CN114908060B - Hybridoma cell strain capable of secreting zoxamide monoclonal antibody and application of hybridoma cell strain - Google Patents

Hybridoma cell strain capable of secreting zoxamide monoclonal antibody and application of hybridoma cell strain Download PDF

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CN114908060B
CN114908060B CN202210515630.7A CN202210515630A CN114908060B CN 114908060 B CN114908060 B CN 114908060B CN 202210515630 A CN202210515630 A CN 202210515630A CN 114908060 B CN114908060 B CN 114908060B
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zoxamide
monoclonal antibody
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刘丽强
程媛
胥传来
匡华
徐丽广
孙茂忠
吴晓玲
马伟
朱建平
郝昌龙
宋珊珊
胡拥明
吴爱红
郭玲玲
胥欣欣
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Abstract

The invention relates to a hybridoma cell strain secreting zoxamide monoclonal antibody and application thereof, and belongs to the technical field of immunodetection. The hybridoma cell strain CNT for secreting the zoxamide monoclonal antibody is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45116. The zoxamide monoclonal antibody secreted by the strain is used for analyzing and detecting zoxamide residue in food safety detection. The zoxamide monoclonal antibody cell strain obtained by the invention can be used for immunoassay detection, and has good detection sensitivity and specificity (IC) for zoxamide 50 The value is 1.09ng/mL, the cross rate of the para-zoxamide analogue is less than 1%, and the cross rate = (IC of zoxamide) 50 IC of/analogue 50 )×100％。

Description

Hybridoma cell strain capable of secreting zoxamide monoclonal antibody and application of hybridoma cell strain
Technical Field
The invention belongs to the technical field of immunodetection, and particularly relates to a hybridoma cell strain secreting zoxamide monoclonal antibodies and application thereof.
Background
Zoxamide is a benzamide type protective fungicide, has a novel action mechanism, is unique in oomycete fungi, inhibits sclerotic division through tubulin-subunit binding and disruption of the microtubule cytoskeleton, does not affect motility, cyst formation or germination of zoospores, and inhibits elongation of germ tubes accompanying the first cycle of sclerotic division, thereby preventing penetration of pathogens into host plants. Zoxamide is the only effective agent for oomycetes in the mitosis-resistant bactericide, can effectively control diseases caused by oomycetes fungi on crops such as fruit trees, vegetables and the like including grapes and potatoes, such as potato and tomato late blight, cucumber downy mildew, grape downy mildew and the like, has special effect on grape downy mildew, has longer lasting period and good rain erosion resistance, has a novel action mechanism, is wide in bactericidal spectrum and high in activity, and has wide application prospect and market prospect in accordance with the pesticide development direction in China. At present, the residual limit of the zoxamide in various agricultural products such as vegetables and fruits is specified in the United states, so that the research and establishment of a residual detection method of the zoxamide are of great significance.
For detecting the zoxamide residue, a high performance liquid chromatography, a gas chromatography-mass spectrometry or a liquid chromatography-mass spectrometry combined method is usually adopted. However, the methods have the defects of complex sample pretreatment, long detection time and the like, are not suitable for rapid detection of a large number of samples, and in order to maintain the benefits of wide consumers, an efficient and rapid detection method for zoxamide 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, is suitable for field rapid detection of a large number of samples, and is widely applied to antibiotic residue analysis. The precondition for detecting zoxamide by using an enzyme linked immunosorbent assay is that a monoclonal antibody with high specificity and high sensitivity to zoxamide is obtained, so that the method for preparing the monoclonal antibody with high specificity and high sensitivity to zoxamide is very critical.
Disclosure of Invention
In order to solve the technical problems, the invention provides a hybridoma cell strain secreting zoxamide monoclonal antibody and application thereof. The zoxamide monoclonal antibody secreted by the hybridoma cell strain has better specificity and detection sensitivity (IC) on zoxamide 50 The value is 1.09 ng/mL), can be used for establishing an immunological detection method of zoxamide and detecting the residue of zoxamide in food.
The invention aims to provide a hybridoma cell strain secreting zoxamide monoclonal antibody, which is characterized in that the hybridoma cell strain is preserved in China general microbiological culture Collection center (CGMCC), china academy of sciences, 3, north Cheng Xilu No. 1, beijing, the area facing the sun, is classified and named as a monoclonal cell strain, the preservation date is 2022, 03 months and 03 days, and the preservation number is CGMCC No.45116.
