CN110643578B

CN110643578B - Anti-avilamycin A monoclonal antibody hybridoma cell strain and application thereof

Info

Publication number: CN110643578B
Application number: CN201911022259.5A
Authority: CN
Inventors: 陈敏
Original assignee: Zhejiang Gongshang University
Current assignee: Zhejiang Gongshang University
Priority date: 2019-10-25
Filing date: 2019-10-25
Publication date: 2021-02-19
Anticipated expiration: 2039-10-25
Also published as: CN110643578A

Abstract

The invention discloses an anti-avilamycin A monoclonal antibody hybridoma cell strain and an application thereof, belonging to the technical field of food safety immunological detection. The cell line of the present invention, which is classified and named as avilamycin A-resistant hybridoma cell line 2GC8, has been deposited in the General Microbiology Center of the China Microorganism Culture Collection and Management Committee, and the deposit number is CGMCC NO.17485. In the present invention, a succinic anhydride method is used to construct a conjugate of aviramycin A and bovine serum albumin as an immunogen, and BALB/c mice are immunized; the immunized mouse spleen cells are then fused with myeloma cells, and the selective Culture and subcloning several times to obtain positive hybridoma cell line. The invention realizes the preparation of avilamycin A component monoclonal antibody product for the first time. The monoclonal antibody secreted by the hybridoma cell line has high sensitivity and strong specificity, which fills the blank of domestic products and can be used for avilamycin Rapid and accurate immunodetection and analysis of

Description

Anti-avilamycin A monoclonal antibody hybridoma cell strain and application thereof

Technical Field

The invention relates to a hybridoma cell strain 2GC8 secreting an avilamycin A-resistant monoclonal antibody and application thereof, and belongs to the technical field of (food safety) immunological detection.

Background

Avilamycin (A) and (B)Avilamycin) Is prepared from streptomycin viridisStreptomyces viridoehrongenesThe dichloro-iso-lichen antacid ester produced by fermentation belongs to oligosaccharide antibiotics of the nysfungin family. The Avermectin has four components of A to N, wherein the Avermectin A (C) is₆₁H₈₈Cl₂O₃₂) The component has the strongest activity up to 1451U/mg, and the second is avilamycin B (C)₅₉H₈₄Cl₂O₃₂) The component is 908U/mg, and the activity of other components is weaker. The avilamycin has good inhibition effect on gram-positive bacteria, clostridium aeroginosum, staphylococcus aureus, streptococcus (except lactobacillus) and part of gram-negative bacteria, has the effects of promoting growth, preventing animal diseases and the like, and is a novel digestion enhancer (digestive enhancer) and metabolic regulator (metabolic modulator).

The avilamycin serving as a novel veterinary antibiotic has a growth-promoting and regulating mechanism which is used for building a good intestinal environment for a host by inhibiting or promoting the growth metabolic process of intestinal bacteria of animals. Because of its ability to promote the absorption of nutrients by animals, it is widely used as an Antibiotic Growth Promoter (AGP) added to feed. In recent years, the rapid increase in antibiotic resistance, particularly the emergence of multidrug-resistant (MDR) microorganisms, has led to the reevaluation of these veterinary antibiotics. Although avilamycin is used advantageously in the production of livestock and poultry, once it is abused in the feed or the period of withdrawal is shortened, it can lead to drug residues in the animal's body and thus in the food. Long-term consumption of food containing antibiotic residues can cause MDR pathogens to enter the body and reduce the effect of antibiotics on bacterial infection diseases, thereby leading to more deaths. In 11 months in 2017, the ministry of agriculture in China organizes and finishes the work of repairing the maximum residue limit standard of veterinary drugs in animal food and formulating part of limit standard according to the stipulation of food safety laws, thereby forming the maximum residue limit (promulgation draft) of veterinary drugs in animal food according to the national standard of food safety, and increasing 13 limit varieties, wherein the varieties comprise avilamycin. The specific standard is as follows: the target tissues of pigs, chickens, turkeys and rabbits are limited to 200. mu.g/kg muscle, 200. mu.g/kg skin and fat, and 300. mu.g/kg liver by using dichloroisovaleric acid (DIA) as a marker residue, and avilamycin is prohibited during the laying period. Although the amount of avilamycin used as a feed additive is not specified by the regulations, it is critical to control the residual amount of DIA in the target cells of the animal by controlling the amount of avilamycin taken up from the source, i.e., the amount and time of avilamycin added to the feed.

