CN112813036A - Triamcinolone acetonide monoclonal antibody hybridoma cell strain and application thereof - Google Patents
Triamcinolone acetonide monoclonal antibody hybridoma cell strain and application thereof Download PDFInfo
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Abstract
A triamcinolone acetonide monoclonal antibody hybridoma cell strain and application thereof belong to the field of food safety immunodetection. The hybridoma cell strain STD6B11 secreting triamcinolone acetonide monoclonal antibody is classified and named as monoclonal cell strain, the preservation date is 2020, 9 and 27 days, and the preservation number is CGMCC No. 20783. The monoclonal antibody secreted by the cell strain has the effect on triamcinolone acetonideHas better specificity and detection sensitivity. The invention synthesizes triamcinolone acetonide hapten, prepares a triamcinolone acetonide complete antigen, mixes and emulsifies the triamcinolone acetonide complete antigen and equivalent Freund's adjuvant completely, immunizes a BALB/c mouse through back subcutaneous injection, and obtains a hybridoma cell strain STD6B11 through screening and three times of subcloning by an indirect competitive enzyme-linked immunosorbent assay. The monoclonal antibody secreted by the cell strain has better specificity and detection sensitivity (IC) on triamcinolone acetonide500.49 ng/mL). The results of the invention can be used for establishing an immunoassay method for the triamcinolone acetonide content in pork and other food containing triamcinolone acetonide, and have practical application value.
Description
Technical Field
The invention relates to a triamcinolone acetonide monoclonal antibody hybridoma cell strain and application thereof, and belongs to the field of food safety immunodetection.
Background
Triamcinolone acetonide is a common glucocorticoid and has the functions of resisting allergy, resisting inflammation, treating skin diseases and the like. Meanwhile, the glucocorticoid can greatly improve the growth rate of animals, so that the glucocorticoid is often used for the breeding of economic animals by illegal farmers in large quantity, and the abuse behaviors can cause triamcinolone acetonide to have residues in the animal bodies to different degrees. Research shows that the health risk of consumers caused by the residual triamcinolone acetonide in food-borne animal tissues is great, and the metabolic disturbance of the organism can be caused by long-term consumption of animal-borne food containing the residual triamcinolone acetonide. Therefore, the method for effectively detecting the triamcinolone acetonide residue is established, and has profound significance for effectively controlling the triamcinolone acetonide residue in pork and other products and guaranteeing the health of the masses.
For the veterinary drug triamcinolone acetonide residue, a residue detection method is lacked at present, and in order to maintain the benefits of wide consumers, an efficient and rapid detection method for triamcinolone acetonide is needed to be established. The enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive and rapid detection method, has the advantages of simple pretreatment of a sample during detection, few purification steps, large analysis capacity, low detection cost and simple and convenient operation, and is suitable for the field rapid detection of a large number of samples, so the ELISA is widely applied to veterinary drug residue analysis. The precondition for detecting triamcinolone acetonide by using an enzyme linked immunosorbent assay is that a monoclonal antibody with high specificity and high sensitivity to triamcinolone acetonide is obtained, so that the key is to find a method for preparing the monoclonal antibody with high specificity and high sensitivity to triamcinolone acetonide.
Disclosure of Invention
The invention aims to provide a hybridoma cell strain of a triamcinolone acetonide monoclonal antibody and application thereof.
The invention aims to provide a triamcinolone acetonide monoclonal antibody hybridoma cell strain STD6B11 which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and is named as a monoclonal cell strain by classification, wherein the preservation date is 2020, 9 months and 27 days, and the preservation number is CGMCC No.20783, and the institute for microorganisms of China academy of sciences No. 3, Xilu No. 1, North Cheng, of the Chaoyang region, Beijing.
The second purpose of the invention is to provide a triamcinolone acetonide monoclonal antibody which is secreted and produced by the hybridoma cell strain STD6B11 with the preservation number of CGMCC No. 20783.
