Hybridoma cell strain secreting nifursolol residue marker monoclonal antibody

Abstract

A hybridoma cell strain secreting a nifursol residue marker monoclonal antibody prepared in the following way: BALB/c mice are subjected to the first immunization with a complete Freund's adjuvant, subjected to booster immunization with an incomplete Freund's adjuvant for four times, and subjected to rush immunization once with nifursol residue marker complete antigen without a Freund's adjuvant so that the BALB/c mice are immunized; the spleen cells of the immunized mice with high titer and low IC50 were fused with mouse myeloma cells by a PEG method, and the fused cells are screened through indirect competitive ELISA and subcloned three times. The monoclonal antibody secreted by this cell strain has good specificity and detection sensitivity (IC50 value of 2 g/L) to the nifursol residue marker and can be used for residue detection of the nifursol residual marker in food.

Claims

1. A method of making a hybridoma cell strain secreting a nifursol residue marker monoclonal antibody comprising: step 1: preparing a nifursol residue marker (DNSH) hapten, synthesizing a DNSH complete antigen through the DNSH hapten, mixing the DNSH complete antigen with an oil, and then adding an emulsifier for emulsifying to obtain an incomplete Freund's adjuvant, and adding mycobacteria to the incomplete Freund's adjuvant to obtain a complete Freund's adjuvant, wherein the oil is a paraffin oil or a vegetable oil, the emulsifier is lanolin or polysorbate 80, and the mycobacteria comprise dead BCG; step 2: subcutaneously injecting the prepared Freund's adjuvant into BALB/c mice in back for multiple immunizations, wherein the complete Freund's adjuvant is used in first immunization and the incomplete Freund's adjuvant is used in booster immunization; step 3: collecting blood from the mice subjected to the immunization process, and detecting the serum immunopotency and immunosuppressive ability of the mice by indirect enzyme-linked immunosorbent assay (ELISA), and selecting the immunized mice with a high DNSH antibody content in serum; step 4: performing the final booster immunization on the selected mice with the incomplete Freund's adjuvant, and then performing further immunization by intraperitoneal injection, wherein the further immunization is carried out using the DNSH complete antigen without a Freund's adjuvant; and step 5: fusing spleen cells of the BALB/c mice subjected to the further immunization with myeloma cells; culturing the fused cells through a medium, detecting positive cell wells by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), and further determining the inhibitory effect of the positive cell wells by ic-ELISA; subcloning the positive cell wells with the best inhibitory effect by a limiting dilution method; and finally screening to obtain the hybridoma cell strain capable of secreting a nifursol residue marker monoclonal antibody; wherein the molecular formula of the DNSH hapten in step 1 is as follows: ##STR00009## the molecular formula of the DNSH complete antigen in step 1 is as follows: ##STR00010##

2. The method of claim 1 further comprising: dissolving p-carboxybenzaldehyde in water, and adding HCl solution to obtain a first mixed solution; adding N,N-dimethylformamide (DMF) and a nifursol residue marker (DNSH) to the first mixed solution, stirring and refluxing to obtain a second mixed solution; centrifuging, washing and drying the obtained second mixed solution to obtain a crude product; and separating the crude product to obtain the DNSH hapten.

3. The method of claim 1 further comprising: dissolving a nifursol residue marker hapten, EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) in DMF, and stirring for activation to obtain a solution A; dissolving KLH in a carbonate buffer solution (CB solution) to obtain a solution B; and adding the solution A to the solution B, and stirring for reaction to obtain the nifursol residue marker complete antigen.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In the drawings:

(2) FIG. 1 is a standard curve of inhibition of the nifursol residue marker monoclonal antibody of the invention against the nifursol residue marker.

