CN113717949B - Hybridoma cell strain secreting ketoconazole monoclonal antibody and application thereof - Google Patents
Hybridoma cell strain secreting ketoconazole monoclonal antibody and application thereof Download PDFInfo
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Abstract
The invention provides a hybridoma cell strain secreting ketoconazole monoclonal antibody and application thereof, wherein the hybridoma cell strain is preserved in China general microbiological culture Collection center (CGMCC) with the address of the institute of microbiological research, national academy of sciences of China, no. 3, north Chenxi Lu No. 1, chaoyang, beijing, and the preservation date of 2021, 05 month and 13 days. The ketoconazole monoclonal antibody secreted by the hybridoma cell strain has better specificity and detection sensitivity (IC) to ketoconazole 50 The value is 0.7ng/mL, the cross rate of the ketoconazole analogue is less than 1 percent, the detection limit of the ketoconazole in a sample is 0.1ng/g, and the detection linear range is 0.1-2.5ng/g. Can be used for establishing an immunological detection method of ketoconazole and detecting the residue of ketoconazole in food.
Description
Technical Field
The invention belongs to the technical field of immunochemistry, and particularly relates to a hybridoma cell strain secreting ketoconazole monoclonal antibody and application thereof.
Background
Ketoconazole (KCZ) is an imidazole antifungal agent, commercially available under the trade names of Jindakrein, mycokanolamine and cotinine, which inhibits ergosterol biosynthesis on fungal cell membranes by highly selectively interfering with the activity of cytochrome P-450 in fungi. Is effective for superficial and deep fungal infection, and can inhibit fungal growth and transformation of spores into mycelium, and prevent further infection. Low concentration antibacterial and high concentration sterilizing. Has antibacterial effect against Candida, trichosporon, coccidioides, histoplasma, sporotrichum, and Trichophyton. Is suitable for treating tinea manus, tinea pedis, tinea cutaneum, tinea corporis, tinea cruris, thrush, tinea versicolor and candidiasis of skin clinically. However, ketoconazole has certain toxicity, and long-term eating of food or feed containing ketoconazole drug residues can have serious influence on human or animals. Residual ketoconazole can cause a significant number of sensitive bacteria to be inhibited or killed and interfere with the proportion and quantity of normal bacteria in the human or animal digestive tract. When this ratio or number balance is broken, the resistant strain or condition will lead to a high bacterial reproduction opportunity, a dyspepsia in humans or animals and a reduced resistance to bacterial food poisoning.
For the detection of ketoconazole residues, high performance liquid chromatography, gas chromatography or liquid chromatography-mass spectrometry are often used. However, these methods have the disadvantages of complex pretreatment of samples and long detection time, and are not suitable for rapid detection of a large number of samples, and in order to maintain the interests of consumers, it is necessary to establish an efficient and rapid detection method for ketoconazole.
The enzyme-linked immunosorbent assay (ELISA) is a very efficient, sensitive and rapid detection method, and has the advantages of simple pretreatment on samples during detection, less purification steps, large analysis capacity, low detection cost and simple and convenient operation, is suitable for the on-site rapid detection of a large number of samples, and is widely applied to the analysis of antibiotic residues. On the premise of detecting ketoconazole by using an enzyme-linked immunosorbent assay, a monoclonal antibody with high specificity and high sensitivity to ketoconazole is obtained, so that the method for preparing the monoclonal antibody with high specificity and high sensitivity to ketoconazole is very critical.
CN109305963 a discloses a ketoconazole hapten, an artificial antigen, a preparation method and application thereof, and shows that the artificial antigen can be used for the preparation of ketoconazole antibodies and the detection of ketoconazole residual drugs, and the ketoconazole hapten adopted is obtained by the reaction of the analogue of ketoconazole, namely acetylketoconazole, with succinic anhydride; however, the sensitivity of the ketoconazole antibody in this patent is still to be further improved.