The third purpose of the invention is to provide the application of the triamcinolone acetonide monoclonal antibody, establish an immunoassay method of the triamcinolone acetonide content, and apply the triamcinolone acetonide content in food to be detected.
In one embodiment of the invention, the application is used for detecting pork and other food containing triamcinolone acetonide.
The fourth purpose of the invention is to provide a preparation method of the triamcinolone acetonide hapten, which comprises the following steps: adding triamcinolone acetonide into succinic anhydride dissolved in anhydrous pyridine, and carrying out water bath reaction; stopping heating, cooling to room temperature, transferring the reactant into cooled hydrochloric acid in advance, and separating out a large amount of white precipitate; washing with deionized water for several times, and drying to obtain white solid, namely the hapten of the triamcinolone acetonide.
Further, the specific steps are as follows: adding 570mg of triamcinolone acetonide into 5mL of anhydrous pyridine in which 600mg of succinic anhydride is dissolved, and carrying out water bath reaction at 60 ℃ for 6 hours; stopping heating, cooling to room temperature, transferring the reactant to 20mL of hydrochloric acid with the mass concentration of 10% and cooling to 4 ℃ in advance, and separating out a large amount of white precipitate; washing with deionized water for several times, and drying to obtain white solid, namely the hapten of the triamcinolone acetonide.
The preparation method of the triamcinolone acetonide complete antigen comprises the following steps: weighing triamcinolone acetonide hapten, dissolving the triamcinolone acetonide hapten in dimethyl formamide DMF, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC and N-hydroxysuccinimide NHS, and stirring at room temperature to obtain a reaction liquid A1; weighing BSA, and dissolving in borate buffer solution to obtain solution B1; and then, dropwise adding the A1 solution into the B1 solution, reacting at room temperature to obtain a conjugate triamcinolone acetonide-BSA mixed solution, and separating a complete antigen and an unconjugated small molecule hapten through dialysis to obtain the immunogen triamcinolone acetonide-BSA.
Further, the preparation method comprises the following specific steps: weighing 1.89mg of triamcinolone acetonide hapten, dissolving the triamcinolone acetonide hapten in 450 mu L of DMF, adding 2.92mg of EDC and 1.75mg of NHS, and stirring the mixture at room temperature for 6 hours to obtain a reaction solution A1; weighing 5mg BSA, and dissolving in 0.1M borate buffer solution to obtain solution B1; and then, dropwise adding the A1 solution into the B1 solution, reacting for 8 hours at room temperature to obtain a conjugate triamcinolone acetonide-BSA mixed solution, and separating a complete antigen and an unconjugated small molecule hapten through dialysis to obtain the immunogen triamcinolone acetonide-BSA.
The preparation method of the triamcinolone acetonide complete antigen comprises the following steps: dissolving triamcinolone acetonide hapten, 1-ethyl carbodiimide hydrochloride and N-hydroxysuccinimide in anhydrous N, N-dimethylformamide to obtain solution A2, and stirring at room temperature for reacting for 6 h; weighing chicken egg white albumin OVA, dissolving the OVA in borate buffer solution to obtain B2 solution, dropwise adding A2 solution into B2 solution at room temperature, reacting at room temperature to obtain a conjugate triamcinolone acetonide-OVA mixed solution, and dialyzing to separate the coating antigen and the unconjugated small molecule hapten to obtain the coating antigen triamcinolone acetonide-OVA.
Further, the preparation method comprises the following steps: weighing 2.68mg of triamcinolone acetonide hapten, 3.94mg of 1-ethyl carbodiimide hydrochloride and 2.50mg of N-hydroxysuccinimide, dissolving the 2.68mg of triamcinolone acetonide hapten, 1-ethyl carbodiimide hydrochloride and the N-hydroxysuccinimide hydrochloride in 450 mu L of anhydrous N, N-dimethylformamide to obtain solution A2, and stirring the solution at room temperature for reacting for 6 hours; weighing 5mg of chicken ovalbumin OVA, dissolving the OVA in 3mL of 0.1M borate buffer solution to obtain a B2 solution, dropwise adding the A2 solution into the B2 solution at room temperature, reacting at room temperature for 8 hours to obtain a conjugate triamcinolone acetonide-OVA mixed solution, and separating the coating antigen and the unconjugated small molecule hapten through dialysis to obtain the coating antigen triamcinolone acetonide-OVA.