At present, a standard detection method for the avilamycin in the feed is not established in China. The maximum residual limit of veterinary drugs in animal food (written reports) in national standards for food safety has limited the residual amount of the metabolite DIA of avilamycin in target cells of animals. Therefore, in order to enhance the monitoring of the drug addition in the feed, the establishment and standardization of the detection method of the avilamycin in the feed and the establishment of the corresponding industry standard are urgent. At present, methods for detecting avilamycin mainly comprise an instrumental analysis method (a liquid chromatography-mass spectrometry combined detection method and a high performance liquid chromatography) and a microbiological method. And the instrumental analysis method has expensive equipment, complex operation and high requirement on detection personnel, and is not suitable for rapid screening of large-scale samples on site. The microbiological method does not need special equipment, has the advantages of simple operation, suitability for mass detection and the like, but has poor repeatability and easy interference of subjective conditions on result judgment. The immunoassay method is an immunoassay method based on antigen and antibody, such as an ELISA method, a colloidal gold immunochromatography detection method and the like, and is widely applied to the rapid detection of various antibiotic residues in food raw materials due to the advantages of low cost, rapid detection, high flux, high sensitivity and the like. Therefore, the development of a high-sensitivity anti-avilamycin a monoclonal antibody is urgently required. The key point of the preparation of the avilamycin A monoclonal antibody is the preparation of an avilamycin A artificial antigen. The avilamycin A has small molecular weight and weak immunogenicity, so that the animal needs to be immunized after being coupled with the carrier protein to prepare the monoclonal antibody.

Disclosure of Invention

The invention aims to provide a hybridoma cell strain for secreting an avilamycin A-resistant monoclonal antibody and application thereof.

In order to achieve the above purpose, the invention provides the following technical routes:

a hybridoma cell strain of an avilamycin A resistant monoclonal antibody is named as a hybridoma cell strain 2GC8, and is preserved in China general microbiological culture Collection center in 29 th of 2019 in 03 th month with the preservation number of CGMCC NO.17485 and the preservation address of Beijing China.

A preparation method of a hybridoma cell strain secreting an avilamycin A resistant monoclonal antibody comprises the following steps: (1) preparing avilamycin A hapten; (2) preparation of artificial antigen (immunogen and coating antigen); (3) immunizing BALB/c mice with the immunogen, and then obtaining the antibody with the titer larger than 1:64000 spleen cells of immunized BALB/c mice were cell fused with myeloma cell SP 2/0; and finally, screening and subcloning for multiple times by using a HAT culture medium to obtain a hybridoma cell strain.

Preferably, the ratio of the avilamycin A to the succinic anhydride in the preparation process of the avilamycin A hapten is 10:1 (mass ratio)

An avilamycin A resisting monoclonal antibody is secreted by hybridoma cell strain 2GC8 with the preservation number of CGMCC NO.17485 or its subcultured cell strain.

The preparation method of the monoclonal antibody comprises the steps of inducing ascites or culturing cells of a hybridoma cell strain 2GC8, precipitating caprylic acid-ammonium sulfate salt and purifying Protein G to obtain the monoclonal antibody.

Preferably, 0.5 mL of sterile paraffin oil is injected intraperitoneally into BALB/c mice, and 7X 10 is injected intraperitoneally one week later⁵Ascites were collected at 0.5 mL/mL hybridoma cells for about 7 days. Purification by the caprylic-ammonium sulfate method followed by affinity chromatography with Protein G was carried out at-20 ℃.

The application of the hybridoma cell strain is to use the monoclonal antibody secreted by the hybridoma cell strain in an immunodetection product.

As a preferred scheme, the monoclonal antibody is prepared into an enzyme linked immunosorbent assay kit for detecting avilamycin residues.

Preferably, the monoclonal antibody is prepared into a colloidal gold immunochromatographic test paper card for detecting the residual avilamycin.

Compared with the prior art, the invention has the advantages that: (1) the preparation method of the avilamycin A hybridoma cell strain is simple and convenient. (2) The hybridoma cell strain of the avilamycin A obtained by the invention can secrete a monoclonal antibody with high specificity. (3) The monoclonal antibody secreted by the hybridoma cell can be effectively used for detecting the avilamycin, and the monoclonal antibody can be used for preparing an ELISA kit for detecting the avilamycin, a colloidal gold immunochromatographic test paper, a sensor and the like. The detection of the avilamycin is of great significance for import and export inspection and quarantine departments and food health departments.