Disclosure of Invention
In order to solve the technical problems, the invention directly generates ester bonds from the ketoconazole original structure and methyl acrylate, and then obtains final antigens through hydrolysis, and on the basis of completely retaining the ketoconazole chemical structure, one side chlorine in the ketoconazole structure is replaced by carboxyl for binding protein, thereby preparing the immunogen. Compared with two hapten structures, the hapten disclosed by the invention has the advantages that the original structure of ketoconazole is reserved to a greater extent, and the specific recognition site of ketoconazole is easier to be exposed after the carboxyl of the side is coupled with the protein, so that the antibody with higher sensitivity to ketoconazole is generated.
The first object of the present invention is to provide a hybridoma cell strain secreting ketoconazole monoclonal antibody, which has been deposited at the China general microbiological center of the culture Collection center of China, the national academy of sciences of China, including North Chen West Lu No. 1, no. 3, of the area of Korea of Beijing, and has a classification designation of monoclonal cell strain, a date of deposit of 2021, day 05, month 13, and a deposit number of CGMCC No.22327.
The second object of the invention is to provide a ketoconazole monoclonal antibody which is secreted by a hybridoma cell strain with the preservation number of CGMCC No.22327.
The third object of the invention is to provide the application of the hybridoma cell strain or the ketoconazole monoclonal antibody in detecting ketoconazole.
The fourth object of the invention is to provide a kit, which contains the hybridoma cell strain or the ketoconazole monoclonal antibody.
A fifth object of the invention is to provide the use of said kit for the detection of ketoconazole.
The sixth object of the present invention is to provide a ketoconazole hapten, which has the structural formula:
the seventh object of the invention is to provide a ketoconazole complete antigen, which has the structural formula:
the seventh object of the present invention is to provide a method for preparing the ketoconazole monoclonal antibody-secreting hybridoma cell strain by using the ketoconazole complete antigen, comprising the following steps:
s1, preparing complete ketoconazole antigens into complete Freund 'S adjuvant containing antigen and incomplete Freund' S adjuvant containing antigen;
s2, performing first subcutaneous immunization on the mice by using the obtained complete Freund adjuvant containing the antigen, performing multiple times of subcutaneous immunization on the mice by using the incomplete Freund adjuvant containing the antigen, and screening the immunized mice with high ketoconazole antibody content in serum;
s3, performing primary boosting subcutaneous immunization on the screened mice obtained by immunization by adopting an incomplete Freund adjuvant containing the antigen, and performing sprint immunization by adopting the incomplete Freund adjuvant containing the antigen;
s4, taking spleen cells and myeloma cells of the mice subjected to sprint immunization in the S3, carrying out cell fusion, and screening to obtain the hybridoma cell strain.
Further, the first subcutaneous immunization dose was 100. Mu.g/dose, the booster subcutaneous immunization dose was 50. Mu.g/dose, and the sprint immunization dose was 25. Mu.g/dose.
Further, the immunization process included 1 first subcutaneous immunization, 4 booster subcutaneous immunization, and 1 sprint immunization.
Further, the interval between the first subcutaneous immunization and the enhanced subcutaneous immunization is 30 days, the interval between the enhanced subcutaneous immunization is 21 days, and the interval between the enhanced subcutaneous immunization and the sprint immunization is 18-21 days.
Further, cell fusion was performed 3 days after termination of sprint immunization.
Further, cell fusion was performed by the polyethylene glycol (PEG 4000) method.
Further, the hybridoma cell line after cell fusion is cultured by adopting RPMI-1640 culture medium.
Compared with the prior art, the technical scheme of the invention has the following advantages:
the ketoconazole monoclonal antibody secreted by the hybridoma cell strain has better specificity and detection sensitivity (IC) to ketoconazole 50 The value is 0.7ng/mL, the cross rate of the ketoconazole analogue is less than 1 percent, the detection limit of the ketoconazole in a sample is 0.1ng/g, and the detection linear range is 0.1-2.5ng/g. Can be used for establishing an immunological detection method of ketoconazole and detecting the residue of ketoconazole in food.