The screening method of the triamcinolone acetonide monoclonal antibody hybridoma cell strain STD6B11 mainly comprises the following steps:
(1) immunization of mice: mixing and emulsifying an immunogen triamcinolone acetonide-BSA and an equivalent amount of Freund adjuvant, and then injecting the mixture to immunize a BALB/c mouse through back subcutaneous injection; complete Freund adjuvant is used for the first immunization, incomplete Freund adjuvant is used for the multiple times of boosting immunization, the interval between the first immunization and the second boosting immunization is 28 days, the interval between the multiple times of boosting immunization is 21 days, and the final immunization is carried out by using triamcinolone acetonide-BSA complete antigen (without adjuvant) in a puncture way; detecting serum titer and inhibition by an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA);
(2) cell fusion and cell line establishment: fusing mouse spleen cells and mouse myeloma cells by a polyethylene glycol (PEG 4000) method, culturing by HAT culture medium, detecting positive cell holes by an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), further determining the inhibition effect of the positive cell holes by the ic-ELISA, carrying out three times of subcloning on the positive cell holes with the best inhibition effect by a limiting dilution method, and finally screening to obtain a triamcinolone acetonide monoclonal antibody hybridoma cell strain STD6B 11;
(3) and (3) identification of the properties of hybridoma cell strains: sensitivity and specificity were determined by ic-ELISA.
The invention has the beneficial effects that: the triamcinolone acetonide monoclonal antibody hybridoma cell strain STD6B11 provided by the invention secretesThe monoclonal antibody has better specificity and detection sensitivity (IC) on triamcinolone acetonide50The value is 0.49ng/mL), and an immunological method is provided for detecting the content of the triamcinolone acetonide in pork and other food containing the triamcinolone acetonide. The triamcinolone acetonide monoclonal antibody hybridoma cell strain and the monoclonal antibody secreted by the triamcinolone acetonide monoclonal antibody hybridoma cell strain can be prepared into a kit for detecting triamcinolone acetonide, and have practical application values.
Biological material sample preservation: a triamcinolone acetonide monoclonal antibody hybridoma cell strain STD6B11 is deposited in China general microbiological culture Collection center (CGMCC), China academy of sciences, China institute of microbiology, No. 3, West Lu 1 institute of North Chen, south China, Kyoho, Beijing, and is classified and named as a monoclonal cell strain, the preservation date is 2020, 9 months and 27 days, and the preservation number is CGMCC No. 20783.
Drawings
FIG. 1 Standard inhibition Curve of the STD6B11 triamcinolone acetonide monoclonal antibody against triamcinolone acetonide.
Detailed Description
The following examples are provided as further illustration of the invention and are not to be construed as limitations or limitations of the invention. The invention is further illustrated by the following examples.
According to the invention, a mouse is immunized by the triamcinolone acetonide complete antigen, cell fusion and HAT selective culture medium culture are carried out, and cell supernatant is screened by ic-ELISA, so that the monoclonal antibody hybridoma cell strain STD6B11 with good specificity and sensitivity to triamcinolone acetonide is finally obtained.