The second purpose of the invention is to provide the preparation method of the hybridoma cell strain, the zoxamide hapten used in the preparation process is ZOX-COOH, and the molecular structural formula is as follows:
Figure BDA0003641322880000021
in one embodiment of the invention, the zoxamide complete antigen used in the preparation process is ZOX-COOH-BSA, the molecular structural formula of which is:
Figure BDA0003641322880000031
the invention also provides a preparation method of the hybridoma cell strain secreting the zoxamide monoclonal antibody, which specifically comprises the following steps:
(1) Preparing a zoxamide complete antigen by using a zoxamide hapten, and preparing the obtained zoxamide complete antigen into an antigen-containing Freund adjuvant and an antigen-containing incomplete Freund adjuvant;
(2) Injecting the obtained Freund adjuvant containing the antigen into a BALB/c mouse body through back subcutaneous injection for multiple times of immunization, wherein the complete Freund adjuvant containing the antigen is adopted for the first immunization, and the incomplete Freund adjuvant containing the antigen is adopted for the boosting immunization;
(3) Collecting blood from the mice subjected to the immunization process, detecting the serum immune titer and the immune suppression capacity of the mice through indirect ELISA, and screening the mice with high content of zoxamide antibody in the serum to obtain the immune mice;
(4) Carrying out the last boosting immunization on the screened mice by using an incomplete Freund adjuvant containing antigen, and then carrying out the sprint immunization by intraperitoneal injection, wherein the sprint immunization is carried out by adopting a zoxamide complete antigen without the Freund adjuvant;
(5) Fusing splenocytes and myeloma cells of the BALB/c mice after the sprint immunization, culturing the fused cells through a culture medium, detecting positive cell holes by using indirect ELISA, further determining the inhibition effect of the positive cell holes by using an indirect competition ELISA method, subcloning the positive cell holes with the best inhibition by using a limiting dilution method, and finally screening to obtain a hybridoma cell strain capable of secreting the zoxamide monoclonal antibody.
In one embodiment of the present invention, the interval between the first immunization and the booster immunization in the steps (2) and (4) is one month, the interval between the booster immunization is 21 days, and the interval between the booster immunization and the sprint immunization is 18 to 21 days.
In one embodiment of the present invention, the first immunization dose of the steps (2) and (4) is 100. Mu.g/mouse, the boosting immunization dose is 50. Mu.g/mouse, and the sprint immunization dose is 25. Mu.g/mouse.
In one embodiment of the present invention, the immunization process of steps (2) and (4) comprises 1 first immunization, 4 booster immunizations and 1 sprint immunization.
In one embodiment of the present invention, the blood collection in step (3) is performed on day 7 after the 3 rd immunization course.
In one embodiment of the present invention, the cell fusion in step (5) is performed 3 days after the completion of the spike immunization.
In one embodiment of the present invention, the cell fusion in step (5) is performed by a polyethylene glycol (PEG 4000) method.
In one embodiment of the present invention, the medium in the step (5) is RPMI-1640 medium.
In one embodiment of the present invention, the number of subclones in step (5) is 3.
In one embodiment of the invention, the zoxamide complete antigen is prepared by the following method: dissolving zoxamide hapten and N-hydroxysuccinimide (NHS) in an organic solvent for reaction to obtain a zoxamide hapten ZOX-COOH solution; simultaneously, dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) in an organic solvent, adding the solution into a zoxamide hapten ZOX-COOH solution, and mixing and reacting to obtain a solution A; diluting bovine serum albumin BSA with a buffer solution to obtain a solution B; and adding the solution A into the solution B for reaction to obtain a reaction solution, and dialyzing the reaction solution by using a buffer solution to obtain the zoxamide complete antigen ZOX-COOH-BSA.
In one embodiment of the invention, the organic solvent is DMF.
In one embodiment of the invention, the buffer solution is a carbonate buffer solution; the buffer solution is phosphate buffer solution.