DESCRIPTION OF PREFERRED EMBODIMENTS

(3) Media involved in the following embodiments are as follows:

(4) RPMI-1640 medium (mg/L): L-arginine 290, L-asparagine 50, L-aspartate 20, L-cystine dihydrochloride 65.15, L-glutamic acid 20, glycine 10, L-histidine 15, L-hydroxyproline 20, L-isoleucine 50, L-leucine 50, L-lysine hydrochloride 40, L-methionine 15. L-phenylalanine 15, L-proline 20, L-serine 30, L-threonine 20, L-tryptophan 5, L-tyrosine 23.19, L-valine 20, p-aminobenzoic acid 1, calcium nitrate 100, anhydrous magnesium sulfate 48.84, anhydrous sodium dihydrogen phosphate 676.13, potassium chloride 400, sodium chloride 6000, glucose 2000, reduced glutathione 1, phenol red 5, L-glutamine 300, biotin 0.2, D-calcium pantothenate 0.25, folic acid 1, i-inositol 35, nicotinamide 1, choline chloride 3, pyridoxine hydrochloride 1, riboflavin 0.2, thiamine hydrochloride 1, vitamin B12 0.005, sodium bicarbonate 2000.

(5) Reagents involved in the following embodiments are as follows:

(6) Carbonate buffer solution (CBS): 1.59 g of Na.sub.2CO.sub.3 and 2.93 g of NaHCO.sub.3 are weighed and separated dissolved in a small amount of double distilled water; the two solutions are mixed; double distilled water is added to the mixed solution till about 800 mL and the mixed solution is mixed to be uniform; the pH is adjusted to 9.6, and double distilled water is added till the mixed solution reaches 1000 mL and the obtained solution is stored at 4 C. for later use.

(7) Phosphate buffer solution (PBS): 8.00 g of NaCl, 0.2 g of KCl, 0.2 g of KH.sub.2PO.sub.4, 2.9 g of Na.sub.2HPO.sub.4.12H.sub.2O are dissolved in 800 mL of pure water, the pH is adjusted to 7.2-7.4 with NaOH or HCl, and the solution is maintained at a constant volume of 1000 mL.

(8) Wash solution (PBST): 0.5 mL of TWEEN 20 (polysorbate 20) is added to 1000 mL of a 0.01 mol/L PBS solution (pH 7.4).

(9) PBST: PBS containing 0.05% TWEEN 20 (polysorbate 20).

(10) Antibody dilution: a wash buffer solution containing 0.1% gelatin.

(11) TMB developing solution: Solution A: 18.43 g of Na.sub.2HPO.sub.4.12H.sub.2O and 9.33 g of citric acid are added with pure water to 100 mL; B solution: 60 mg of TMB is dissolved in 1000 mL of ethylene glycol. The solution A and the solution B are mixed at a ratio of 1:5 to obtain TMB (a developing solution, mixed when necessary).

(12) Detection methods involved in the following embodiments are as follows:

(13) Method for detecting the inhibition rate of a nifursol residue marker standard: DNSH standard solutions with concentrations of 0 g/L, 0.5 g/L, 1 g/L, 2 g/L, 5 g/L, 10 g/L, 20 g/L and 40 g/L are prepared respectively with the PBS; the standard solutions are added to a closed ELISA plate, 50 L per well, 2 wells per solution, 50 L of an anti-DNSH monoclonal antibody diluted at a ratio of 1:32000 is then added to each well and reacts at 37 C. for 0.5 h, and then the plate is washed and patted dry; 100 L of an HRP-labeled goat anti-mouse IgG secondary antibody diluted with the PBS containing 0.1% gelatin at a ratio of 1:3000 is added to each well, and reacts at 37 C. for 0.5 h, and then the plate is washed and patted dry; 100 L of the TMB developing solution is added to each well; after development at 37 C. for 15 min, 50 L of 2M H.sub.2SO.sub.4 stop solution is added to each well, and the absorbance (OD) is measured at 450 nm; an inhibition standard curve is drawn using Origin plotting software (results shown in FIG. 1).

Example 1

(14) Synthesis of a Nifursol Residue Marker Hapten

(15) 0.5 g of p-carboxybenzaldehyde is dissolved in 10 ml of water, 3 ml of HCl solution with a concentration of 1 mol/L is added to obtain a mixed solution 1; and DMF and 1.02 g of a nifursol residue marker (DNSH) are added to the mixed solution while magnetically stirring, and then the mixed solution is stirred magnetically at 60 C. and refluxed overnight to obtain a mixed solution 2; the obtained mixed solution 2 is centrifuged, washed and dried to obtain a crude product; and column chromatography separation is carried out on the crude product to obtain the nifursol residue marker hapten.

