen cells three times with RPMI-1640 medium, after the last time the centrifugal, spleen cells were diluted to a certain volume, count, and standby application.

(32) Collect sp2/0 cells: Sp2/0 tumor cells were cultured in 5% CO2 culture box with RPMI-1640 medium containing 10% FBS (fetal bovine serum) between 7 and 10 days before fusion. Before fusion, the number of sp2/0 tumor cells was required to reach 1-4107, ensuring that sp2/0 tumor cells were in the logarithmic growth stage. At the time of fusion, tumor cells were collected and suspended in rpm-1640 basic medium for cell counting.

(33) The fusion process lasted for 7 min. During the first min. 1 mL of PEG1500 was added to the cells from slow to fast. For the second minute, there was stewing. For the three and four minutes, culture medium of 1 ml RPMI-1640 was added within 1 min. For the five and six minutes, Culture medium of 2 m RPMI-1640 was added within 1 min. For the seven minute, 1 mL rpm-1640 culture medium was added every 10 s. Then the cells were under warm bath at 37 C. 5 min, abandoned supernatant through centrifugation (800 rpm, 8 min), resuspended with 20% fetal bovine serum. And then 2% of the 50HAT RPMI-1640 filter medium added to 96 hole cell plate according to the 200 L/hole, at 37 C. and 5% CO2 incubator to cultivate.

(34) Cell Screening and Cell Line Establishment

(35) On the third day of cell fusion, the fusion cells were partially replaced with the rpm-1640 screening medium, and on the fifth day, the cells were fully replaced with the rpm-1640 transition medium containing 20% fetal bovine serum and 1% 100HT, and the supernatant was taken on the seventh day for screening.

(36) Screening is divided into two steps: the first step was to screen out the positive cells by indirect ELISA; in the second step, Melo was selected as the standard product, and the inhibitory effect of positive cells was measured by indirect competitive ELISA. Cell pores that had good inhibition on all meroxicam standard products were selected, and subclone was conducted by finite dilution method. The same method was used for detection, and the cell lines were obtained after repeated for three times.

Example 5

Preparation of Meloxicam Monoclonal Antibody

(37) BALB/c mice with 8 to 10 weeks, were intraperitoneally injected with paraffin oil 1 mL each. After 7 days, each mouse intraperitoneally is injected with 1106 hybrid tumor cells secreting meroxicam monoclonal antibody. From 7 days start collecting ascites, purified by bitter-ammonium sulfate law, in the condition of partial acid; n-caprylic acid can precipitate other heterologous proteins except IgG immunoglobulin in ascites, and then the precipitation was discarded after centrifuge. The monoclonal antibody of IgG type was precipitated with ammonium sulfate solution of equal saturation, and then the supernatant was discarded after centrifuge. After dissolving precipitate with 0.01 MPBS solution (pH7.4), being desalination through dialysis, finally, the monoclonal antibody was obtained after purification and preserved at 20 C.

Example 6

Identification of Meroxicam Monoclonal Antibody

(38) coating: coating antigen Melo-OVA had reacted for 2 h after serial dilution of the pH of 9.6 of 0.05 m carbonate buffer from 1 g/mL, 100 L/hole at 37 C.

(39) Washing: the solution in the plate was poured out and wash it 3 times with wash solution, 3 min each.

(40) Sealing: After pat dry, 200 L/hole sealing fluid was added to reaction within 2 h at 37 C., drying after washing.

(41) Sample adding: Antiserum (After the blood was collected from the tail of the mice, the antiserum was diluted with antibody diluent) was diluted from the ratio of 1:1000, and was added into the degree of the dilution of coating hole, 100 L/hole, and had reacted at 70 C. for 30 min. After fully washing, HRPGoat anti Mouse IgG that had diluted with the ratio of 1:3000 was added and reacted at 37 C. for 30 min, 100 L/hole;

(42) Coloration: The enzyme label plate was taken out, after washing, each hole was added into TMB Color liquid, and reacted at 37 C. for 15 min avoiding light.

(43) Termination and determination: To terminate the reaction, 50 L termination solution was added to each hole, and then the value of OD450 of each hole was measured with an enzyme marker.

(44) The IC50 of the monoclonal antibody Melo was 0.3 ng/mL and the minimum detection limit was 0.1 ng/mL detected by ic-ELISA, indicating a good sensitivity to Melo and can be used for Melo immunoassay. (the standard curve of inhibition of meloxicam by meroxicam monoclonal antibody was shown in FIG. 2).

(45) The above description is only a preferred method of implementation of the invention, and is not used to limit the invention. It should be noted that, for ordinary technical personnel in the field of technology, some improvements and variations can be made under the technical principles of the invention. These improvements and variations should also be considered as the scope of protection of the invention.