HYBRIDOMA CELL STRAIN THAT SECRETS ANTI-DINITOLMIDE MONOCLONAL ANTIBODIES AND THE APPLICATION OF HYBRIDOMA CELL STRAIN

Abstract

A hybridoma cell strain secreting anti-dinitolmide monoclonal antibodies deposited at China General Microbiological Culture Collection Center (CGMCC) at No. 1 West Beichen Road, Chaoyang District, Beijing 100101, China on Nov. 28, 2019, with deposit number of CGMCC No. 19165. It is classified as a monoclonal cell strain. The monoclonal antibody secreted by the hybridoma cell strain DAS3H10 has a good affinity and high sensitivity to dinitolmide. Because of IC50 to dinitolmide up to 9.01 ng/mL, the monoclonal antibody could be used to prepare dinitolmide immunoassay kit and colloidal gold test strip and provides a powerful detection method and means for the detection of dinitolmide in animal-derived foods.

Description

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to a hybridoma cell strain that secretes anti-dinitolmide monoclonal antibodies and its application, and belongs to the field of food safety immunoassay.

2. Background Art

Dinitolmide (DTM) is a poultry feed additive that is widely used as a nitroamide anticoccidial drug because of its effective treatment and prevention of coccidiosis. Stability, low toxicity, low resistance and low cost are also the reasons for its widespread use in animal feed. However, the use of excessive and long-acting drugs may lead to residues of veterinary drugs in poultry tissue, and lead to harm to people's health. In addition, the overuse also results in the excretion of veterinary drugs ingested by poultry and animals in the form of metabolites or prototype drugs, which is harmful to the environment. As a result, the Ministry of Agriculture of China and the U.S. Food and Drug Administration have established strict regulations on the maximum residual limits of dinitolmide in animal tissues: no more than 2,000 μg·kg⁻¹ in fat, no more than 6,000 μg·kg⁻¹ in liver and kidneys, and no more than 3,000 μg·kg⁻¹ in muscles.

The conventional detection methods of dinitolmide contain high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS), ultra performance liquid chromatography (UPLC), ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), gas chromatography tandem mass spectrometry (GC-MS/MS) and so on. However, there are many drawbacks of these methods in different degrees such as time-consuming, expensive instruments, and a mass of sample pretreatment procedures. Therefore, these methods above are not suitable for on-site detection of high-throughput analysis of dinitrotolamine. What's more, the content of dinitolmide has been reported to be measured by surface-enhanced Raman spectroscopy. However, nanotechnology is highly sensitive and reliable, and also highly dependent on accurate data acquisition, so an analysis system is required, which means that the application of these methods in on-site detection is limited. Therefore, it is necessary to further develop a rapid detection method to meet the requirements of government supervision as well as self-inspection of enterprises.

The immunoassay is characterized by low cost, high throughput, high sensitivity and low relative requirements for technicians, so it is suitable for rapid screening of large numbers of samples. The purpose of the present invention is to provide a preparation method for monoclonal antibody hybridoma cell lines with high affinity and detection sensitivity to dinitolmide. The present invention lays the foundation for the for the development and promotion of indirect competition ELISA kits and colloidal gold test strips.

SUMMARY OF THE INVENTION

Based on the technical problems of the background technology, the purpose of the invention is to provide a hybridoma cell strain that secretes anti-dinitolmide monoclonal antibodies and its application. The monoclonal antibody secreted by the cell strain has better affinity and sensitivity to dinitolmide, which can be used to establish an enzyme-linked immunosorbent assay (ELISA) for dinitolmide, or to establish a rapid detection method of colloidal gold immunodialysis test strips.

The technical scheme of the present invention is to provide a hybridoma cell strain DAS3H10 that secretes anti-dinitolmide monoclonal antibodies, which has been deposited in Comprehensive Microbiology Center of China Microbial Culture Collection Management Committee (CGMCC), addressed in No. 1 Hospital No. 3 Institute of Microbiology of the Chinese Academy of Sciences, North Chenxi Road, Beijing Chaoyang District in Beijing. It is classified as a monoclonal cell strain. The deposit date is 28, Nov., 2019, and its deposit number MCCC No. 19165.