Drawings
In order that the invention may be more readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings, in which
FIG. 1 is a standard curve of inhibition of ketoconazole by a ketoconazole monoclonal antibody of the invention.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
The following examples relate to the following media:
RPMI-1640 medium (mg/L): l-arginine 290, L-asparagine 50, L-aspartic acid 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 B12.005, sodium bicarbonate 2000.
The reagents involved in the following examples were as follows:
carbonate Buffer (CBS): weighing Na 2 CO 3 1.59g，NaHCO 3 2.93g, respectively dissolving in a small amount of double distilled water, mixing, adding double distilled water to about 800mL, mixing, adjusting pH to 9.6, adding double distilled water to 1000mL, and storing at 4deg.C for use;
phosphate Buffer (PBS): 8.00g NaCl,0.2g KCl,0.2g KH 2 PO 4 ，2.9g Na 2 HPO 4 ·12H 2 O is dissolved in 800mL of pure water, pH is regulated to 7.2-7.4 by NaOH or HCl, and volume is regulated to 1000mL;
PBST: PBS containing 0.05% Tween 20;
antibody dilution: PBS was added to 0.1% gelatin.
TMB color development liquid: and (3) solution A: na (Na) 2 HPO 4 ·12H 2 18.43g of O, 9.33g of citric acid and pure water to 1000mL; and (2) liquid B: 60mg of TMB was dissolved in 100mL of ethylene glycol. A. The volume ratio of the solution B is 5:1 to obtain TMB color development liquid, and mixing immediately.
The detection method involved in the following examples is as follows:
the ketoconazole inhibition rate detection method comprises the following steps: the most appropriate antigen and antibody concentrations in the ic-ELISA were selected by a checkerboard assay. Diluting antigen to 0.03,0.1,0.3 and 1 μg/mL with Carbonate Buffer (CBS) and usingAntibody dilutions antibodies were diluted to 0.03,0.1,0.3 and 1 μg/mL. After selecting the optimal working point, the ketoconazole standard substance is diluted to 0,0.068,0.20,0.61,1.85,5.55,16.66 and 50ng/mL, and the concentrations are equal, according to the IC-ELISA operation step, the OriginPro 8.5 is finally used for drawing (the result is shown as figure 1), the ketoconazole standard inhibition curve is obtained, and the IC is calculated 50 。
Example 1: synthesis of ketoconazole hapten
Because the ketoconazole small molecule has no immunogenicity, can not stimulate mice to generate immune response, and then generate antibodies, the ketoconazole is coupled to protein through protein connection technology, so that the ketoconazole has immunogenicity; the active groups commonly used in the protein coupling technology include amino, carboxyl, hydroxyl, mercapto and the like, and the carboxyl needs to be derived from the ketoconazole molecular structure in view of the fact that the ketoconazole molecular structure does not contain amino, carboxyl and hydroxyl.
The ketoconazole hapten derived by the invention has the following structure:
the derivatization process includes three reactions:
1. weighing ketoconazole 1 and Pd as raw materials 2 (dba) 3 Cesium carbonate in a Schlenk tube, PPh3 was added to the Schlenk tube followed by methyl acrylate 2, solvent 1, 4-dioxane. Complete oxygen removal was performed by lyophilization with liquid nitrogen. The reaction was carried out at 120℃under Ar atmosphere for 5 hours. After the reaction, ethyl acetate was used for extraction, and anhydrous Na was used 2 SO 4 Dried, concentrated and transferred to a round bottom flask for direct further experiments.
2. The weighed Pd/C was added to a round bottom flask containing product 3, and after sealing the flask was replaced with H 2 The solvent methanol was added to the mixture in the atmosphere, and the mixture was reacted at room temperature for about 10 hours. After the completion of the reaction, the reaction was carried out with celiteFiltering, concentrating and then directly carrying out the next reaction.
