Anti-phenacetin monoclonal antibody hybridoma cell strain ad and its preparation method and application

Abstract

The invention discloses an anti-phenacetin monoclonal antibody hybridoma cell strain AD, a preparation method and application thereof, and relates to the technical field of food safety immunodetection. The monoclonal antibody hybridoma cell strain is named monoclonal cell strain AD and the number CGMCC19681. The Phe-BA obtained by the hydrolysis of the reaction product of the phenacetin metabolite acetaminophen and ethyl 4-bromobutyrate is used as the hapten, and the hapten is coupled with the carrier protein to prepare the immunogen Phe-BA-BSA. After the mice were immunized with the immunogen Phe-BA-BSA, they were fused with myeloma cells by PEG method, screened by indirect competitive enzyme-linked immunosorbent assay and subcloned five times to obtain hybridoma cell lines. The monoclonal antibody secreted by the cell line can be made into a phenacetin detection kit, which has good affinity and detection sensitivity for phenacetin, and can be used for immunodetection of phenacetin residues in food.

Claims

1. A hybridoma cell strain AD with anti-phenacetin monoclonal antibody, deposited at the China General Microbiological Culture Collection Center with Accession Number CGMCC No. 19681.

2. An anti-phenacetin monoclonal antibody secreted by the monoclonal hybridoma cell strain AD of claim 1.

3. A method for analyzing and detecting phenacetin residues in food comprising: adding the an anti-phenacetin monoclonal antibody of claim 2 to a subject to form a detection subject, and using the detection subject for analyzing and detecting phenacetin residues in a food sample, wherein the detection subject is a reagent, a detection plate, or a kit.

Description

BRIEF DESCRIPTION THE DRAWINGS

(1) FIG. 1 is the subtype identification of monoclonal antibody AD;

(2) FIG. 2 is the affinity determination of monoclonal antibody AD;

(3) FIG. 3 is a standard curve for the inhibition of phenacetin by monoclonal antibody AD.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

(4) The present invention will be further described below with reference to the accompanying drawings and embodiments.

EXAMPLE

(5) The present invention can be better understood from the following examples. However, those skilled in the art can easily understand that the specific material ratios, process conditions and results described in the examples are only used to illustrate the present invention and should not and will not limit the present invention described in detail in the claims.

(6) In the present invention, the mice immunized with the phenacetin complete antigen are subjected to cell fusion, cultured in a HAT selective medium, and the cell supernatant is screened by icELISA, and finally a monoclonal antibody hybridoma cell strain with high sensitivity to phenacetin is obtained.

(7) 1. Synthesis of Hapten

(8) (1) Synthesis of Hapten Phe-BA:

(9) The synthetic route is as follows:

(10) ##STR00002##

(11) Dissolve 0.20 g of acetaminophen in 2 mL of dimethyl sulfoxide, add 1.0 g of K.sub.2CO.sub.3, and stir for 0.5 h. Then, 0.77 g of ethyl 4-bromobutyrate was added under stirring. The mixture was heated to reflux at 110 C. for 24 h, and monitored by TLC. When the reaction is complete, stop heating and concentrated until the temperature of the reaction solution cooled down to room temperature; Then, 2 mL of 1 mol/L NaOH was added into the above solution, heating to 80 C. for 1 h under stirring. When the temperature calmed down to room temperature, the reaction solution which was adjusted to pH 4 with 1.0 mol L.sup.1 aqueous HCl., The solution was then washed with ethyl acetate three times, and the organic phase was collected, dried over anhydrous Na.sub.2SO.sub.4 and concentrated., After purification by silica gel column and concentration, a white solid was obtained, that is the hapten Phe-BA.

(12) 2. Preparation of Complete Antigen: The Hapten Phe-BA Prepared in the Above Step 1 is Coupled with Bovine Serum Albumin (BSA) to Obtain the Complete Antigen Phe-BA-BSA.

(13) The preparation method of the complete antigen Phe-BA-BSA is as follows:

(14) a. 2.85 mg of the hapten Phe-BA obtained in step (1) was dissolved in 200 L of anhydrous N,N-dimethylformamide, and then 1.6 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and 1.0 mg of N-hydroxysuccinimide (NHS) were added, and then the mixture was stirred at room temperature for 0.5 h to obtain liquid A. In addition, 10 mg of bovine serum albumin (BSA) was dissolved in 2 mL of boric acid buffer solution to obtain solution B. Then, the solution A was slowly added dropwise to solution B with constant stirring at room temperature for 2 h to obtain a mixture containing Phe-BA-BSA.

