Hybridoma cell line that secrets cyproheptadine monoclonal antibodies and preparation method thereof

Abstract

A hybridoma cell line of secreting cyproheptadine monoclonal antibodies with a preservation number of CGMCC No. 14699 belongs to the field of food safety immunological detection. 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 cyproheptadine 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 cyproheptadine (value of IC50 is 0.37 ng/ml), being suitable for detection of cyproheptadine in food.

Claims

1. A hybridoma cell line that secretes cyproheptadine monoclonal antibodies deposited with the general microbial center of the China General Microbial Culture Collection Committee (No. 3, yard 1, beichen west road, chaoyang district, Beijing) under Accession Number CGMCC No. 14697 on Sep. 5, 2017.

2. A cyproheptadine monoclonal antibody obtained through the hybridoma cell line that secretes cyproheptadine monoclonal antibodies according to claim 1.

3. The cyproheptadine monoclonal antibody according to claim 2 wherein the antibody is applicable in specifically identifying cyproheptadine.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

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

(2) FIG. 2 shows the UV characterization of cyproheptadine complete antigen

(3) FIG. 3 shows the standard curve of inhibition of cyproheptadine by cyproheptadine monoclonal antibody.

DESCRIPTION OF PREFERRED EMBODIMENTS

(4) 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.

Example 1

(5) Synthesis of Cyproheptadine Hapten

(6) 5 g compound 1 of concentration of 17.4 mmol was dissolved in 50 mL methanol solution, and 3 g ethyl chloroformate of concentration of 34.8 mmol was added for stirring under 120° C. for the night to obtain mixture. the cooled mixture mentioned above was added HCL, extracted three times with 50 mL ethyl acetate, the extracted organic layer was dried with anhydrous Na.sub.2SO.sub.4, after vacuum concentration the 4 g compound 2 was obtained. 4 g compound 2 of concentration of 11.6 mmol was dissolved in 64 mL ethyl alcohol and added dropwise potassium hydroxide aqueous solution of equivalent of 15 to stir under 30° C. for the night to get mixture, which the aqueous solution of potassium hydroxide is 10.3 g potassium hydroxide dissolved in 16 mL H.sub.2O to form a solution with a concentration of 174 mmol. the mixture was extracted three times with 50 mL ethyl after acetatevacuum concentration, and the extracted organic layer was dried with anhydrous Na.sub.2SO.sub.4, and the 2.5 g compound 3 was obtained after vacuum concentration. 2.5 g compound 3 of the concentration of 9.2 mmol was dissolved in the mixture of 30 mL tetrahydrofuran THF solution containing 1 g 3-chloropropionic acid of the concentration of 9.2 mmol, the 1 g triethylamine of the concentration of 9.2 mmol was added stirring in 60° C. under for the night N.sub.2 to get mixture. the mixture was 30 mL extracted three times with ethyl acetate after vacuum concentration, the extracted organic layer was dried with anhydrous Na.sub.2SO.sub.4 and vacuum concentrated, and the crude product of cyproheptadine haptens was obtained. After purification by silica gel chromatography (DCM/MeOH=20:1), 400 mg haptens were obtained.

Example 2

(7) Synthesis of Cyproheptadine Complete Antigen

(8) 5 mg cyproheptadine haptens was taken into brown flask adding 1 mL MES buffer solution of pH4.7 and the concentration of 0.1M to dissolve, 10 mg EDC and 6 mg NHS was added stirring for 6-8 h to get the activated liquid; the keyhole limpet haemocyanin KLH was dissolved in CB solution, the activated liquid was added adjusting pH to 9.0 with NaOH of the concentration of 1M, keeping in the dark and reacting for night. After isolating complete antigens and uncoupled small molecules, and identified by uv method, the cyproheptadine complete antigen was obtained (the uv characterization effect of cyheptadine complete antigen of the present invention is shown in FIG. 2).

Example 3

(9) Preparation of hybrid tumor cell lines that secrete the monoclonal antibody of cyproheptadine

(10) Animal Immunization

(11) After the cyproheptadine complete antigen and equivalent freund's adjuvant were mixed and emulsified, BALB/c mice were immunized by subcutaneous injection in the neck and back. For the first immunization, complete freund's adjuvant was used. For two times booster immunization, incomplete freund's adjuvant was used. The blood collection is performed on the seventh day after the end of the third immune process. Blood samples were taken from the mice after the above immune process, and the serum immune titer and immunosuppressive ability were detected by indirect ELISA to select the mice with high cyproheptadine antibody content in serum. The selected mice were subjected to 4th booster immunization with incomplete freund's adjuvant, and then, the rush immunization is performed via intraperitoneal injection, using cyproheptadine complete antigen without freund's adjuvant. (the interval between the booster immunization is 21 days, and the interval between the booster immunization and the rush immunization is 18 days).

(12) 2. Cell Fusion

(13) 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:

(14) The spleen of the mouse was taken aseptically, spleen cell suspension was obtained by grinding and screening with 200 mesh cells and then Counting cells

(15) Sp2/0 cells was collected, and suspended in rpm-1640 basic medium for cell counting.

(16) Spleen cells and sp2/0 cells were mixed in a ratio of 1:10, centrifuged and fused with 50% PEG for 1 mi, the basic culture medium RPMI-1640 was added from slow to fast, after the centrifugal cells suspended in RPMI-1640 filter medium containing 20% fetal bovine serum and 2% of the 50×HAT, and added to the 96 cell culture plate at 37° C. and cultivating in 5% CO2 incubator.

(17) Cell Screening and Cell Line Establishment

(18) 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% 100×HT, and the supernatant was taken on the seventh day for screening.