3. To a round bottom flask containing product 4 was added aqueous NaOH and ethanol in sequence. The reaction was carried out under reflux for 1h. After the reaction, the aqueous phase is extracted twice by ethyl acetate to wash off byproducts, then the reaction system is adjusted to be acidic by hydrochloric acid solution, ethyl acetate is used for extraction again, and anhydrous Na is used after the organic phase is combined 2 SO 4 Drying, concentrating, separating by column chromatography, and separating by column to obtain ketoconazole hapten.
Example 2: synthesis of Ketoconazole complete antigen
8mg of ketoconazole hapten (KCZ-HS), 5mg of N-hydroxysuccinimide (NHS) and dissolved in 300 mu L N and N-Dimethylformamide (DMF) are weighed and stirred at room temperature for reaction for 10min; then, 7mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) was weighed out, dissolved in 100. Mu.L of DMF, added to the KCZ-HS solution, and reacted at room temperature with stirring for 4 to 6 hours (referred to as solution A). 5mg BSA was diluted to 2mg/mL (referred to as solution B) with 0.01M Carbonate Buffer (CBS), and solution A was slowly added dropwise to solution B, followed by reaction overnight at room temperature; then, the solution is dialyzed by 0.01M PBS to remove unreacted small molecule hapten, thus obtaining complete antigen KCZ-HS-BSA, and the complete antigen KCZ-HS-BSA is identified by an ultraviolet absorption scanning method.
Example 3: synthesis of Ketone Kangbao quilt
Dissolving 4mg of ketoconazole hapten (KCZ-HS) and 2.5 mgN-hydroxysuccinimide (NHS) in 300 mu L of anhydrous N, N-Dimethylformamide (DMF), and stirring and reacting for 10min at room temperature to obtain ketoconazole hapten (KCZ-HS) solution; after 4.2mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) is dissolved in 100 mu L of anhydrous DMF, the solution is added into KCZ-HS solution, and the solution is stirred at room temperature for reaction for 4 to 6 hours to obtain solution A; diluting 5mg chicken Ovalbumin (OVA) with 1mL of Carbonate Buffer (CBS) with concentration of 0.01mmol/L to obtain solution B; slowly adding the solution A into the solution B dropwise to react to obtain a reaction solution; the reaction solution was dialyzed against PBS to remove unreacted small molecule hapten, and to obtain the coating antigen (KCZ-HS-OVA).
Example 4: preparation of hybridoma cell strain secreting ketoconazole monoclonal antibody
1. Animal immunization was obtained: mixing and emulsifying ketoconazole complete antigen and equivalent Freund's adjuvant, and performing subcutaneous multipoint injection immunization (except sprint immunization) on the back of the neck of a BALB/c mouse; the first immunization is carried out by using complete Freund's adjuvant, and the dosage is 100 mug/dose; multiple boosting with incomplete Freund's adjuvant and halving the dose to 50 μg/dose; the sprint immunity does not need adjuvant, and the dosage is halved to 25 mug/patient after the sprint immunity is directly diluted by normal saline for intraperitoneal injection; one month is separated from the first immunization and the second immunization, 21 days is separated from the multiple times of the immunization, and 18-21 days is separated from the sprint immunization and the last immunization; observing the immune effect of the mice by an indirect competition enzyme-linked immunosorbent assay (ic-ELISA), namely detecting the titer and inhibition of the serum of the mice;