(15) b. Dialysis: The 10 cm dialysis bag was boiled in boiling water for 5 min, rinsed with deionized water at 60 C. for 3 min, and stored in deionized water at 4 C. for later use. The mixed solution containing Phe-BA-BSA in step (a) was placed in a dialysis bag, and 0.01 mol/L PBS was used as the dialysate, dialyzed at 4 C. for 3 d, and the dialysate was replaced three times a day to separate the complete antigen and unconjugated haptens and other small molecules. The complete antigen includes immunogen and coating antigen; wherein, the immunogen Phe-BA-BSA is used for the next step of mouse immunization; the coating antigen is used for the detection in the subsequent step 7.

(16) In the same way, the complete antigen Phe-BA-OVA is obtained by coupling the hapten Phe-BA and ovalbumin (OVA), including the immunogen and the coating antigen. The immunogen is used for the next step of mouse immunization, and the coating antigen is used for the detection in the subsequent step 7.

(17) In the present Experimental example, the subsequent immunization and detection-related steps are specifically described by using the immunogen Phe-BA-BSA and the coating original Phe-BA-OVA as examples.

(18) 3. Immunization of Mice: The Injection was Formed by Emulsification of Phe-BA-BSA Immunogen Mixed with an Equal Volume of Freund's Adjuvant, and Subcutaneously Injected into BALB/c Mice Through the Back of the Neck.

(19) For the first immunization (100 g/only), an equal volume of mixed emulsion of Freund's complete adjuvant and phenacetin immunogen was used as an injection. For 5 boosting immunizations, an equal volume emulsion of incomplete Freund's adjuvant and phenacetin immunogen was used as an injection. One month between the first immunization and the first booster immunization, and 21 days between multiple booster immunizations. The last sprint immunization was performed with 50 L of water diluted to 0.5 mg/mL Phe-BA-BSA immunogen (25 g/only, no adjuvant). Serum titers and inhibition rates were detected by indirect competitive enzyme-linked immunosorbent assay (icELISA).

(20) 4. Cell Fusion

(21) Three days after the rush immunization, cell fusion is carried out according to the conventional PEG method. The specific steps are as follows:

(22) a. The eyeballs were removed to collect blood, and the Phe-BA-BSA-immunized mice were killed by cervical dislocation, and then immediately put into 75% alcohol for disinfection and soaked for about 5 minutes. The spleen of the mouse was aseptically removed, moderately ground with the tip of a syringe and passed through a 200-mesh cell screen to obtain a spleen cell suspension. The suspension was collected, centrifuged (1200 rpm, 8 min), and the splenocytes were washed three times with RPMI-1640 medium. After the last centrifugation, the splenocytes were diluted to a certain volume, counted, and used for later use.

(23) b. Collection of murine myeloma SP2/0 cells: 7-10 days before cell fusion, SP2/0 tumor cells were cultured in RPMI-1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO.sub.2 incubator. Before fusion, the number of SP2/0 tumor cells was required to reach 110.sup.7, to ensure that the SP2/0 tumor cells were in the logarithmic growth phase before fusion. At fusion, tumor cells were collected, suspended in RPMI-1640 basal medium, and counted.

(24) c. The fusion process was 7 min. The first minute, 1 mL of PEG 1500 was added dropwise to the cells from slow to fast; the second minute, let stand. On the 3rd and 4th minutes, drop 1 mL of RPMI-1640 medium within 1 minute; on the 5th and 6th minutes, dropwise add 2 mL of RPMI-1640 medium within 1 minute; on the 7th minute, every 10 s Add 1 mL of RPMI-1640 medium dropwise. Then, the above cell mixture was placed in a 37 C. warm bath for 5 min, centrifuged (800 rpm, 8 min), and the supernatant was discarded to obtain a precipitate. 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.

(25) 5. Cell Screening and Cell Strain Establishment

(26) 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.

(27) The screening is divided into two steps: the first step is to select positive cell wells by icELISA, the second step is to use the phenacetin as a standard and measure the inhibitory effect of the positive cells by icELISA. Cell wells with a good inhibitory effect to the phenacetin standard are selected and subcloned by a limiting dilution method. The same method is used for detection and repeat three times to obtain a cell strain AD.

(28) 5. Preparation and Identification of Anti-Phenacetin Monoclonal Antibody

(29) 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. The purified anti-phenacetin monoclonal antibody was finally obtained and stored at 20 C.