EXAMPLE 1 preparation of hybridoma cell line STD6B11
(1) Preparation of complete antigen:
a. the hapten synthetic route is shown as the following formula:
triamcinolone acetonide 570mg was added to succinic anhydride 600mg dissolved in anhydrous pyridine (5mL) and reacted in a water bath at 60 ℃ for 6 hours. Stopping heating, cooling to room temperature, transferring the reactant into 20mL of 10% hydrochloric acid which is placed into a refrigerator in advance for cooling, separating out a large amount of white precipitate, washing with deionized water for a plurality of times, and drying to obtain a white solid, namely the hapten of the triamcinolone acetonide;
b. preparation of immunogen triamcinolone acetonide-BSA: 1.89mg of triamcinolone acetonide hapten is weighed and dissolved in 450 microliter DMF, then 2.92mg of EDC and 1.75mg of NHS are added, and the mixture is stirred for 6 hours at room temperature to obtain a reaction solution A1; weighing 5mg BSA, and dissolving in 0.1M borate buffer solution to obtain solution B1; and then, dropwise adding the A1 solution into the B1 solution, reacting for 8 hours at room temperature to obtain a conjugate triamcinolone acetonide-BSA mixed solution, and separating a complete antigen and an unconjugated small molecule hapten by dialysis to obtain immunogen triamcinolone acetonide-BSA.
(2) Preparation of coated original triamcinolone acetonide-OVA:
weighing 2.68mg of triamcinolone acetonide hapten, 3.94mg of 1-ethyl carbodiimide hydrochloride and 2.50mg of N-hydroxysuccinimide, dissolving the triamcinolone acetonide hapten, the 1-ethyl carbodiimide hydrochloride and the N-hydroxysuccinimide in 450 mu L of anhydrous N, N-dimethylformamide to obtain solution A2, and stirring the solution at room temperature for reacting for 6 hours. Weighing 5mg of chicken ovalbumin OVA, dissolving the OVA in 3mL of 0.1M borate buffer solution to obtain a B2 solution, dropwise adding the A2 solution into the B2 solution at room temperature, reacting at room temperature for 8 hours to obtain a conjugate triamcinolone acetonide-OVA mixed solution, and separating a coating antigen and an unconjugated small molecule hapten through dialysis to obtain the coating antigen triamcinolone acetonide-OVA;
the coating antigen is used for detecting the serum titer and inhibition of the mouse in the monoclonal antibody preparation process, is not directly used for immunizing the mouse, and is necessary for preparing the monoclonal antibody.
(3) Animal immunization: healthy BALB/c mice 6-8 weeks old were selected for immunization. Three triamcinolone acetonide-BSA complete antigens with different molar ratios are mixed and emulsified with an equivalent amount of Freund's adjuvant, and then the mixture is injected subcutaneously into the back of a human body to immunize a BALB/c mouse respectively. Complete Freund's adjuvant was used for the first immunization, followed by incomplete Freund's adjuvant. The interval between the first immunization and the second boosting immunization is 28 days, and the interval between the boosting immunization is 21 days. Blood was collected 7 days after the third immunization (mice were bled with tail-off 5 μ L +995 μ L antibody diluent ═ antiserum), serum titers and inhibition were determined in mice using ic-ELISA, mice with high titers and good inhibition were selected, immunized by puncture 21 days after the fifth immunization, injected intraperitoneally, requiring a halved priming dose without any adjuvant.
(4) Cell fusion: after three days of the spurting immunization, cell fusion is carried out according to a conventional PEG (polyethylene glycol, molecular weight is 4000) method, and the specific steps are as follows:
a. taking eyeballs and blood, killing a mouse by a cervical vertebra dislocation method, immediately putting the mouse into 75% alcohol for disinfection, soaking for about 5min, taking out the spleen of the mouse by aseptic operation, properly grinding by using a rubber head of an injector, passing through a 200-mesh cell screen to obtain a spleen cell suspension, collecting, centrifuging (1200rpm, 8min), washing the spleen cells for three times by using an RPMI-1640 culture medium, diluting the spleen cells to a certain volume after the last centrifugation, and counting for later use;
b. collecting SP2/0 cells: 7-10 days before fusion, SP2/0 tumor cells were cultured in RPMI-1640 medium containing 10% FBS (fetal bovine serum) at 5% CO2Culturing in an incubator. Before fusion, the number of SP2/0 tumor cells is required to reach (1-4) x 107Ensuring that SP2/0 tumor cells are in logarithmic growth phase before fusion. During fusion, tumor cells are collected and suspended in RPMI-1640 basic culture solution for cell counting;
c. the fusion process is 7 min: 1min, 1mL of PEG 4000 was added to the cells dropwise from slow to fast; standing for 2 min; dropping 1mL of RPMI-1640 culture medium within 1min at 3min and 4 min; dropping 2mL of RPMI-1640 culture medium within 1min at 5min and 6 min; at 7min, 1mL of RPMI-1640 culture medium is added dropwise every 10 s; then, the cells were incubated at 37 ℃ for 5min, centrifuged (800rpm, 8min), the supernatant was discarded, resuspended in RPMI-1640 screening medium containing 20% fetal bovine serum and 2% 50 XHAT, applied to 96-well cell plates at 200. mu.L/well, and incubated at 37 ℃ with 5% CO2Culturing in an incubator.