The invention also provides a synthetic method of the zoxamide peridium antigen, which comprises the following steps:
dissolving zoxamide hapten (ZOX-COOH) and N-hydroxysuccinimide (NHS) in N, N-Dimethylformamide (DMF), and stirring at room temperature for reaction to obtain a zoxamide hapten (ZOX-COOH) solution; dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) in an organic solvent, adding the solution into ZOX-COOH solution, and stirring at room temperature to react for 4-6h to obtain a solution A; diluting egg albumin (OVA) with Carbonate Buffer Solution (CBS) to obtain solution B; slowly adding the solution A into the solution B dropwise for reaction to obtain a reaction solution; the reaction solution was dialyzed against PBS solution to remove unreacted small molecule hapten, and thus a coating antigen (ZOX-COOH-OVA) was obtained.
The third purpose of the invention is to provide a zoxamide monoclonal antibody, wherein the zoxamide monoclonal antibody is secreted by the hybridoma cell strain.
The fourth purpose of the invention is to provide a composition, wherein the composition contains the hybridoma cell strain and/or the zoxamide monoclonal antibody.
The fifth purpose of the invention is to provide a kit, wherein the kit contains one or more of the hybridoma cell strain, the zoxamide monoclonal antibody and the composition.
The sixth purpose of the invention is to provide a test strip, which contains one or more of the hybridoma cell strain, the zoxamide monoclonal antibody and the composition.
The seventh purpose of the invention is to provide a chip, wherein the chip contains one or more of the hybridoma cell strain, the zoxamide monoclonal antibody and the composition.
The eighth purpose of the invention is to provide the application of the hybridoma cell strain, the zoxamide monoclonal antibody, the composition, the kit, the test strip or the chip in detecting zoxamide.
Compared with the prior art, the technical scheme of the invention has the following advantages:
the invention has the beneficial effects that: the zoxamide monoclonal antibody obtained by the invention can be used for immunoassay detection, and has good detection sensitivity and specificity (IC) for zoxamide 50 A value of 1.09ng/mL, a crossing rate of the zoxamide analogue of less than 1%, and a crossing rate = (IC of zoxamide) 50 IC of/analogue 50 )×100％。
Biological material sample preservation: a hybridoma cell strain CNT for secreting zoxamide monoclonal antibody is deposited in China general microbiological culture Collection center (CGMCC), china academy of sciences, no. 3 of China academy of sciences, hospital, no. 1 of Xilu, beijing, chaoyang, the address of the microorganism, is classified and named as a monoclonal cell strain, the preservation date is 2022 years, 03 months and 03 days, and the preservation number is CGMCC No.45116.
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In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the embodiments of the present disclosure taken in conjunction with the accompanying drawings, in which
FIG. 1 is a standard curve of the inhibition of the zoxamide monoclonal antibody of the invention on zoxamide.
Detailed Description
The present invention is further described below in conjunction with the drawings and the embodiments so that those skilled in the art can better understand the present invention and can carry out the present invention, but the embodiments are not to be construed as limiting the present invention.
The culture medium used in the examples of the present invention is as follows:
RPMI-1640 medium: l-arginine 290mg/L, L-asparagine 50mg/L, L-aspartic acid 20mg/L, L-cystine dihydrochloride 65.15mg/L, L-glutamic acid 20mg/L, glycine 10mg/L, L-histidine 15mg/L, L-hydroxyproline 20mg/L, L-isoleucine 50mg/L, L-leucine 50mg/L, L-lysine hydrochloride 40mg/L, L-methionine 15mg/L, L-phenylalanine 15mg/L, L-proline 20mg/L, L-serine 30mg/L, L-threonine 20mg/L, L-tryptophan 5 mg/83 zxft 83-tyrosine 78 mg/5478 zxft 5478 mg/L-valine/7439 mg/7438 zxft 7439 mg 1mg/L of p-aminobenzoic acid, 100mg/L of calcium nitrate, 48.84mg/L of anhydrous magnesium sulfate, 8624 mg/L of anhydrous sodium dihydrogen phosphate, 400mg/L of potassium chloride, 6000mg/L of sodium chloride, 2000mg/L of glucose, 1mg/L of reduced glutathione, 5 mg/9696 mg/L of phenol red, 300mg/L of L, L-glutamine, 0.2 mg/3235 mg/L of biotin, L, D-calcium pantothenate, 1 mg/3292 mg/L of folic acid, L, i-inositol, 35mg/L of nicotinamide, 3mg/L of choline chloride, 1mg/L of pyridoxine hydrochloride, 0.2mg/L of riboflavin, 1mg/L of thiamine hydrochloride, 120.005 mg/L of vitamin B, and 2000mg/L of sodium bicarbonate.