Drawings

FIG. 1 is a full wavelength scan of an artificial antigen of the A component of avilamycin;

FIG. 2 is an electrophoretogram of Protein G purified antibody;

FIG. 3 is a graph showing the standard inhibition curves of the monoclonal antibodies of the A component of avilamycin.

Detailed Description

The following examples are provided to facilitate a better understanding of the objects, solutions and advantages of the present invention, and are set forth further in the accompanying drawings and are not intended to limit the invention.

The main experimental materials used in the present invention are as follows: myeloma cells SP2/0 were obtained from China center for type culture Collection (university of Wuhan), and 6-8 week-old BALB/c female mice were purchased from Shanghai Spiker laboratory animals, Inc. Fetal bovine serum was purchased from Vickers Biotechnology Inc., cell fusion was with PEG 1450, 50 XHAT stock, 50 HT stock, BSA, OVA were purchased from sigma, RPMI-1640 medium was purchased from Gibco, USA. QuickAntibody^TMImmunoadjuvant (beijing boolong immuno-technology ltd) PBS buffer was purchased from bio-engineering (shanghai) ltd.

Example 1: preparation of avilamycin artificial antigen and hybridoma cell strain

1. Preparation of Avermectin A-hemisuccinic acid (AVI-A-HS): 200mg of avilamycin A is weighed and added with succinic anhydride according to the mass ratio of 5:1, 10:1, 20:1 and 40:1 respectively, and the mixture is divided into 4 groups respectively. Firstly, dissolving avilamycin A in 2mL of pyridine, then adding succinic anhydride in proportion, and stirring for reacting for 48 hours; after the reaction is finished, the temperature is reduced to 4 ℃, and then 10% of glacial hydrochloric acid is slowly dripped into the reaction solution until the precipitate is completely separated out, and the reaction solution is dried. Monitored and detected by thin layer TLC (developing solvent chloroform: isopropanol = 94: 6).

2. Preparation of immunogen Avermemycin A-BSA: weighing 50mg of the hapten, dissolving the hapten in 1ml of dimethylformamide, adding dicyclohexylcarbodiimide and N-hydroxysuccinimide according to the mass ratio of 5:2:4, and stirring for reacting for 8 hours in a dark place to obtain solution A. 15mg/mL Bovine Serum Albumin (BSA) solution was used as solution B, and solution A was slowly added dropwise to 5mL of solution B under stirring, and dialyzed overnight at 4 ℃ for 48 hours.

3. Preparation of coating antigen avilamycin A-OVA: the above BSA may be replaced by OVA.

4. Animal immunization: BALB/c mice 6-8 weeks old are selected for immunization. Taking immunogen avilamycin A-BSA (1 mg/mL) and equal amount of QuickAntibody^TMAfter the immune adjuvants are mixed evenly, 100 mu L of the mixture is injected into the hind leg muscle of each mouse. 21dThen, a second immunization is carried out, and the immunization dose is the immunogen avilamycin A-BSA (1 mg/mL) and the equivalent QuickAntibody^TMThe immunoadjuvants were mixed well and injected into the hind leg muscle of each mouse at 50. mu.L.

5. Determination of serum titer and inhibition rate: 14d after the second immunization, blood is taken by adopting an eyeball blood taking method, the serum titer is measured by using indirect ELISA, the immunosuppressive property of the serum to the avilamycin A is measured by using indirect competition ELISA, the mouse titer can reach 128000 when the mouse titer is measured, but the serum inhibition rates are different, one group of mice select AVI-A and succinic anhydride with the ratio of 5:1, the serum inhibition rate is 39.12-51.53%, the other group of mice select AVI-A and succinic anhydride with the ratio of 10:1, the serum inhibition rate is 50.76-71.64%, the other group of mice select AVI-A and succinic anhydride with the ratio of 20:1, the serum inhibition rate is 42.76-58.27%, the other group of mice select AVI-A and succinic anhydride with the ratio of 40:1, and the serum inhibition rate is 27.76-40.87%. The mice with the best immunosuppression (namely AVI-A and succinic anhydride with the ratio of 10: 1) are selected for boosting immunization, namely, the immunogen avilamycin A-BSA is directly injected into the abdominal cavity, and the spleen of the mice is taken for cell fusion after 3 d.

6. Cell fusion

(1) Collection of myeloma cells: SP2/0 cells were collected, resuspended in culture after centrifugation, and cell counted.