Example 2

(16) Synthesis of a Nifursol Residue Marker Complete Antigen

(17) 4.5 mg of a nifursol residue marker hapten, 2.0 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 1.0 mg of NHS (N-hydroxysuccinimide) are dissolved in DMF (N,N-dimethylformamide), and stirred for activation at room temperature for 4 h to obtain a solution A; 5 mg of KLH is dissolved in 2 mL of the CB solution with a concentration of 0.05 mol/L and a pH of 9.6 to obtain a solution B; and the solution A is dropwise added to the solution B, and stirred at room temperature for reaction overnight to obtain a nifursol residue marker complete antigen.

Example 3

(18) Synthesis of a Nifursol Residue Marker Coating Antigen

(19) 5 mg of thiamethoxam hapten (TMX-COOH) and 4.8 mg of N-hydroxysuccinimide (NETS) are dissolved in 300 L of anhydrous N,N-dimethylformamide (DMF), and the solution is stirred at room temperature to react for 10 min to obtain a TMX-COOH solution; 7.6 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) is dissolved in 100 L of anhydrous DMF and then added to the TMX-COOH solution, and stirred at room temperature to react for 6-8 h to obtain a solution A; 10 mg of chicken OVA is diluted with 1 mL of a phosphate buffer solution (PBS) with a concentration of 0.01 mmol/L and a pH of 7.4 to obtain a solution B; the solution A is slowly added dropwise to the solution B to react to obtain a reaction solution; the reaction solution is dialyzed with the PBS solution to remove the unreacted small molecular hapten to obtain a coating antigen (TMX-COOH-OVA).

Example 4

(20) Preparation of a Hybridoma Cell Strain Secreting a Nifursol Residue Marker Monoclonal Antibody

(21) 1. Acquisition of Immunity in Animals

(22) Healthy 6-8 week old BALB/c mice are selected for immunization. After a DNSH complete antigen (1 mg/mL) is emulsified uniformly with an equal amount of a Freund's adjuvant, BALB/c mice are immunized with the emulsified DNSH complete antigen by subcutaneous injection at a dose of 100 L for each mouse. The first immunization is performed with a complete Freund's adjuvant, the booster immunization is performed with an incomplete Freund's adjuvant, and the immunization dose for the rush immunization is half of the dose for previous immunization; the Freund's adjuvants are directly used through intraperitoneal injection after being mixed with normal saline; the immunization interval of the immunizations is three weeks. After the third immunization, blood samples are taken at intervals of one week to detect serum titer and inhibitory effect; the mice with the best inhibitory effect are selected and are subject to rush immunization 21 days after the fifth immunization.

(23) 2. Cell Fusion

(24) Three days after the rush immunization, cell fusion is carried out according to the conventional PEG (polyethylene glycol, with a molecular weight of 4000) method. The specific steps are as follows:

(25) (1) The mouse spleens are aseptically taken, ground and screened through a 200-mesh cell sieve to obtain a spleen cell suspension, and cells are counted;

(26) (2) SP2/0 cells are collected, suspended in a RPMI-1640 basal medium, and subjected to cell counting;

(27) (3) Spleen cells and SP2/0 cells are mixed at a ratio of 1:10, centrifuged and fused with 50% PEG for 1 min, then the RPMI-1640 basal medium is added from slow to fast, the solution is centrifuged and then suspended in an RPMI-1640 screening medium containing 20% fetal bovine serum and 2% 50HAT, and then added to a 96-well cell culture plate and cultured in an incubator at 37 C. in a 5% CO.sub.2 atmosphere.

(28) 3. Cell Screening and Cell Strain Establishment

(29) The medium of the fused cells is semi-changed with an RPMI-1640 screening medium on the third day of cell fusion, and then completely changed with an RPMI-1640 transition medium containing 20% fetal bovine serum and 1% 100HT on the 5th day. The cell supernatant is taken for screening on the 7th day.

(30) The screening is divided into two steps: the first step is to select positive cell wells by ic-ELISA, the second step is to use a nifursol residue marker as a standard and measure the inhibitory effect of the positive cells by ic-ELISA.

(31) Cell wells with a good inhibitory effect to the nifursol residual marker standard are selected and subcloned by a limiting dilution method. The same method is used for detection and repeated three times to obtain a cell strain.

