CN110950962A - Hybridoma cell strain A11S for secreting bimesomepheniul monoclonal antibody and application thereof - Google Patents
Hybridoma cell strain A11S for secreting bimesomepheniul monoclonal antibody and application thereof Download PDFInfo
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Abstract
A hybridoma cell strain A11S secreting bimesomephenil monoclonal antibody and application thereof belong to the field of food safety immunodetection. The hybridoma cell strain A11S secreting the bimesomepheniumide monoclonal antibody is obtained by screening, is preserved in the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 18517. The monoclonal antibody secreted by the cell strain has better specificity and detection sensitivity (IC) on the imibenuron50The value is 2 ng/mL), can realize the detection of the residual amount of the imibenclamide in the beef, the pork and the feed, provides raw materials for the immunodetection of the residual amount of the imibenclamide in the food, and has practical application value.
Description
Technical Field
The invention relates to a hybridoma cell strain A11S secreting bimesomephenil monoclonal antibody and application thereof, and belongs to the field of food safety immunodetection.
Background
Diminazene urea (IMI), also known as Imidocarb, is an internationally recognized anti-scorching drug nowadays and has the characteristics of high curative effect, low toxicity and small dosage. The babesiesiosis which is seriously infected to cattle and sheep is treated by the imidazole phenylurea, the disease condition can be quickly controlled, and the relapse is not seen after three months of treatment. The product can also be used for preventing trichodiniasis. Clinical data also prove that the medicine has a definite curative effect on bovine trypanosomiasis, eperythrozoonosis and artificially infected swine trypanosomiasis evansi and eperythrozoonosis, can control epidemic situations, but cannot thoroughly kill eperythrozoonosis.
Imidazofenoxuron has been widely used in pig and cattle breeding, feed and other fields. Therefore, some residual is generally present in beef, pork and feed, and excessive intake of the imibenuron by human body causes metabolic burden to human organs such as liver and kidney, and if residual is present in animal protein (beef, pork and milk), the residual is harmful to human health. The world health organization food additive experts' joint committee stipulates that the limit of imibenclamide in cattle muscle and milk is 50 μ g/kg. However, the standards are all detected by a high performance liquid chromatography method, the detection method is complex and complicated, in order to maintain the benefits of wide consumers, a high-efficiency and rapid detection method aiming at IMI is needed to be established, the enzyme-linked immunosorbent assay (ELISA) pretreatment is simple, the cost is low, rapid detection of a large number of samples can be realized, and the requirement on the purity of the samples during detection is not high. Therefore, it is necessary to establish an efficient immunological detection method, and an important prerequisite for establishing the method is to screen a high specificity monoclonal monomer aiming at the diminazene urea.
Disclosure of Invention
The invention aims to provide a hybridoma cell strain A11S secreting bimesomephenil monoclonal antibody and application thereof, the monoclonal antibody prepared by the cell strain has good specificity and detection sensitivity to bimesomephenil, and can be used for establishing an immunological detection method of bimesomephenil.
The technical scheme of the invention is that a hybridoma cell strain A11S secreting bimbazamide monoclonal antibody has been preserved in China general microbiological culture Collection center (CGMCC), China academy of sciences, China institute of microbiology, No. 3, of Suzuolu No.1, North Cheng, west, of the Korean area, Beijing, and is classified and named as monoclonal cell strain, the preservation date is 2019, 10 months and 14 days, and the preservation number is CGMCC No. 18517.
The monoclonal antibody of the bimesoxim is produced by secreting hybridoma cell strain A11S which secretes the bimesoxim monoclonal antibody with the preservation number of CGMCC No. 18517.
The application of the imibenclamide monoclonal antibody is used for analyzing and detecting the residual imibenclamide in food safety detection.