The reagents involved in the following examples are as follows:
carbonate Buffer (CBS): weighing Na 2 CO 3 1.59 g，NaHCO 3 2.93 g, respectively dissolving a small amount of double distilled water, mixing, adding the double distilled water to about 800mL, uniformly mixing, adjusting the pH value to 9.6, adding the double distilled water to a constant volume of 1000mL, and storing at 4 ℃ for later use;
phosphate Buffered Saline (PBS): 8.00gNaCl,0.2g KCl,0.2g KH 2 PO 4 ，2.9gNa 2 HPO 4 ·12H 2 Dissolving 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 1000mL;
PBST: PBS containing 0.05% tween 20;
antibody dilution: PBS was added with 0.1% gelatin.
TMB color development liquid: solution A: na (Na) 2 HPO 4 ·12H 2 18.43g of O, 9.33g of citric acid and pure water to reach the constant volume of 1000mL; 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.
The detection methods referred to in the following examples are as follows:
the method for detecting the zoxamide inhibition rate comprises the following steps: the most suitable antigen and antibody concentrations in the ic-ELISA were selected by a checkerboard assay. The antigen was diluted to 0.03,0.1,0.3 and 1 μ g/mL with Carbonate Buffer (CBS) and the antibody was diluted to 0.03,0.1,0.3 and 1 μ g/mL with antibody diluent. After the optimal working point is selected, the zoxamide standard substance is diluted 0,0.041,0.123 and 0370,1.11,3.33,10And the concentration of 30ng/mL, according to the operation steps of the IC-ELISA, finally drawing by using originPro 8.5 (the result is shown in figure 1), obtaining a zoxamide standard inhibition curve, and calculating IC 50 。
Example 1: synthesis of zoxamide hapten
The zoxamide small molecule has no immunogenicity, and can not stimulate a mouse to generate immune response so as to generate an antibody, so that the zoxamide is coupled to a protein by a protein connection technology to obtain the immunogenicity; active groups commonly used in the protein coupling technology comprise amino, carboxyl, hydroxyl, sulfydryl and the like, and the zoxamide does not contain amino, carboxyl and hydroxyl in the molecular structure, so that the carboxyl needs to be derived from the structure.
The structure of the derived zoxamide hapten is as follows:
Figure BDA0003641322880000081
the derivation process is as follows:
dissolving 100mg of zoxamide in 5mL of pyridine, adding 200mg of CMO, performing magnetic stirring reaction in a 70 ℃ water bath for 6 hours, drying the reaction solution by using nitrogen, dissolving the precipitate in a 2m L trichloromethane solution, and extracting with ultrapure water of the same volume for three times. Collecting an organic phase, drying by nitrogen and obtaining a precipitate which is a derivative product.
Example 2: synthesis of zoxamide complete antigen
Weighing 6mg of zoxamide hapten (ZOX-COOH) and 4.5mg of N-hydroxysuccinimide (NHS), dissolving in 300 mu LN, N-Dimethylformamide (DMF), and stirring at room temperature for reaction for 10min; 6.5mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) was weighed out and dissolved sufficiently in 100. Mu.L of DMF, and then added to the ZOX-COOH solution, followed by reaction with stirring at room temperature for 4 to 6 hours (referred to as solution A). Taking 5mg BSA, diluting to 2mg/mL (called as solution B) by using 0.01M Carbonate Buffer Solution (CBS), slowly adding the solution A into the solution B dropwise, and reacting at room temperature overnight; then dialyzing with 0.01M PBS solution, removing unreacted small molecule hapten to obtain a complete antigen ZOX-COOH-BSA, and identifying by an ultraviolet absorption scanning method.
Example 3: synthesis of zoxamide peridium
Dissolving 4mg of zoxamide hapten (ZOX-COOH) and 2.8mg of N-hydroxysuccinimide (NHS) in 300 mu L of anhydrous N, N-Dimethylformamide (DMF), and stirring and reacting at room temperature for 10min to obtain a zoxamide hapten (ZOX-COOH) solution; dissolving 4.4mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) in 100 mu L of anhydrous DMF, adding the solution into ZOX-COOH solution, and stirring at room temperature to react for 4-6h to obtain solution A; diluting 5mg of chicken Ovalbumin (OVA) with 2mL of 0.01M Carbonate Buffer Solution (CBS) to obtain solution B; slowly adding the solution A into the solution B dropwise for reaction to obtain a reaction solution; the reaction solution was dialyzed against PBS solution to remove unreacted small molecule hapten, and thus a coating antigen (ZOX-COOH-OVA) was obtained.