The anti-dinitramine monoclonal antibody is secreted by the hybridoma cell strain DAS3H10 with the deposit number CGMCC No. 19165.

The steps of producing complete immunogen of dinitolmide are as follows: 4.5 mg of 3,5-dinitro-2-methylbenzoic acid, as well as 5.0 mg EDC and 3.7 mg NHS are dissolved in DMF with stirring and activating at room temperature for 6 h to obtain the activating solution. Another 15 mg BSA was dissolved in 3 mL, 0.05M, pH 9.6 carbonate buffer solution. The activating solution was added dropwise to the BSA solution, stirred at room temperature overnight, and then the immunogen was taken out and dialyzed with PBS for 3 days. At last, the immunogen was aliquoted storage in −20° C., which is the very complete immunogen of dinitolmide.

The application of anti-dinitolmide monoclonal antibodies is used for the detection of dinitolmide residues in food.

The basic steps for the preparation of the DAS3H10 cell line provided by the invention are:

• (1) Preparation and identification of immunogen: 3,5-dinitro-2-methylbenzoic acid, which is a structure analogue of dinitolmide, was used as a raw material and connected to the amino group of the protein carrier with the activated ester method. After the reaction was completed, the complete immunogen and the unconnected small molecule hapten were separated by dialysis. And the complete immunogen was identified by ultraviolet absorption scanning.
• (2) Immunization of mice: BALB/c mice aged 6-8 weeks were selected for immunization. The immunogen, which was emulsified with Fuchs adjuvant, was injected to the BALB/c mice by subcutaneous injection at multiple points. The Faust complete adjuvant was used for the first immunization, the Faust incomplete adjuvant was used for the booster immunization, and the commixture of the immunogen and normal saline was used for the sprint immunization by intraperitoneal injection. Each immunization dose was half of the previous immunization dose, and the interval between each immunization was three weeks. After the third immunization, blood collection was collected every one week for the detection of serum titer and inhibition.
• (3) Cell fusion and cell line establishment: the cell fusion of mouse spleen cells and mouse myeloma cells was accomplished by polyethylene glycol (PEG 2000). After cultured in HAT medium, there was detection of positive cells hole by indirect ELISA, and further detection of the inhibition effect of positive cell holes by indirect competition ELISA. Positive cell holes with the best inhibition effect were subcloned three times by limiting dilution assay, and finally the hybridoma cell strain DAS3H10 was finally screened.
• (4) Identification of the properties of hybridoma cell strains: the identification of hybridoma cell strains were determined by HRP, and IC50, cross-reaction rates and affinity were determined by ELISA.

The beneficial effect of the present invention: (1) The anti-dinitolmide monoclonal antibody obtained by the present invention has better detection sensitivity and affinity for dinitolmide, and (2) it is a new method of synthesis of dinitolmide immunogen with more simplified and effective synthesis steps, which provides the idea and method for the synthesis of immunogen for future research.

BIOLOGICAL PRESERVATION INSTRUCTIONS

The hybridoma cell strain that secretes anti-dinitolmide monoclonal antibodies, DAS3H10, has been deposited in Microbiology Center of China Microbial Culture Collection Management Committee CGMCC in Beijing, China, on Nov. 28, 2019, addressed in No. 1 Hospital No. 3 Institute of Microbiology of the Chinese Academy of Sciences, North Chenxi Road, Chaoyang District in Beijing. It is classified as a monoclonal cell strain. And its deposit number MCCC No. 19165.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the chemical structure of hapten and complete immunogens; a, hapten; b, complete immunogen.

FIG. 2 is the UV absorption spectrum characterization of the immunogens.

FIG. 3 is the standard inhibition curve of dinitolmide monoclonal antibodies.

DESCRIPTION OF PREFERRED EMBODIMENTS

The detailed implementation of the invention is further described as follows. The following embodiments are used to illustrate the invention, but not to limit the scope of the invention. Except for special instructions, the experimental methods used in the following embodiments are all conventional methods. Except for special instructions, the materials and reagents etc. used in the inventation can be obtained from commercial sources.