The preparation of the bimesomephenil monoclonal antibody hybridoma cell strain A11S provided by the invention comprises the following basic steps:
(1) selection of hapten: the hapten has the following structural formula:
2- (3-aminophenyl) imidazoline is derived by adopting a raw imidafenamide drug; the reaction equation is as follows:
(2) preparation of complete antigen IMI-BSA: weighing 4.38mg IMI derivatives, dissolving in 300 mu L N N-dimethylformamide DMF, and reacting for 10min under the condition of ice-water bath with stirring; under the condition of ice-water bath, 48 mu L of HCl with the concentration of 1M is dripped to carry out acidification for 0.5h, so as to obtain an acidified IMI derivative solution; weighing 150mg of sodium nitrite, and fully dissolving with 500 microliter of pure water to obtain a sodium nitrite solution; adding 5.56 mu L of sodium nitrite solution into the acidified IMI derivative solution, and reacting for 0.5-1h under the condition of ice-water bath to obtain solution A; 6mg of BSA was dissolved in 2mL of 0.01M, pH =9.0 carbonate buffer CB, referred to as solution B; slowly adding the solution A into the solution B drop by drop, and reacting for 4 hours under the ice-water bath condition; dialyzing with 0.01M PBS solution, removing unreacted small molecule hapten to obtain complete antigen IMI-BSA, and identifying by ultraviolet absorption scanning method;
(3) immunization of mice: after IMI-BSA complete antigen was mixed and emulsified with an equal amount of Freund's adjuvant, BALB/c mice were immunized by subcutaneous multipoint injection at the back of the neck (except for the booster immunization). Complete Freund adjuvant is used for the first immunization, and the dosage is 100 mug/mouse; incomplete Freund's adjuvant is used for multiple times of boosting immunization, and the dosage is reduced by half to be 50 mu g/mouse; the thorny immunity does not use an adjuvant, and the thorny immunity is directly diluted by normal saline and injected into the abdominal cavity, and the dosage is reduced by half to obtain 25 mu g/mouse. The interval between the first immunization and the second boosting immunization is one month, the interval between the multiple boosting immunizations is 21 days, and the interval between the sprint immunization and the last boosting immunization is 18-21 days. The immune effect of the mouse is observed by an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), namely the titer and inhibition of the mouse serum are detected;
(4) cell fusion and cell line establishment: fusing mouse spleen cells and mouse myeloma cells by a polyethylene glycol (PEG 4000) method, screening hybridoma cells by using a selective medium (HAT medium), and performing cell culture by using an HT medium. And detecting the positive cell holes by using an ic-ELISA method after one week of fusion, further determining the inhibition effect of the positive cell holes by using the ic-ELISA method, performing subcloning on the positive cell holes with better inhibition by using a limiting dilution method, and detecting, selecting and subcloning again after one week. Carrying out subcloning for three times according to the method to obtain a monoclonal hybridoma cell strain A11S of IMI high-secretion specific antibody;
(5) 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 monoclonal antibody secreted by the cell strain A11S provided by the invention has better specificity and detection sensitivity (IC) on IMI50The value is 2 ng/mL), the detection of the IMI residual quantity in beef, pork and feed can be realized, raw materials are provided for the immunodetection of the IMI residual quantity in food, and the method has practical application value.
Biological material sample preservation: a hybridoma cell strain A11S secreting bimbazamide monoclonal antibody is deposited in China general microbiological culture collection center (CGMCC), China academy of sciences (China institute of microbiology, No. 3, Xilu No.1, Beijing, the south of the republic of China, and is classified and named as monoclonal cell strain, the preservation date is 2019, 10 and 14 days, and the preservation number is CGMCC No. 18517.
Drawings
FIG. 1 shows standard curves for inhibition of the monoclonal antibody to Imidazobiurea.
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.
The invention finally obtains the hybridoma cell strain with high secretion specificity antibody aiming at the diminazene through immunizing mice with the diminazene complete antigen, fusing cells, culturing in HAT selective culture medium and screening cell supernatant through ic-ELISA.