2. cell fusion: three days after sprint immunization, cell fusion was performed according to the conventional PEG (polyethylene glycol, molecular weight 4000) method, as follows:
a. taking blood from the tail, killing a mouse by a cervical 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, moderately grinding the spleen by using the rubber head of a syringe, obtaining spleen cell suspension by a 200-mesh cell screen, collecting, centrifuging (1200 rpm,8 min), washing the spleen cells for three times by using an RPMI-1640 culture medium, and diluting the spleen cells to a certain volume after the last centrifuging, and counting for later use;
b. collecting SP2/0 cells: SP2/0 tumor cells were cultured in 10% FBS (fetal bovine serum) RPMI-1640 medium at 5% CO 7-10 days prior to fusion 2 In the incubator, the number of SP2/0 tumor cells required to reach (1-4) x 10 before fusion 7 Ensuring that SP2/0 tumor cells are in logarithmic growth phase before fusion, collecting tumor cells during fusion, suspending in RPMI-1640 basic culture solution, and performing cell count;
c. fusion process 7min: 1min, 1mL of PEG 1500 was added dropwise to the cells from slow to fast; standing for 2 min; dripping 1mL of RPMI-1640 culture medium in the period of 1min for 3min and 4 min; dripping 2mL of RPMI-1640 culture medium in the period of 1min at the 5 th and 6 th min; dripping 1mL of RPMI-1640 medium every 10s for 7 min; then carrying out warm bath at 37 ℃ for 5min; centrifugation (800 rpm,8 mi)n), discarding the supernatant, re-suspending in an RPMI-1640 screening medium containing 20% fetal bovine serum and 2% 50 XHAT, adding 200. Mu.L/well to 96-well cell plates, and placing at 37℃and 5% CO 2 Culturing in an incubator;
3. cell screening and cell strain establishment: half-changing the RPMI-1640 screening culture solution of the fused cells on the 3 rd day of cell fusion, performing full-changing with the RPMI-1640 transitional culture solution containing 20% of fetal bovine serum and 1% of 100 XHT on the 5 th day, and taking the cell supernatant on the 7 th day for screening;
screening is carried out in two steps: firstly, screening positive cell holes by using an ic-ELISA method, secondly, selecting ketoconazole as a standard substance, and measuring the inhibition effect of positive cells by using the ic-ELISA method;
selecting a cell hole with better inhibition on a ketoconazole standard substance, subcloning by adopting a limiting dilution method, and detecting by using the same method after seven days;
and performing subcloning for three times according to the method to finally obtain the ketoconazole monoclonal antibody cell strain 3C11.
Example 5: preparation and identification of ketoconazole monoclonal antibody
Taking 8-10 week old BALB/c mice, and injecting 1mL of sterile paraffin oil into the abdominal cavity of each mouse; intraperitoneal injection of 1X 10 per mouse after 7 days 6 Ketoconazole hybridoma cells, collecting ascites from the seventh day, and purifying the ascites by an octanoic acid-saturated ammonium sulfate method;
under the condition of meta-acid, the n-octanoic acid can precipitate other hetero proteins except IgG immunoglobulin in ascites, and then the mixture is centrifuged and the precipitate is discarded; precipitating IgG type monoclonal antibody with equal amount of saturated ammonium sulfate solution, centrifuging, discarding supernatant, dissolving with 0.01M PBS solution (pH 7.4), dialyzing for desalting, and storing at-20deg.C.