Example 4: preparation of hybridoma cell strain secreting zoxamide monoclonal antibody
1. Obtaining animal immunity: mixing and emulsifying a zoxamide complete antigen and an equivalent amount of Freund's adjuvant, and performing neck and back subcutaneous multipoint injection immunization (except for sprint immunization) on a BALB/c mouse; 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, the thorny immunity is directly diluted by normal saline and then injected into the abdominal cavity, and the dosage is reduced by half to obtain 25 mu g/mouse; one month is separated between the first immunization and the second boosting immunization, 21 days are separated between the multiple boosting immunizations, and 18-21 days are separated between the sprint immunization and the last boosting immunization; 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;
2. cell fusion: after three days of the spurt immunization, cell fusion is carried out according to a conventional PEG (polyethylene glycol, molecular weight 4000) method, and the specific steps are as follows:
a. cutting tail, taking blood, killing the mouse by cervical dislocation, 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 by using a rubber head of an injector, passing through a 200-mesh cell screen to obtain spleen cell suspension, collecting, centrifuging (1200rpm, 8min), washing the spleen cells for three times by using 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, the SP2/0 tumor cells are treated with a medium containing 10% FBS (fetal bovine serum) RPMI-1640 in 5% CO 2 In an incubator, the number of SP2/0 tumor cells is required to reach (1-4) multiplied by 10 before fusion 7 Ensuring that SP2/0 tumor cells are in logarithmic growth phase before fusion, collecting the tumor cells during fusion, suspending the tumor cells in RPMI-1640 basic culture solution, and counting the cells;
c. the fusion process is 7min: 1min, 1mL of PEG 1500 is added to the cells from slow to fast; standing for 2 min; dropping 1mL of RPMI-1640 culture medium within 1min at 3min and 4 min; at 5min and 6min, 2mL of RPMI-1640 culture medium is added dropwise within 1 min; at 7min, 1mL of RPMI-1640 culture medium is added dropwise every 10 s; then carrying out warm bath at 37 ℃ for 5min; centrifuging (800rpm, 8min), discarding the supernatant, resuspending in RPMI-1640 screening medium containing 20% fetal bovine serum and 2% 50 XHAT, adding to a 96-well cell plate at 200. Mu.L/well, incubating at 37 ℃ and 5% CO 2 Culturing in an incubator;
3. cell screening and cell strain establishment: on day 3 of cell fusion, performing RPMI-1640 screening culture medium half-exchange on fused cells, performing total-exchange on day 5 by using RPMI-1640 transition culture medium containing 20% fetal calf serum and 1% 100 × HT, and on day 7, taking cell supernatant for screening;
the screening is divided into two steps: firstly, screening out positive cell holes by using an ic-ELISA method, secondly, selecting zoxamide as a standard substance, and determining the inhibition effect of positive cells by using the ic-ELISA method;
selecting a cytopore which has better inhibition to a zoxamide standard substance, carrying out subcloning by adopting a limiting dilution method, and detecting by using the same method after seven days;
and carrying out subcloning for three times according to the method to finally obtain the zoxamide monoclonal antibody cell strain CNT.
Example 5: preparation and identification of zoxamide monoclonal antibody
Collecting 8-10 week-old BALB/c mice, and abdominal cavity of each mouseInjecting 1mL of sterile paraffin oil; 7 days later, 1X 10 injections were administered intraperitoneally to each mouse 6 Zoxamide 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 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 (pH 7.4), dialyzed and desalted to finally obtain a purified monoclonal antibody, which was stored at-20 ℃.