(5) Cell screening and cell strain establishment: on day 3 of cell fusion, the fused cells were subjected to RPMI-1640 screening medium half-replacement, on day 5, to total-replacement with RPMI-1640 medium containing 20% fetal bovine serum and 1% 100 XHT, and on day 7, cell supernatants were collected and screened. The screening is divided into two steps: firstly, screening positive cell holes by using ic-ELISA, secondly, selecting triamcinolone acetonide as a standard substance, and measuring the inhibition effect of the positive cells by using the ic-ELISA. And selecting cell holes with good inhibition on triamcinolone acetonide standard products, subcloning by adopting a limiting dilution method, and detecting by using the same method. This was repeated three times to obtain cell line STD6B 11.
(6) Preparation and identification of monoclonal antibody: taking BALB/c mice 8-10 weeks old, and injecting 1mL of sterile paraffin oil into the abdominal cavity of each mouse; 7 days later, each mouse was injected intraperitoneally with 1X 106Hybridoma cells, ascites fluid was collected from the seventh day, and the ascites fluid was purified by the octanoic acid-ammonium sulfate method. Under the condition of partial acid, the caprylic acid can precipitate other hybrid proteins except IgG immunoglobulin in the ascites, then the centrifugation is carried out, and the precipitate is discarded; then, the IgG type monoclonal antibody was precipitated with an ammonium sulfate solution of the same saturation, centrifuged, the supernatant was discarded, dissolved in a 0.01M PBS solution (pH 7.4), dialyzed and desalted to finally obtain a purified monoclonal antibody, which was stored at-20 ℃.
Example 2 IC of triamcinolone acetonide monoclonal antibody50Measurement of (2)
Carbonate Buffer (CBS): weighing Na2CO3 1.59g，NaHCO32.93g of the mixture is respectively dissolved in a small amount of double distilled water and then mixed, the double distilled water is added to about 800mL and mixed evenly, the pH value is adjusted to 9.6, the double distilled water is added to the volume of 1000mL, and the mixture is stored for later use at 4 ℃;
phosphate Buffered Saline (PBS): 8.0g NaCl, 0.2g KCl, 0.2g KH2PO4，2.9g Na2HPO4·12H2Dissolving O in 800mL of pure water, adjusting the pH value to 7.2-7.4 by using NaOH or HCl, and fixing the volume to 1000 mL;
wash solution (PBST): adding 0.5mL of Tween-20 into 1000mL of 0.01mol/L PBS solution with pH7.4; PBST: PBS containing 0.05% Tween-20;
antibody dilution: wash buffer containing 0.1% gelatin;
TMB color development liquid: solution A: na (Na)2HPO4·12H218.43g of O, 9.33g of citric acid and pure water to reach the constant volume of 1000 mL; and B, liquid B: 60mg of TMB was dissolved in 100mL of ethylene glycol. A. Mixing the liquid B according to the volume ratio of 1:5 to obtain the TMB color developing solution, and mixing the liquid B at the present time.