The invention obtains a monoclonal antibody hybridoma cell strain with better affinity and sensitivity to dinitolmide by immunization of mice with complete immunogen of dinitolmide, cell fusion, growth in HAT medium, and screening of the hybridoma cell culture supernatant by indirect ELISA and indirect competition ELISA.

Implementation 1. Preparation of DAS3H10, a Monoclonal Antibody Hybridoma Cell Strain

1. The synthesis of complete immunogens: 3,5-dinitro-2-methylbenzoic acid, which is a structure analogue of dinitolmide, was used as a raw material and connected to the amino group of the protein carrier with the activated ester method. After the reaction was completed, the complete immunogen and the unconnected small molecule hapten were separated by dialysis. And the complete immunogen was identified by ultraviolet absorption scanning.

The structure of hapten is shown in FIG. 1a, the structure of the complete immunogen is shown in FIG. 1b, and the UV absorption spectroscopic characterization is shown in FIG. 2.

4.5 mg of 3,5-dinitro-2-methylbenzoic acid, 5.0 mg EDC (1-(3-dimethylamphetamine)-3-ethyl carbon diamide hydrochloride) and 3.7 mg NHS (N-hydroxyamide) were dissolved in DMF (N,N-dimethyl methamphetamine) with stirring and activating at room temperature for 6 h to obtain the activating solution. Another 15 mg BSA (bovine serum protein) was dissolved in 3 mL, 0.05M, pH 9.6 CB (carbonate buffer solution). The activating solution was added dropwise to the BSA solution, stirred at room temperature overnight, and then the immunogen was taken out and dialyzed with PBS for 3 days. At last, the immunogen was aliquoted storage in −20° C., which is the very complete immunogen of dinitolmide.

2. Immunization of mice: BALB/c mice aged 6-8 weeks were selected for immunization. The immunogen (1 mg/mL), which was emulsified with Fuchs adjuvant, was injected to the BALB/c mice by subcutaneous injection at multiple points, with 100 μL for every mice. The Faust complete adjuvant was used for the first immunization, the Faust incomplete adjuvant was used for the booster immunization, and the commixture of the immunogen and normal saline was used for the sprint immunization by intraperitoneal injection. Each immunization dose was half of the previous immunization dose, and the interval between each immunization was three weeks. After the third immunization, blood collection was collected every one week for the detection of serum titer and inhibition. The mice with the best inhibition effect were selected for sprint immunity 18 days after the fifth immunization, for the ready of fusion.

3. Cell fusion: three days after the sprint immunity, the cell fusion was accomplished in accordance with the conventional PEG ASSAYs (polyethyl glycol, molecular weight of 2000), the specific steps are as follows:

• (1) The spleen of mice was obtained in aseptic conditions, grinded and then screened through cell strainers with the screen mesh number of 200 mesh, to get the spleen cell suspension which was used for cell counting;
• (2) SP2/0 cells were collected and suspended in RPMI-1640 basic medium for cell counting;
• (3) Spleen cells and SP2/0 cells were mixed in accordance with the count ratio of 2˜10:1. After centrifugation, the cell fusion was carried on with PEG for 1 min. RPMI-1640 basic medium was added from slow to fast. The mixture was centrifuged and the centrifugal suspension was suspended in the RPMI-1640 screening medium containing 20% fetal bovine serum and 2% 50×HAT which was then added to a 96-well cell culture plate and cultured in a incubator with 5% CO₂ on 37° C.

4. Cell screening and cell line establishment: during the cell fusion, half of the RPMI-1640 screening medium of the fusion cell was changed on the 3rd day, and all was replaced by the RPMI-1640 screening medium with 20% fetal bovine serum, 1% of 100×HT on the fifth day. The cell supernatant was used for screening on the 7th day, which is divided into two steps: the first step is to screen out the positive cell holes with indirect ELISA, the second step is to detect the inhibition effect of positive cell holes by indirect competition ELISA with dinitolmide used as the standard substance. The cell holes with good inhibition effect of dinitrotolamine were selected to be subcloned with finite dilution assay, and the inhibition effect was detected in the same way. Repeat three times to obtain the cell line DAS3H10.

