CN114908060A

CN114908060A - Hybridoma cell strain secreting zoxamide monoclonal antibody and application thereof

Info

Publication number: CN114908060A
Application number: CN202210515630.7A
Authority: CN
Inventors: 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
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2022-05-12
Filing date: 2022-05-12
Publication date: 2022-08-16
Anticipated expiration: 2042-05-12
Also published as: CN114908060B

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 secreting the zoxamide monoclonal antibody has been preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No. 45116. The zoxamide monoclonal antibody secreted and obtained by the strain is used for analyzing and detecting zoxamide residue in food safety detectionAnd (6) measuring. 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 percent, and the cross rate is equal to (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 bactericide, has a novel action mechanism, is unique in oomycete fungi, inhibits sclerotic division through tubulin-subunit combination and microtubule cytoskeleton rupture, does not affect the motility, cyst formation or germination of zoospores, and inhibits the elongation of germ tubes along with the first cycle of sclerotic division, thereby preventing germs from penetrating through 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 1.09ng/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 (China institute of sciences, 3, North West Lu No. 1 Hospital, Chaoyang, Beijing), is classified and named as monoclonal cell strain, and has a preservation date of 2022 years, 03 months and 03 days and a preservation number of CGMCC No. 45116.

The second purpose of the invention is to provide a preparation method of the hybridoma cell strain, wherein a zoxamide hapten used in the preparation process is ZOX-COOH, and the molecular structural formula is as follows:

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 as follows:

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 antigen-containing incomplete Freund adjuvant, 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 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 18-21 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 sprint 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; meanwhile, 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, and stirring at room temperature to react for 4-6h to obtain 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, thus giving a coating antigen (ZOX-COOH-OVA).

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 The value is 1.09ng/mL, the cross rate of the para-zoxamide analogue is less than 1 percent, and the cross rate is equal to (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.

Drawings

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 was 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 5mg/L, L-tyrosine 23.19mg/L, L-valine 20mg/L, p-aminobenzoic acid 1mg/L, calcium nitrate 100mg/L, anhydrous magnesium sulfate 48.84mg/L, anhydrous sodium dihydrogen phosphate 676.13mg/L, potassium chloride 400mg/L, sodium chloride 6000mg/L, glucose 2000mg/L, reduced glutathione 1mg/L, phenol red 5mg/L, L-glutamine 300mg/L, biotin 0.2mg/L, D-calcium pantothenate 0.25mg/L, folic acid 1mg/L, i-inositol 35mg/L, nicotinamide 1mg/L, choline chloride 3mg/L, pyridoxine hydrochloride 1mg/L, riboflavin 0.2mg/L, thiamine hydrochloride 1mg/L, vitamin B120.005 mg/L, and sodium bicarbonate 2000 mg/L.

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 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 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 1000 mL;

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 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.

The detection methods referred to in the following examples are as follows:

the method for detecting the inhibition rate of zoxamide 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. mu.g/mL with Carbonate Buffer (CBS) and the antibody was diluted to 0.03, 0.1, 0.3 and 1. mu.g/mL with antibody diluent. After selecting the optimal working point, the zoxamide standard substance is diluted to the concentration of 0, 0.041, 0.123, 0370, 1.11, 3.33,10 and 30ng/mL and the like, according to the operation steps of the IC-ELISA, and finally an originPro 8.5 is used for drawing (the result is shown in figure 1), so as to obtain the zoxamide standard inhibition curve, and the IC is calculated ₅₀ 。

Example 1: synthesis of zoxamide hapten

The zoxamide micromolecule has no immunogenicity, and cannot stimulate a mouse to generate immune response so as to generate an antibody, so that the zoxamide is coupled to protein through 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:

the derivation process is as follows:

dissolving 100mg of zoxamide in 5mL of pyridine, adding 200mg of CMO, magnetically stirring in a 70 ℃ water bath for reaction for 6 hours, drying the reaction solution by using nitrogen, dissolving the precipitate in a 2m L trichloromethane solution, and extracting with ultrapure water with 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 10 min; 6.5mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) was weighed out and dissolved in 100. mu.L of DMF, and then added to ZOX-COOH solution, followed by stirring at room temperature for 4-6 hours (referred to as solution A). 5mg of BSA is taken and diluted to 2mg/mL (called solution B) by 0.01M Carbonate Buffer Solution (CBS), and solution A is slowly added into solution B dropwise and reacted at room temperature overnight; then dialyzing with 0.01M PBS solution to remove unreacted small molecule hapten to obtain complete antigen ZOX-COOH-BSA, and identifying by 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 for 10min at room temperature 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, and stirring at room temperature to react for 4-6h to obtain solution A; diluting 5mg of egg albumin (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, thus giving a coating antigen (ZOX-COOH-OVA).