Determination of IC of Ketoconazole monoclonal antibody Using Indirect competition ELISA 50 The value is 0.7ng/mL, the cross rate of the ketoconazole analogue is less than 1%, which shows that the ketoconazole analogue has good sensitivity to ketoconazole and can be used for the immunoassay detection of the ketoconazole. (Cross Rate= (IC of Ketoconazole) 50 IC of analog 50 )×100％)。
Example 6: application of ketoconazole monoclonal antibody
The monoclonal antibody prepared from hybridoma cell strain 3C11 through in vivo ascites is applied to an ELISA (enzyme-linked immunosorbent assay) additive recovery test of ketoconazole, and the specific steps are as follows:
(1) Coating 96-well ELISA plates with coating raw materials diluted by Carbonate Buffer Solution (CBS) and having a concentration of 0.3 mug/mL, wherein each well is 100 mug, coating at 37 ℃ for 2 hours, washing the plates with PBST washing liquid three times, each well is 200 mug, each time is 3min, and beating to dry;
(2) Blocking with CBS containing 0.2% gelatin, blocking at 37deg.C for 2 hr, washing the plate with PBST wash solution three times, 200 μl each time, 3min each time, and drying;
(3) Preparing 0,0.068,0.20,0.61,1.85,5.55,16.66 and 50ng/mL ketoconazole standard solutions respectively by using Phosphate Buffer Solution (PBS), adding the standard solutions and the sample extracting solution to be detected into the sealed ELISA plates respectively, repeating 3 holes for each sample by 50 mu L per hole, adding 50 mu L of anti-ketoconazole monoclonal antibody diluted with 1:32000 per hole, reacting for 0.5h at 37 ℃, washing the plates, and beating to dryness;
(4) 100 μl of HRP-labeled goat anti-mouse IgG secondary antibody diluted 1:3000 with PBS containing 0.1% gelatin was added to each well, reacted at 37deg.C for 0.5h, and then washed with plates and dried;
(5) 100. Mu.L of TMB developing solution was added to each well, and after developing at 37℃for 15min, 50. Mu.L of 2M H was added to each well 2 SO 4 Stop solution, measuring light absorption value at 450 nm;
the inhibition standard curve of ketoconazole monoclonal antibody against ketoconazole is shown in figure 1, and IC-ELISA is used for determining IC of ketoconazole monoclonal antibody 50 The detection limit of ketoconazole in a sample is 0.1ng/g, and the detection linear range is 0.1-2.5ng/g. The antibody has better sensitivity to ketoconazole, and can be used for the immunoassay detection of ketoconazole.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.
Claims (6)
1. A hybridoma cell strain secreting ketoconazole monoclonal antibody is characterized by being preserved in the China general microbiological culture Collection center, the national academy of sciences of China, national academy of sciences of China, no. 1, north Chen, west Lu, and No. 3 of the area of the Beijing, and the preservation date of 2021, 05, 13 days and the preservation number of CGMCC No.22327.
2. Use of the hybridoma cell line of claim 1 for detecting ketoconazole for non-diagnostic and non-therapeutic purposes.
3. The ketoconazole monoclonal antibody is characterized by being secreted by a hybridoma cell strain with a preservation number of CGMCC No.22327.
4. Use of a ketoconazole monoclonal antibody according to claim 3 for detecting ketoconazole for non-diagnostic, non-therapeutic purposes.
5. A kit comprising the ketoconazole monoclonal antibody according to claim 3.
6. Use of the kit of claim 5 for the detection of ketoconazole for non-diagnostic, non-therapeutic purposes.
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| CN114480296B (en) * | 2022-01-28 | 2023-09-15 | 北京壹拾智检生物科技有限公司 | Hybridoma cell strain, monoclonal antibody, detection kit and detection method |
| CN114717197A (en) * | 2022-03-10 | 2022-07-08 | 长春博迅生物技术有限责任公司 | Hybridoma cell strain monoclonal antibody capable of secreting anti-human IgG1 monoclonal antibody and application |
| CN114807051B (en) * | 2022-05-11 | 2023-08-04 | 江南大学 | Hybridoma cell strain of anti-dechloridone monoclonal antibody and application thereof |
| CN116376847B (en) * | 2023-05-15 | 2024-05-24 | 江南大学 | Hybridoma cell strain secreting famoxadone monoclonal antibody and application thereof |
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| CN111304174A (en) * | 2020-04-16 | 2020-06-19 | 江南大学 | Triazolone monoclonal antibody hybridoma cell strain B11S and application thereof |
| CN112280744A (en) * | 2020-10-26 | 2021-01-29 | 江南大学 | Hybridoma cell strain secreting monoclonal antibody of hypnone and application thereof |
| CN112280745A (en) * | 2020-10-26 | 2021-01-29 | 江南大学 | Hybridoma cell strain capable of secreting pyridaben monoclonal antibody and application thereof |
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