Embodiment 5: Preparation and Identification of a Nifursol Residue Marker Monoclonal Antibody

(32) 8-10 weeks old BALB/c mice are taken and each intraperitoneally injected with 1 mL of sterile paraffin oil, and 7 days later, intraperitoneally injected with a 110.sup.6 hybridoma cell strain; ascites is collected from the 7th day and purified by an octanoic acid-ammonium sulfate method.

(33) Under an acidic condition, n-octanoic acid can precipitate heterologous proteins other than IgG immunoglobulin in ascites, then centrifugation is performed to remove the precipitate; the IgG monoclonal antibody is then precipitated with an ammonium sulfate solution with the equivalent saturability, centrifuged to remove the supernatant, dissolved in 0.01 M PBS solution (with a pH of 7.4), and dialyzed for desalting to finally obtain the purified monoclonal antibody, and the purified monoclonal antibody is stored at 20 C.

(34) By using an indirect competitive ELISA, the IC.sub.50 of the monoclonal antibody is determined to be 2 g/L, indicating a good sensitivity to DNSH. Thus the monoclonal antibody can be used for nifursol residue immunoassay.

Embodiment 6: Application of the Nifursol Residue Marker Monoclonal Antibody

(35) The monoclonal antibody prepared from the hybridoma cell strain via in-vivo ascites is applied to the ELISA addition recovery test of DNSH, and the specific steps are as follows:

(36) (1) the coating antigen with a coating concentration of 0.3 g/mL diluted with the CBS is used to coat a 96-well ELISA plate, 100 L per well, at 37 C. for 2 h; the plate is then washed with the PBST wash solution three times, 200 L per well, 3 min each time, and patted dry;

(37) (2) the plate is closed with the CBS containing 0.2% gelatin, 200 L per well, at 37 C. for 2 h; the plate is washed with the PBST wash solution three times, 200 L per well, 3 min each time, and patted dry;

(38) (3) the DNSH standard solutions with concentrations of 0 g/L, 0.5 g/L, 1 g/L, 2 g/L, 5 g/L, 10 g/L, 20 g/L and 40 g/L are prepared respectively with the PBS; the standard solutions and to-be-tested samples are respectively added to the closed ELISA plate, 50 L per well, 3 wells for each sample; 50 L of an anti-DNSH monoclonal antibody diluted at a ratio of 1:32000 is then added to each well and reacts at 37 C. for 0.5 h, and then the plate is washed and patted dry;

(39) (4) 100 L of an HRP-labeled goat anti-mouse IgG secondary antibody diluted with the PBS containing 0.1% gelatin at a ratio of 1:3000 is added to each well, and reacts at 37 C. for 0.5 h, and then the plate is washed and patted dry;

(40) (5) 100 L of the TMB developing solution is added to each well; after development at 37 C. for 15 min, 50 L of 2M H.sub.2SO.sub.4 stop solution is added to each well, and the absorbance (OD) is measured at 450 nm;

(41) (6) Addition recovery and sample preparation:

(42) three parts of 20.02 g homogenized chicken are weighed and placed in a 50 ml centrifuge tube respectively, and then added with 15 ml of a mixed solution of methanol and water (1:1), shaken for 10 min, and centrifuged at 4000 r/min for 5 min to remove liquid; the residue is added with 10 ml of 0.2 mol/L hydrochloric acid, homogenized at 10000 r/min for 1 min, and then added with 5 ppb, 10 ppb, 100 ppb DNSH respectively, mixed uniformly by vortexing for 30 s, and then shaken for 30 min. 2 ml of 0.3 mol/L potassium phosphate is added and the pH is adjusted to 7.4 with 0.2 mol/L NaOH, 10 ml of ethyl acetate is added, and the mixture is shaken for extraction for 10 min, centrifuged at 10,000 r/min for 10 min, and the ethyl acetate layer is collected. The ethyl acetate layer is dried at 40 C. with N2, dissolved in 1 ml of a 0.1% aqueous solution of formic acid, and then defatted with 3 ml of acetonitrile saturated n-hexane, and the aqueous phase at the bottom is used as an ELISA sample extract.

(43) The addition recovery test is carried out by indirect competitive ELISA, and the recovery rates are 85%, 86%, and 90%, respectively.

(44) Although the invention has been disclosed in the above preferred embodiments, the invention is not limited thereto, and any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. The scope of the invention should be defined by the claims.