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 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 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. cutting the tail, taking blood, immediately putting the mouse into 75% alcohol for disinfection after the mouse is killed by a cervical vertebra dislocation method, soaking for about 5min, taking out the spleen of the mouse through aseptic operation, properly grinding the spleen 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% CO ₂ In an incubator, the number of SP2/0 tumor cells is required to reach (1-4) x 10 before fusion ⁷ Ensuring SP2/0 tumor cells to be 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 7 min: 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; 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 carrying out warm bath at 37 ℃ for 5 min; centrifuging (800rpm, 8min), discarding the supernatant, resuspending in RPMI-1640 screening medium containing 20% fetal bovine serum, 2% 50 XHAT, adding to 96-well cell plates at 200. mu.L/well,placing at 37 ℃ and 5% CO ₂ Culturing in an incubator;

3. cell screening and cell strain establishment: performing RPMI-1640 screening culture medium half-exchange on fused cells on the 3 rd day of cell fusion, performing total-exchange with RPMI-1640 transitional culture medium containing 20% fetal calf serum and 1% 100 XHT on the 5 th day, and taking cell supernatant on the 7 th day 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 cell hole which has better inhibition on a zoxamide standard substance, carrying out subcloning by adopting a limiting dilution method, and carrying out detection 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

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 10 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 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 (pH7.4), dialyzed and desalted to finally obtain a purified monoclonal antibody, which was stored at-20 ℃.

IC of zoxamide monoclonal antibody was determined using indirect competition ELISA ₅₀ The value is 1.09ng/mL, the cross rate of the zoxamide analogue is less than 1%, and the results are shown in a table, so that the zoxamide analogue has good sensitivity and can be used for immunoassay detection of the zoxamide. (crossover rate ═ IC of zoxamide 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 for three times, wherein 200 mu L of the washing liquor in each well is used for 3min, and drying by beating;

(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) respectively preparing 0, 0.041ng/mL, 0.123ng/mL, 0370ng/mL, 1.11ng/mL, 3.33ng/mL, 10ng/mL and 30ng/mL zoxamide standard solutions by using Phosphate Buffered Saline (PBS), respectively adding the standard solutions and the extract of a sample to be detected into the closed enzyme label plate, wherein each well is 50 mu L, each sample is repeatedly provided with 3 wells, 50 mu L of zoxamide-resistant monoclonal antibody diluted by 1:32000 is added into each well, and after the reaction is carried out at 37 ℃ for 0.5h, the plate is washed and dried;

(4) adding 100 mu L of HRP-labeled goat anti-mouse IgG secondary antibody diluted by PBS containing 0.1% gelatin at a ratio of 1:3000 into each well, reacting at 37 ℃ for 0.5h, washing the plate and drying;

(5) adding 100 μ L of TMB developing solution into each well, developing at 37 deg.C for 15min, and adding 50 μ L of H2M into each well 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. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the scope of the invention.

Claims

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. The method for preparing hybridoma cell strain according to claim 1, wherein the zoxamide hapten used in the preparation process is ZOX-COOH, the molecular structural formula of which is as follows:

3. the method for preparing hybridoma cell strain according to claim 2, wherein the zoxamide complete antigen used in the preparation process is ZOX-COOH-BSA, and the molecular structural formula is as follows:

4. the method for preparing the hybridoma cell strain according to claim 3, wherein the zoxamide complete antigen is prepared by the following method: dissolving a zoxamide hapten and N-hydroxysuccinimide NHS in an organic solvent for reaction to obtain a zoxamide hapten ZOX-COOH solution; meanwhile, 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.

5. A zoxamide monoclonal antibody, which is secreted by the hybridoma cell line as claimed in claim 1.

6. A composition, which comprises the hybridoma cell line of claim 1 and/or the zoxamide monoclonal antibody of claim 5.

7. A kit, which is characterized by comprising one or more of the hybridoma cell strain of claim 1, the zoxamide monoclonal antibody of claim 5 and the composition of claim 6.

8. A test strip is characterized by comprising one or more of the hybridoma cell strain disclosed in claim 1, the zoxamide monoclonal antibody disclosed in claim 5 and the composition disclosed in claim 6.

9. A chip, which is characterized by comprising one or more of the hybridoma cell line as defined in claim 1, the zoxamide monoclonal antibody as defined in claim 5 and the composition as defined in claim 6.

10. The hybridoma cell strain of claim 1, the zoxamide monoclonal antibody of claim 5, the composition of claim 6, the kit of claim 7, the test strip of claim 8 or the chip of claim 9 for detecting zoxamide.