The method comprises the following specific steps:
(1) coating: diluting the coated original triamcinolone acetonide-OVA by 0.05M of carbonate buffer solution with pH 9.6 from 1 mu g/mL in a multiple ratio, and reacting at 37 ℃ for 2h at 100 mu L/hole;
(2) washing: the plate solution was decanted and washed 3 times for 3min each with washing solution;
(3) and (3) sealing: after patting to dryness, 200. mu.L/well blocking solution was added and reacted at 37 ℃ for 2 hours. Drying for later use after washing;
(4) sample adding: diluting antiserum (which is obtained by diluting the antiserum by a corresponding multiple with an antibody diluent after tail-cutting blood collection of a mouse) from 1:1000 by multiple, adding the diluted antiserum into coated holes of each dilution, reacting at the temperature of 100 mu L/hole for 30 min; after fully washing, adding HRP-goat anti-mouse IgG diluted by 1:3000, 100 mu L/hole, and reacting for 30min at 37 ℃;
(5) color development: taking out the ELISA plate, fully washing, adding 100 mu L of TMB color development solution into each hole, and reacting for 15min at 37 ℃ in a dark place;
(6) termination and measurement: the reaction was stopped by adding 50. mu.L of a stop solution to each well, and the OD of each well was measured by a microplate reader450The value is obtained.
The standard inhibition curve of STD6B11 triamcinolone acetonide monoclonal antibody to triamcinolone acetonide is shown in FIG. 1, and IC-ELISA was used to determine the IC of the monoclonal antibody to triamcinolone acetonide50The concentration is 0.49ng/mL, which indicates that the sensitivity to triamcinolone acetonide is good, and the method can be used for the immunoassay detection of triamcinolone acetonide.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A triamcinolone acetonide monoclonal antibody hybridoma cell strain STD6B11 is deposited in China general microbiological culture Collection center (CGMCC), China academy of sciences, China institute of microbiology, No. 3, West Lu 1 institute of North Chen, south China, Kyoho, Beijing, and is classified and named as a monoclonal cell strain, the preservation date is 2020, 9 months and 27 days, and the preservation number is CGMCC No. 20783.
2. A triamcinolone acetonide monoclonal antibody is characterized in that: the hybridoma cell strain STD6B11 with the preservation number of CGMCC No.20783 as set forth in claim 1.
3. The preparation method of the triamcinolone acetonide hapten is characterized by comprising the following steps: adding triamcinolone acetonide into succinic anhydride dissolved in anhydrous pyridine, and carrying out water bath reaction; stopping heating, cooling to room temperature, transferring the reactant into cooled hydrochloric acid in advance, and separating out a large amount of white precipitate; washing with deionized water for several times, and drying to obtain white solid, namely the hapten of the triamcinolone acetonide.
4. The method for preparing triamcinolone acetonide hapten according to claim 3, characterized by the following steps: adding 570mg of triamcinolone acetonide into 5mL of anhydrous pyridine in which 600mg of succinic anhydride is dissolved, and carrying out water bath reaction at 60 ℃ for 6 hours; stopping heating, cooling to room temperature, transferring the reactant to 20mL of hydrochloric acid with the mass concentration of 10% and cooling to 4 ℃ in advance, and separating out a large amount of white precipitate; washing with deionized water for several times, and drying to obtain white solid, namely the hapten of the triamcinolone acetonide.
5. The preparation method of the triamcinolone acetonide complete antigen is characterized by comprising the following steps of: weighing triamcinolone acetonide hapten, dissolving the triamcinolone acetonide hapten in dimethyl formamide DMF, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC and N-hydroxysuccinimide NHS, and stirring at room temperature to obtain a reaction liquid A1; weighing BSA, and dissolving in borate buffer solution to obtain solution B1; and then, dropwise adding the A1 solution into the B1 solution, reacting at room temperature to obtain a conjugate triamcinolone acetonide-BSA mixed solution, and separating a complete antigen and an unconjugated small molecule hapten through dialysis to obtain the immunogen triamcinolone acetonide-BSA.