(19) 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 cyproheptadine 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.

(20) 4. Cell Lines are Frozen

(21) The hybrid tumor cell line named monoclonal cell line has been deposited with the general microbial center of China General Microbial Culture Collection Committee (No. 3 yard 1 beichen west road chaoyang district Beijing) under Accession Number CGMCC No. 14697 on Sep. 5, 2017.

Example 4 Preparation and Identification of Cyproheptadine Monoclonal Antibody

(22) 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 1×106 hybrid tumor cells secreting cyproheptadine monoclonal antibody. From 7 days start collecting ascites, the monoclonal antibody was obtained after purification and preserved at −20° C.

(23) Using indirect competition ELISA, the IC50 value of the cyproheptadine monoclonal antibody was measured to be 0.37 ng/mL and the crossover value of the cyproheptadine analogue was measured to be less than 10%, which indicated that it has good sensitivity to cyproheptadine and could be used for cyproheptadine immunoassay.

Example 5

(24) Application of Cyproheptadine Monoclonal Antibody

(25) Monoclonal antibodies prepared by hybrid tumor cell lines were applied to the addition and recovery test of ELISA about cyproheptadine. The specific steps are as follows:

(26) The Solution Configuration

(27) Carbonate buffer solution (CBS): Weigh Na.sub.2CO.sub.3 1.59 g and NaHCO.sub.31.59 g, then mix 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), store at 4° C.;

(28) Phosphate Buffer solution (PBS): 8.00 g NaCl, 0.2 g KCl, 0.2 g KH.sub.2PO.sub.4, 2.9 g Na.sub.2HPO.sub.4.12H.sub.2O, dissolved in 800 mL pure water, pH was adjusted to 7.2-7.4 with NaOH or HCl, then the capacity was constant to 1000 mL;

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

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

(31) TMB Substrate: Mixture of solution A and solution B with 1:5; Solution A: Na.sub.2HPO.sub.4.12H.sub.2O 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.

(32) (2) Coating: coating antigen CLA-CMO-OVA had reacted for 2 h after serial dilution of the carbonate buffer (pH 9.6, 0.05 m) from 1 μg/mL, 100 μL/hole at 37° C.

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

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

(35) (5) Sample adding: antiserum was diluted from the ratio of 1:1000, and was added into the degree of the dilution of coating hole, 100 μL/hole, and had reacted at 70° C. for 30 min. After fully washing, HRP—Goat anti Mouse IgG that had diluted with the ratio of 1:3000 was added and reacted at 37° C. for 30 min, 100 μL/hole;

(36) (6) 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.

(37) (7) Termination and determination: To terminate the reaction, 50 μL termination solution was added to each hole, and then the value of OD.sub.450 of each hole was measured with an enzyme marker.

(38) (8) Reading result: The ELISA titer of serum was the highest dilution factor corresponding to the serum with the OD.sub.450 value greater than or equal to 2.1 times of the negative control hole (i.e., P/N≥2.1).

(39) The IC50 of the monoclonal antibody was 0.37 ng/mL, indicating a good sensitivity to cyproheptadine and can be used for cyproheptadine immunoassay.

(40) Serum Test Results of Mice after Three Immunizations:

(41) TABLE-US-00001 The concentra- The dilution tion of standard ratio of The concentration of coating antigen ng/ml mice serum 1 μg/ml 0.3 μg/ml 0.1 μg/ml  0 ng/ml 1k 2.983 2.623 1.278 3k 2.618 2.134 0.844 9k 1.973 1.328 1.497 20 ng/ml 1k 2.163 1.28 0.419 3k 1.597 0.751 0.248 9k 0.746 0.338 0.136 OD OD (Standard addition Small molecule screening Cell line (without concentration to be measured process number standard) Xng/ml) cyproheptadine After the 4A11 1.768 0.282(5 ng/ml).sup.  fusion 1 g 2H12 1.338 0.078(2 ng/ml).sup.  2 g 4B1 1.244 0.468(0.5 ng/ml) 3 g 3E′ 10 1.379 0.612(0.5 ng/ml)

Contrast Example 1: Preparation of Hybrid Tumor Cell Lines Secreting Cyproheptadine Monoclonal Antibody

(42) Replacing the coating antige in the above embodiments with BSA, it was found that cyproheptadine conjugated OVA was more effective as the coating antigen.

(43) TABLE-US-00002 The concentra- The concentra- The coating tion of tion of IC50 antigen coating antibody ODmax (ng/ml) CHP-OVA 40:1 0.3 μg/ml 0.3 μg/ml 1.457 0.37 CHP-BSA 60:1 0.3 μg/ml 0.3 μg/ml 1.895 0.514

Contrast Example 2: Preparation of Hybrid Tumor Cell Lines Secreting Cyproheptadine Monoclonal Antibody

(44) In the above embodiment, the number of addition and exemption was changed to the serum test results of mice after the Secondary immunization and exemption:

(45) TABLE-US-00003 The concentra- The dilution tion of standard ratio of The concentration of coating antigen ng/ml mice serum 1 μg/ml 0.3 μg/ml 0.1 μg/ml  0 ng/ml 1k 2.683 2.323 0.978 3k 2.318 1.834 0.544 9k 1.673 1.028 0.297 20 ng/ml 1k 2.141 1.318 0.519 3k 1.647 0.815 0.359 9k 0.864 0.479 0.018

(46) Compared with the three times booster immunizations, the titer and inhibition of serum after the two times booster immunizations were not as good as those after the three times booster immunizations.

(47) 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.