(30) Using the mouse monoclonal antibody subtype identification kit to identify the immunoglobulin subtype of the anti-phenacetin monoclonal antibody purified from ascites, its subtype is IgG2b type, and it was detected by the mouse monoclonal antibody subtype identification kit. The chain type is the kappa type, as shown in FIG. 1.

(31) The affinity of anti-phenacetin monoclonal antibody determined by indirect ELISA was 5.3610.sup.8 L/mol, as shown in FIG. 2. The sensitivity to phenacetin was detected by icELISA, as shown in FIG. 3. According to the standard equation y=0.3479 x+0.6733 (R.sup.2=0.9863), the IC.sub.50 was calculated to be 3.0 g/L.

(32) The above results show that the prepared anti-phenacetin monoclonal antibody has high affinity and sensitivity and can be used for phenacetin immunoassay detection and the preparation of affinity columns.

(33) 7. Antibody Application

(34) The anti-phenacetin monoclonal antibody prepared by the hybridoma cell strain AD through in vivo ascites was applied to the addition and recovery test of phenacetin, and the specific steps were as follows: 7.1 Coating: The coating antigen Phe-BA-OVA obtained in the previous step 2 was diluted to 0.1 g/mL with 0.05 mol L1 pH 9.6 carbonate buffer, 100 L/well, at 37 C. for 2 h. 7.2 Washing: The solution in the plate was poured out and washed three times with washing solution, 3 min each time. 7.3 Closure: the plate is closed with blocking solution, 200 L per well, at 37 C. for 2 h; the plate is washed and dried for later use; 7.4 Add sample:

(35) 100 L of PBS was added to the positive control wells; 100 L of phenacetin standard solution with a concentration of 0.350 g/L was added to the detection wells. Then, the anti-phenacetin monoclonal antibody was diluted with 0.01 mol L.sup.1 PBS to 0.1 g/mL, and added to the coated wells of each dilution, 100 L/well, and reacted at 37 C. for 30 min; Then the plate is thoroughly washed. 100 L of HRP-goat anti-mouse IgG diluted at a ratio of 1:3000 is added to each well and reacts at 37 C. for 30 min. 7.5 Color rendering: The ELISA plate was taken out, washed thoroughly, added 100 L of TMB color developing solution to each well, and reacted at 37 C. for 15 min in the dark. 7.6 Termination and determination: 50 L of stop solution was added to each well to stop the reaction, and then the OD450 value of each well was measured with a microplate reader.

(36) The IC.sub.50 of anti-phenacetin monoclonal antibody determined by icELISA was 3.0 g/L. It shows that it has high sensitivity to phenacetin and can be used for phenacetin immunoassay detection.

(37) In the above steps, the configuration of each solution is as follows:

(38) 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.

(39) 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.Math.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.

(40) PBST: PBS Containing 0.05% Tween 20.

(41) 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).

(42) The above is only the preferred embodiment of the invention. It should be pointed out that for ordinary technicians in the technical field, several improvements and refinements can be made without departing from the principle of the invention, and these improvements and refinements should also be regarded as the protection scope of the invention.

(43) The referenced hybridoma cell strain AD (CGMCC No. 19681) was deposited on Apr. 23, 2020, in the China General Microbiological Culture Collection Center (CGMCC) and is identified by Accession Number of CGMCC No. 19681.

(44) The China General Microbiological Culture Collection Center (CGMCC), which is located at No. 3, Yard. 1, Beichen West Road, Chaoyang District, Beijing, China, 100101, is an International Depository Authority under the provisions of the Budapest Treaty.

(45) The referenced deposit was made under the provisions of the Budapest Treaty. The deposits will be replaced by Applicant if they should become non-viable or non-replicable. (i) During the pendency of this application, access to the invention will be afforded to the Commissioner upon request; (ii) all restrictions upon availability to the public will be irrevocably removed upon granting of the patent based on this application, with Applicant reserving the right to employ the reporting conditions allowed by the patent owner as expressly recited within 37 C.F.R. 1.808(b), pursuant to 37 C.F.R. 1.808(a)(2); (iii) the deposit will be maintained in a public depository for a period of 30 years or 3 years after the last request or for the effective life of the patent, whichever is longer; (iv) a test of the viability of the biological material at the time of deposit was performed in accordance with 37 C.F.R. 1.807; and (v) the deposit is capable of reproduction under 37 C.F.R. 1.806.