(2) Preparation of splenocytes: the spleens of the mice were removed aseptically, ground and centrifuged through a 200 mesh cell screen to obtain a spleen cell suspension, which was then resuspended in RPMI-1640 basal medium and subjected to cell counting.

(3) Cell fusion: spleen cells were mixed with SP2/0 cells at a ratio of 8:1, centrifuged to remove supernatant, and 1mL of 50% PEG was added over 1 min and gently shaken. Then adding 10 mL of RPMI-1640 basic culture solution from slow to fast, centrifuging to remove PEG, suspending in HAT culture medium, spreading on 96-well cell culture plate, placing at 37 deg.C and 5% CO₂Culturing in an incubator.

7. Screening hybridoma cell strains: supplementing liquid to the cells after three days of cell fusion, half replacing liquid after five days, and observing whether new cell clusters are generated or not; taking cell culture medium supernatant after seven days for detection, detecting the positive and immunosuppressive property of the cell culture medium by using an indirect ELISA method and an indirect competitive ELISA method during detection, selecting a cell strain with high positive value and good immunosuppressive property for subcloning, and obtaining a monoclonal cell strain after three times of subcloning.

Example 2: preparation and identification of avilamycin A resisting monoclonal antibody

1. Preparing and purifying ascites: injecting 0.5 mL of sterile paraffin oil into the abdominal cavity of BALB/c mice, injecting 7 x 10 into the abdominal cavity after one week⁵Ascites were collected at 0.5 mL/mL hybridoma cells for about 7 days. Purification by the caprylic-ammonium sulfate method followed by affinity chromatography with Protein G was carried out at-20 ℃.

2. And (3) antibody subtype identification: identifying the antibody obtained in the step 1 by using a monoclonal antibody subtype detection kit, diluting the coating antigen into 7 mu g/mL by using a carbonate buffer solution with the concentration of 0.01mol/L and the pH value of 9.6, coating an enzyme label plate, and standing overnight at 4 ℃; discard the coating solution, wash the plate with PBST for 5 times, 30S each time. Adding CBS blocking solution containing 5% skimmed milk powder 0.1mL per well, incubating at 37 deg.C for 1h, discarding the blocking solution, and washing the plate with PBST for 5 times. Purified antibody was purified using PBST with 0.1% gelatin at 1: after 1000 dilution, 0.1 mL/well of the buffer solution was added to the microplate, and the plate was washed after incubation at 37 ℃ for 1 h. And then, adding various anti-antibodies (IgG 1, IgG2a, IgG2b, IgG3, IgM and IgA) marked by horseradish peroxidase in sequence, incubating at 37 ℃ for 1h, washing the plate, adding a freshly prepared TMB color development solution, and reacting at 37 ℃ in a dark place for 20min, wherein each well is 0.1 mL. And finally, adding 2mol/L sulfuric acid into the solution in an amount of 0.05 mL/hole to terminate the reaction, wherein the subtype of the hole corresponding to the reaction solution changed into yellow is the subtype of the monoclonal antibody. The results of the reaction solution showed that the subtype of the antibody secreted by the monoclonal cell line 2GC8 was IgG1, kappa type.

3. Determination of antibody affinity constant

(1) After the coated antigen Er-OVA is diluted in a gradient way (the concentration is 1, 2, 5 and 7 mu g/mL in sequence), the ELISA plate is coated, the concentration is 100 mu L/hole, and the temperature is kept overnight at 4 ℃.

(2) And (3) discarding the coating liquid, washing the board for 5 times by PBST, and covering the board on absorbent paper for drying each time for 30 s. The cells were blocked with CBS containing 5% skimmed milk powder at 200. mu.L/well for 1h at 37 ℃. After blocking was complete, the plates were washed 5 times with PBST, 30S each, and blotted dry with filter paper.

(3) The anti-avilamycin A component monoclonal antibody (diluted by 1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000 times) is added in a dilution of multiple times, 3 of each concentration are used in parallel at 37 ℃ for 1 h.

(4) Discarding the liquid in the hole, adding goat anti-mouse antibody diluted at 1:5000, and acting at 37 deg.C for 45 min.

(5) Discarding liquid in the hole, adding a freshly prepared TMB developing solution, reacting for 15min at 37 ℃ in a dark place for 0.1mL in each hole, and adding 2mol/L sulfuric acid into 0.05mL in each hole to terminate the reaction. The absorbance was read by a microplate reader OD 450nm, and the affinity constant (Kaff) was calculated according to the following formula:

Ka=（n-1）/2（n*Ab1－Ab2）,n=Ag2／Ag1

wherein Ag1 and Ag2 are different coating antigen mass concentrations (mu g/mL); ab1 and Ab2 indicate the antibody concentrations corresponding to the envelope antigens.