EXAMPLE 1 preparation of hybridoma cell line A11S
(1) Synthesis of complete antigen: weighing 4.38mg IMI derivatives, dissolving in 300 mu L N N-dimethylformamide DMF, and reacting for 10min under the condition of ice-water bath with stirring; under the condition of ice-water bath, 48 mu L of HCl with the concentration of 1M is dripped to carry out acidification for 0.5h, so as to obtain an acidified IMI derivative solution; weighing 150mg of sodium nitrite, and fully dissolving with 500 microliter of pure water to obtain a sodium nitrite solution; adding 5.56 mu L of sodium nitrite solution into the acidified IMI derivative solution, and reacting for 0.5-1h under the condition of ice-water bath to obtain solution A; 6mg of BSA was dissolved in 2mL of 0.01M, pH =9.0 carbonate buffer CB, referred to as solution B; slowly adding the solution A into the solution B drop by drop, and reacting for 4 hours under the ice-water bath condition; dialyzing with 0.01M PBS solution, removing unreacted small molecule hapten to obtain complete antigen IMI-BSA, and identifying by ultraviolet absorption scanning method;
(2) animal immunization: after complete antigen IMI-BSA and an equal amount of Freund's adjuvant are mixed and emulsified, BALB/c mice are immunized by subcutaneous multipoint injection at the back of the neck (except for the puncture immunization). Complete Freund adjuvant is used for the first immunization, and the dosage is 100 mug/mouse; incomplete Freund's adjuvant is used for multiple times of boosting immunization, and the dosage is reduced by half to be 50 mu g/mouse; the thorny immunity does not use an adjuvant, and the thorny immunity is directly diluted by normal saline and injected into the abdominal cavity, and the dosage is reduced by half to obtain 25 mu g/mouse. The interval between the first immunization and the second boosting immunization is one month, the interval between the multiple boosting immunizations is 21 days, and the interval between the sprint immunization and the last boosting immunization is 18-21 days. The immune effect of the mouse is observed by an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), namely the titer and inhibition of the mouse serum are detected;
(3) 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. the method comprises the following steps of (1) taking eyeballs of a mouse, taking blood, killing the mouse by a cervical vertebra dislocation method, immediately placing the mouse into 75% alcohol for disinfection, soaking for about 5min, taking out the spleen of the mouse by aseptic operation, properly grinding the spleen by using a syringe rubber head, passing through a 200-mesh cell screen to obtain a spleen cell suspension, collecting, centrifuging (1200 rpm, 8 min), 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. The number of SP2/0 neoplastic cells reached (1-4) x 10 before fusion7Ensuring 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, dripping 1mL of PEG 4000 into cells from slow to fast; standing for 2 min. Dropping 1mL of RPMI-1640 culture medium within 1min at 3min and 4 min; dripping 2mLRPMI-1640 culture medium within 1min at 5min and 6 min; at 7min, 1mL of RPMI-1640 medium was added dropwise every 10 s. Then, the mixture is incubated at 37 ℃ for 5 min. Centrifuging (800 rpm, 10 min), discarding the supernatant, gently tapping the cells, adding RPMI-1640 selective medium (HAT medium) containing 20% fetal bovine serum and 2% 50 XHAT to the supernatant, adding the mixture to a 96-well cell plate at 200. mu.L/well, and standing at 37 ℃ with 5% CO2Culturing in an incubator.
(4) Cell screening and cell strain establishment: half-changing the fused cells with HAT medium on day 3 after cell fusion; on day 5, the whole culture medium was changed with RPMI-1640 medium (HT medium) containing 20% fetal bovine serum and 1% 100 × HT; cell supernatants were taken on day 7 for screening. The screening is divided into two steps: the first step is to screen out positive cell holes by an ic-ELISA method, and the second step is to select the dimidine as a standard substance and to test the inhibition effect of the positive cells by the ic-ELISA method. And selecting a cell hole with good inhibition on the imibenuron standard substance, performing subcloning by adopting a limiting dilution method, and detecting by using the same method after seven days. Subcloning for three times according to the method to finally obtain the bimesomepiride monoclonal antibody cell strain A11S.
(5) 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 106Imidazobium hybridoma cells, fromAscites was collected at seven days, and antibody purification was performed by caprylic acid-saturated 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, and the supernatant was 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 ℃.
5.1 coating: diluting the coating source IMI-OVA by 3 times from 1 mug/mL by using 0.05M carbonate buffer solution with pH9.6, reacting at the temperature of 37 ℃ for 2h, wherein the concentration of the coating source IMI-OVA is 100 mug/hole;
5.2 washing: the plate solution was decanted and washed 3 times for 3min each with washing solution;
5.3 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;
5.4 sample adding: diluting antiserum from 1:1000 times, adding into coated wells of each dilution, reacting at 37 deg.C for 30min at 100 μ L/well; after fully washing, adding HRP-goat anti-mouse IgG diluted by 1:3000, 100 mu L/hole, and reacting for 30min at 37 ℃;
5.5 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;
5.6 termination and determination: the reaction was stopped by adding 50. mu.L of a stop buffer to each well, and the OD 450 value of each well was measured by a microplate reader.
Determination of IC of Imidazobiurea monoclonal antibody by IC-ELISA50Comprises the following steps: 2.0 ng/mL, which shows that the sensitive to the dimidine is good, and the dimidine can be used for immunoassay detection.