Determination of IC of zoxamide monoclonal antibody Using Indirect competitive ELISA 50 The value is 1.09ng/mL, the crossing rate of the zoxamide analogue is less than 1%, and the result is shown in a table, which shows that the zoxamide analogue has good sensitivity and can be used for immunoassay detection of the zoxamide. (Cross-over Rate = (zoxamide IC) 50 IC of/analogue 50 )×100％)。
TABLE 1
Compound (I) IC 50 (ng/mL) Crossover ratio (%)
Zoxamide 1.09 100
Fluoramides >500 <1
Fenhexamid >500 <1
Penthiopyrad >500 <1
Mandipropamid >500 <1
Example 6: application of zoxamide monoclonal antibody
The monoclonal antibody prepared from the hybridoma cell strain CNT through in-vivo ascites is applied to an ELISA addition recovery test of zoxamide, and the method specifically comprises the following steps:
(1) Coating a 96-well enzyme label plate with coating antigen with the concentration of 0.3 mu g/mL diluted by Carbonate Buffer Solution (CBS), coating 100 mu L of the enzyme label plate in each well at 37 ℃ for 2h, washing the plate with PBST washing liquor three times, wherein 200 mu L of the washing liquor is used in each well, and the washing liquor is patted dry after 3min each time;
(2) Sealing with CBS containing 0.2% gelatin, sealing at 37 deg.C for 2 hr at 200 μ L per well, washing the plate with PBST lotion for three times, each time at 200 μ L per well, each time for 3min, and drying;
(3) Preparing 0,0.041ng/mL,0.123ng/mL,0370ng/mL,1.11ng/mL,3.33ng/mL,10ng/mL and 30ng/mL of zoxamide standard solution by using Phosphate Buffered Saline (PBS), adding the standard solution and the extract of a sample to be detected into the closed enzyme label plate respectively, repeating 3 holes in each hole, adding 50 mu L of zoxamide-resistant monoclonal antibody diluted by 1 32000 in each hole, reacting at 37 ℃ for 0.5h, washing the plate, and drying;
(4) mu.L of HRP-labeled goat anti-mouse IgG secondary antibody diluted with PBS containing 0.1% gelatin at 1;
(5) Adding 100 μ L of TMB color developing solution into each well, developing at 37 deg.C for 15min, and collecting the solutionAdd 50. Mu.L of 2M H 2 SO 4 Stopping solution, measuring the light absorption value at 450 nm;
the inhibition standard curve of the zoxamide monoclonal antibody on zoxamide is shown in figure 1, and the IC of the zoxamide monoclonal antibody is determined by using IC-ELISA 50 The value is 1.09ng/mL, which indicates that the antibody has better sensitivity to zoxamide and can be used for immunoassay detection of zoxamide.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. This need not be, nor should it be exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.

Claims (7)

1. The hybridoma cell strain secreting zoxamide monoclonal antibody is characterized in that the hybridoma cell strain is deposited in China general microbiological culture Collection center (CGMCC), china academy of sciences, china institute of microbiology, no. 3, of the West Lu 1 Hospital, north Cheng, the south China area, beijing, is classified and named as monoclonal cell strain, the preservation date is 2022 years, 03 months and 03 days, and the preservation number is CGMCC No.45116.
2. A zoxamide monoclonal antibody, which is secreted by the hybridoma cell line as claimed in claim 1.
3. A composition, which comprises the hybridoma cell line of claim 1 and/or the zoxamide monoclonal antibody of claim 2.
4. A kit, which is characterized by comprising one or more of the hybridoma cell strain of claim 1, the zoxamide monoclonal antibody of claim 2 and the composition of claim 3.
5. A test strip, which is characterized by comprising one or more of the hybridoma cell strain of claim 1, the zoxamide monoclonal antibody of claim 2 and the composition of claim 3.
6. A chip, which is characterized by comprising one or more of the hybridoma cell strain described in claim 1, the zoxamide monoclonal antibody described in claim 2 and the composition described in claim 3.
7. The hybridoma cell strain of claim 1, the zoxamide monoclonal antibody of claim 2, the composition of claim 3, the kit of claim 4, the test strip of claim 5 or the chip of claim 6 for use in detecting zoxamide.
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CN110950962A (en) * 2019-12-20 2020-04-03 江南大学 Hybridoma cell strain A11S for secreting bimesomepheniul monoclonal antibody and application thereof

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CN106282125B (en) * 2016-09-23 2019-06-14 江南大学 One plant of hybridoma cell strain NaN-1 for secreting anti-sulfa antibiotics monoclonal antibody and its application
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CN110423729A (en) * 2019-06-27 2019-11-08 江南大学 One plant of hybridoma cell strain GTY for secreting anti-Mobucin monoclonal antibody and its application
CN110950962A (en) * 2019-12-20 2020-04-03 江南大学 Hybridoma cell strain A11S for secreting bimesomepheniul monoclonal antibody and application thereof

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