Results of affinity assay: the measurement result showed that the monoclonal cell line 2GC8 secreted antibody with an affinity constant of 4.01X 10¹⁰L/M, a high affinity antibody.

Example 3: determination of IC50 value of avilamycin A by hybridoma cell strain monoclonal antibody

1.1 mu g/mL avilamycin-A-OVA is taken as a coating source to coat a 96-well enzyme label plate, each well is 100 mu L, after overnight at 4 ℃, PBST is used for washing the plate for 5 times, each time for 30S, and filter paper is used for drying.

2. Blocking with CBS containing 5% skimmed milk powder, 200 μ L per well, blocking at 37 deg.C for 1h, washing the plate with PBST for 5 times, 30S each time, and drying with filter paper.

3. Preparing 0.00246, 0.00449, 00898, 0.01797, 0.03594, 0.07187, 0.14375, 0.2875, 0.575 and 1.15 mu g/mL of avilamycin standard solution, adding 50 mu L/well into a closed enzyme label plate, wherein each sample is 3 in parallel, and then adding 50 mu L of 1: the monoclonal antibody diluted by 4000 is used as a positive control, and the reaction is carried out for 1h at 37 ℃, and then the plate is washed and dried.

4. Add 1: after 5000-diluted HRP-labeled goat anti-mouse IgG was reacted at 37 ℃ for 1 hour, the plate was washed and patted dry.

5. Adding 100 μ L TMB color developing solution into each well, developing at 37 deg.C for 15min, adding 50 μ L2M concentrated sulfuric acid into each well to terminate reaction, and measuring light absorption value at 450 nm.

6. According to the difference of OD values of different standard substance concentrations, a curve of OD value and standard substance concentration can be established, and the IC50 value is 7.44 ng/mL.

Example 4: sample precision and accuracy testing

The additive recovery tests were carried out on pig feed and chicken breast at 6 concentrations of 0.1, 0.5, 1, 5, 10, 20 mug/kg, respectively, 3 times in parallel, and the recovery rate and precision of avilamycin in the samples were calculated.

Calculating the formula: recovery (%) = actual measurement value/theoretical value × 100%, relative deviation (%) = standard deviation/average value of measurement data.

Adding a standard and recovering a result: the average recovery rate of the added pig feed sample is 85.88-109.35%, and the relative deviation is less than 9%. The average recovery rate of the chicken breast meat sample addition recovery is 74.66-116.41%, and the relative deviation is less than 10%.

Example 5: indirect competitive ELISA method for detecting content of avilamycin in pig feed

The method comprises the following specific steps:

1. coating: a96-well enzyme label plate is coated with coating antigen of avilamycin A-OVA with different concentrations (0.2, 0.4, 0.8, 1.6 and 3.2 mu g/mL), each well is 100 mu L, and the temperature is 4 ℃ overnight. The coating solution was discarded, the plates were washed 5 times with PBST for 30S each, and the filter paper was patted dry.

2. And (3) sealing: blocking with Carbonate Buffer (CBS) containing 1% BSA at 100. mu.L per well for 1h at 37 ℃; the plates were then washed 5 times in PBST, 30S each time, and the filters were blotted dry.

3. Drawing a standard curve: first, 0, 0.005, 0.01, 0.025, 0.05, 0.08, 0.1, 0.2, 0.4, 0.8. mu.g/mL of standard avilamycin A solution was prepared using PBS buffer. Standard solutions of avilamycin at different concentrations were then added to the well-blocked microplate in an amount of 50 μ L per well, repeating three wells per concentration. Then 50. mu.L of monoclonal antibody diluted 1:1000 was added to each experimental well of each column, reacted at 37 ℃ for 1 hour, washed and patted dry. mu.L of HRP-labeled goat anti-mouse IgG diluted 1:3000 was added to each experimental well again, reacted at 37 ℃ for 1h, washed and patted dry. Then, 100. mu.L of TMB developing solution was added to each experimental well, and the mixture was developed at 37 ℃ for 20 min. Finally, 50. mu.L of 2M concentrated sulfuric acid was added to each experimental well to terminate the reaction, and the absorbance was measured at 450 nm. And establishing a standard curve Y = aX + b of the OD value (X) and the standard substance concentration (Y) according to the OD value of different standard substance concentrations.