6. The method for preparing triamcinolone acetonide complete antigen according to claim 5, characterized in that the method for preparing immunogen specifically comprises: weighing 1.89mg of triamcinolone acetonide hapten, dissolving the triamcinolone acetonide hapten in 450 mu L of DMF, adding 2.92mg of EDC and 1.75mg of NHS, and stirring the mixture at room temperature for 6 hours to obtain a reaction solution A1; weighing 5mg BSA, and dissolving in 0.1M borate buffer solution to obtain solution B1; and then, dropwise adding the A1 solution into the B1 solution, reacting for 8 hours at room temperature to obtain a conjugate triamcinolone acetonide-BSA mixed solution, and separating a complete antigen and an unconjugated small molecule hapten through dialysis to obtain the immunogen triamcinolone acetonide-BSA.
7. The preparation method of the triamcinolone acetonide complete antigen is characterized by comprising the following steps of: dissolving triamcinolone acetonide hapten, 1-ethyl carbodiimide hydrochloride and N-hydroxysuccinimide in anhydrous N, N-dimethylformamide to obtain solution A2, and stirring at room temperature for reacting for 6 h; weighing chicken egg white albumin OVA, dissolving the OVA in borate buffer solution to obtain B2 solution, dropwise adding A2 solution into B2 solution at room temperature, reacting at room temperature to obtain a conjugate triamcinolone acetonide-OVA mixed solution, and dialyzing to separate the coating antigen and the unconjugated small molecule hapten to obtain the coating antigen triamcinolone acetonide-OVA.
8. The method for preparing the triamcinolone acetonide complete antigen according to claim 7, which is characterized in that the method for preparing the coating antigen specifically comprises the following steps: weighing 2.68mg of triamcinolone acetonide hapten, 3.94mg of 1-ethyl carbodiimide hydrochloride and 2.50mg of N-hydroxysuccinimide, dissolving the 2.68mg of triamcinolone acetonide hapten, 1-ethyl carbodiimide hydrochloride and the N-hydroxysuccinimide hydrochloride in 450 mu L of anhydrous N, N-dimethylformamide to obtain solution A2, and stirring the solution at room temperature for reacting for 6 hours; weighing 5mg of chicken ovalbumin OVA, dissolving the OVA in 3mL of 0.1M borate buffer solution to obtain a B2 solution, dropwise adding the A2 solution into the B2 solution at room temperature, reacting at room temperature for 8 hours to obtain a conjugate triamcinolone acetonide-OVA mixed solution, and separating the coating antigen and the unconjugated small molecule hapten through dialysis to obtain the coating antigen triamcinolone acetonide-OVA.
9. The use of the triamcinolone acetonide monoclonal antibody of claim 2, characterized in that: an immunoassay method for the content of triamcinolone acetonide is established, and the immunoassay method is applied to the detection of the triamcinolone acetonide in food.
10. The use of the triamcinolone acetonide monoclonal antibody according to claim 9, characterized in that: the detection field is pork and other food containing triamcinolone acetonide.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US4818684A (en) * | 1985-09-10 | 1989-04-04 | The Trustees Of Columbia University In The City Of New York | Auto-anti-idiotypic monoclonal antibodies to steroid receptors and uses thereof |
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| US4818684A (en) * | 1985-09-10 | 1989-04-04 | The Trustees Of Columbia University In The City Of New York | Auto-anti-idiotypic monoclonal antibodies to steroid receptors and uses thereof |
Non-Patent Citations (2)
| Title |
|---|
| GABOR F ET AL.: "Drug-Protein Conjugates: Preparation of Triamcinolone-Acetonide", 《ARCHIV DER PHARMAZIE》 * |
| ZHANG S ET AL.: "Upconversion luminescence nanoparticles-based immunochromatographic assay for quantitative detection of triamcinolone acetonide in cosmetics", 《SPECTROCHIMICA ACTA PART A: MOLECULAR AND BIOMOLECULAR SPECTROSCOPY》 * |
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