Solution preparation:
carbonate Buffer (CBS): weighing Na2CO31.59 g，NaHCO32.93 g 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): 8.00g NaCl, 0.2g KCl, 0.2g KH2PO4，2.9g Na2HPO4·12 H2Dissolving 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;
PBST: PBS containing 0.05% tween 20;
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. And the volume ratio of the solution B to the solution B is 5: 1 to obtain the TMB color developing solution which is mixed at present.
Claims (7)
1. A hybridoma cell strain A11S secreting monoclonal antibody of bimesomepheniumide is deposited in China general microbiological culture Collection center (CGMCC), China academy of sciences and sciences, China institute of microbiology, No. 3, West Lu 1 institute of North Chen, south China, Kyoto China, and is classified and named as monoclonal cell strain with the preservation date of 2019, 10 and 14 days and the preservation number of CGMCC No. 18517.
2. The bimesophenyl urea monoclonal antibody is characterized in that: it is secreted and produced by hybridoma cell strain A11S which secretes the bimesomepheniul monoclonal antibody with the preservation number of CGMCC No.18517 and the preservation number of claim 1.
3. The use of a monoclonal antibody against imidazoylurea as claimed in claim 2, characterized in that: the method is used for analyzing and detecting the residual of the imibenuron in food safety detection.
4. The method for producing the hybridoma cell line A11S according to claim 1, wherein the method comprises the following steps: after a mouse is immunized by a complete antigen, namely, bimesomephenil IMI-BSA, hybrid cells obtained by fusing two different cell mouse splenocytes and mouse myeloma cells are screened out through culture; and screening the cells by an indirect competitive enzyme-linked immunosorbent assay and subcloning for three times to obtain a hybridoma cell strain A11S.
5. The method for preparing hybridoma cell line A11S according to claim 4, wherein the complete antigen Imi-BSA is prepared as follows: weighing 4.38mg hapten-dimidine IMI derivative, dissolving in 300 mu L N N-dimethylformamide DMF, and stirring and reacting for 10min under the condition of ice-water bath; under the condition of ice-water bath, 48 mu L of HCl with the concentration of 1M is dripped to carry out acidification for 0.5h, so as to obtain an acidified IMI derivative solution; weighing 150mg of sodium nitrite, and fully dissolving with 500 microliter of pure water to obtain a sodium nitrite solution; adding 5.56 mu L of sodium nitrite solution into the acidified IMI derivative solution, and reacting for 0.5-1h under the condition of ice-water bath to obtain solution A; 6mg of BSA was dissolved in 2mL of 0.01M, pH =9.0 carbonate buffer CB, referred to as solution B; slowly adding the solution A into the solution B drop by drop, and reacting for 4 hours under the ice-water bath condition; then dialyzing with 0.01M PBS solution, removing unreacted small molecule hapten to obtain complete antigen IMI-BSA, and identifying by a UV absorption scanning method.
6. The method for preparing hybridoma cell strain A11S according to claim 5, wherein the method comprises the following steps: the molar ratio of the hapten-imidazoylurea IMI to the bovine serum albumin BSA is 60: 1.
7. the method for preparing hybridoma cell strain A11S according to claim 5, wherein the method comprises the following steps: the structural formula of the hapten-imidazofenomide IMI is as follows:
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| CN112094815A (en) * | 2020-09-23 | 2020-12-18 | 绿城农科检测技术有限公司 | Hybridoma cell strain secreting methimazole monoclonal antibody, monoclonal antibody and application |
| CN114908060A (en) * | 2022-05-12 | 2022-08-16 | 江南大学 | Hybridoma cell strain secreting zoxamide monoclonal antibody and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109825478A (en) * | 2019-03-08 | 2019-05-31 | 江南大学 | One plant of hybridoma cell strain E for secreting anti-glibenclamide monoclonal antibody and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109825478A (en) * | 2019-03-08 | 2019-05-31 | 江南大学 | One plant of hybridoma cell strain E for secreting anti-glibenclamide monoclonal antibody and its application |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112094815A (en) * | 2020-09-23 | 2020-12-18 | 绿城农科检测技术有限公司 | Hybridoma cell strain secreting methimazole monoclonal antibody, monoclonal antibody and application |
| CN114908060A (en) * | 2022-05-12 | 2022-08-16 | 江南大学 | Hybridoma cell strain secreting zoxamide monoclonal antibody and application thereof |
| CN114908060B (en) * | 2022-05-12 | 2023-04-14 | 江南大学 | Hybridoma cell strain capable of secreting zoxamide monoclonal antibody and application of hybridoma cell strain |
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