4. Sample treatment: weighing 10g of pig feed of different samples into a centrifuge tube, adding 20 mL of methanol, extracting for 2h at 37 ℃, centrifuging and collecting supernatant 1; then adding 20 mL of methanol into the precipitate, extracting for 1h at 37 ℃, centrifuging and collecting a supernatant 2; finally, combining the supernatant 1 and the supernatant 2, blowing the mixture by a nitrogen blowing instrument, adding methanol to a constant volume of 5ml for later use

5. Determination of avilamycin in samples: from the established calibration curve and the OD value of the sample solution, the content of avilamycin in each sample (μ g/kg) = (aX + b) × n × 2/(m × 1000), n is the dilution factor, and m is the sample mass.

Example 6: indirect competitive ELISA method for detecting avilamycin residue in chicken

1. Coating: a96-well enzyme label plate is coated by using 1 mu g/mL Er-A-OVA as a coating source, each well is 100 mu L, and the temperature is kept overnight at 4 ℃. The plates were then washed 5 times in PBST, 30S each time, and the filters were blotted dry.

2. And (3) sealing: blocking with CBS containing 5% skimmed milk powder, 200. mu.L per well, blocking at 37 ℃ for 1 h. After blocking was complete, the plates were washed 5 times with PBST, 30S each, and blotted dry with filter paper.

3. Sample treatment: feeding chicken breast samples for 1 week with feed containing avilamycin, taking 5 samples, taking 30 g of chicken breast, and uniformly stirring by a homogenizer. Weighing 5g of different homogeneous samples in different centrifuge tubes, adding 10 mL of methanol, extracting at 37 ℃ for 2h, centrifuging and collecting supernatant 1; then adding 10 mL of methanol into the precipitate, extracting for 1h at 37 ℃, centrifuging and collecting a supernatant 2; and finally, combining the

supernatants

1 and 2, drying by a nitrogen blowing instrument, and adding methanol to a constant volume of 2ml for later use.

4. Drawing a standard curve: 0, 0.002, 0.004, 0.008, 0.016, 0.032, 0.064, 0.128, 0.256 and 0.512 mu g/mL of avilamycin standard solution are prepared by PBS buffer solution respectively, 50 mu L/hole are added into a closed enzyme label plate, and 3 samples are paralleled. Then 50. mu.L of monoclonal antibody diluted 1:1000 was added to each experimental well, reacted at 37 ℃ for 1 hour, washed and dried. mu.L of HRP-labeled goat anti-mouse IgG diluted at 1:4000 was added to each experimental well again, reacted at 37 ℃ for 60min, and then washed and patted dry. Then, 100. mu.L of TMB developing solution was added to each experimental well, and the mixture was developed at 37 ℃ for 15 min. Finally, 50. mu.L of 2M concentrated sulfuric acid was added to each experimental well to terminate the reaction, and the absorbance at 450nm was measured. And establishing a standard curve Y = aX + b of the OD value (X) and the standard substance concentration (Y) according to the OD value of different standard substance concentrations.

In conclusion, the avilamycin A component monoclonal antibody obtained by the invention has the advantages of strong specificity and high sensitivity, so that the avilamycin A component monoclonal antibody can be used for detecting avilamycin residues, including but not limited to products detected by a colloidal gold test strip method, an enzyme-linked immunosorbent assay, a chemiluminescence assay and the like. The above description is only a part of the embodiments, and is not intended to limit the scope of the present invention. All changes and modifications that come within the spirit of the invention are to be understood as being within the scope of the invention.

Claims

1. A hybridoma cell strain 2GC8 of an avilamycin A resistant monoclonal antibody, the preservation number is CGMCC NO. 17485.

2. The hybridoma cell line 2GC8 or a passaged cell line thereof according to claim 1, secreting and producing an anti-avilamycin A monoclonal antibody.

3. The method for producing a monoclonal antibody according to claim 2, wherein the monoclonal antibody is obtained by ascites induction or cell culture of hybridoma cell line 2GC8, followed by ammonium caprylate-sulfate precipitation and Protein G purification.

4. Use of the anti-avilamycin a monoclonal antibody according to claim 2 in the preparation of an immunoassay product.

5. The use of claim 4, wherein the immunoassay comprises, but is not limited to, a test paper card, a kit, or a biochip.