5. Preparation and identification of monoclonal antibody: 1 mL paraffin oil is injected into the enterocoelia of every BALB/c mice aged 8˜10 weeks, and the hybridoma cell was injected into the enterocoelia after 7 days. Hydroperitoneum was collected and purified with caprylic acid-saturated ammonium sulfate to obtain McAb (monoclonal antibody), which was storaged in −20° C.

The McAb obtained from the purified hydroperitoneum was identified by McAb subtype identification kit to be IgG2a, as shown in Table 1.

TABLE 1
Subtype identification of dinitolmide monoclonal antibodies
Subtype identification OD
IgA                    0.104
IgG1                   0.303
IgG2a                  2.233
IgG2b                  0.221
IgG3                   0.112
IgM                    0.023

The IC₅₀ of the monoclonal antibodies against dinitolmide was determined to be 9.01 ng/mL in the way of indirect competitive ELISA, which also verified the IC₅₀ and the cross-reaction rates of the monoclonal antibodies against dikjuli et al., as shown in Table 2.

TABLE 2
IC50 and cross-reaction rates of dinitolmide monoclonal antibodies
to dinitolmide, dikjuli, torquoly, nicarbazine
	IC50 (ng/mL)	cross-reaction rates
dinitolmide	9.01	100%
dikjuli	>500	<5%
torquoly	>500	<5%
nicarbazine	>500	<5%

6. Antibody application: The McAb obtained from the purified hydroperitoneum of DAS3H10 hybridoma cell strain was used in additive reclamation text of dinitolmide ELISA. The specific steps are as follows:

• (1) 96-ELISA Plate was coat by ENR-OVA composed of 0.1 μg/mL dinitolmide diluted with carbonate buffer (CBS) with 100 μL per hole at 37° C. for 2 h. The Plate was washed with 200 μL PBST per hole each time for 3 min for three times and dried then.
• (2) The plate was closed with 0.2% gelatin CBS, 200 sL per hole at 37° C. for 2 h. The plate was washed with 200 μL PBST per hole each time for 3 min for three times and dried then.
• (3) The standard solution of dinitolmide with phosphate buffer (PBS) is configured with 0, 0.02, 0.05, 0.1, 0.2, 0.5, 1,2 μg/L, respectively. The standard solution and the sample extract was respectively added to the closed ELISA plate, 50 μL per hole. Each sample was added to 3 holes. And then 50 μL anti-dinitolmide monoclonal antibody diluted by 1:16000 was added. After the reaction for 0.5 h at 37° C., The Plate was then washed and dried;
• (4) 100 μL sheep anti-rat IgG abcam which is diluted with PBS with 0.1% gelatin by 1:3000 and marked with HRP was added each hole for 0.5 h at 37° C., the plate was then washed and dried;
• (5) 100 μL TMB (color solution) was added into every hole for color rendering for 15 min at 37° C., and then 50 μL 2M H₂SO₄ was added as termination fluid, and UV absorption was detected. The standard inhibition curve of dinitolmide monoclonal antibody was shown in FIG. 3.
• (6) Adding recovery and sample pre-treatment: three different doses of dinitolmide standard substance, 50 ng, 100 ng, 200 ng, was respectively added into 5 g fresh or warm (refrigerated) milk. The mixture was placed in a 50 mL centrifuge tube with 1 mL 50% potassium hydroxide solution dropped in slowly. Keep on oscillating fully on the vortex mixer for 10 min, with 20 mL ethyl acetate dropped in slowly. After the centrifuge at 3000 r/min for 5 min, 4 mL of supernate was transferred to another centrifuge tube, which was then blowed by dry nitrogen and added in 1 mL PBS with 10% methanol. 50 μL sample was transferred for testing. The recovery rates were 91.2%, 101.5% and 95.6%, respectively, resulted from adding recovery tests with indirect competition ELISA.

Configuration of the Solution:

Carbonate buffer (CBS): Na₂CO₃ 1.59 g and NaHCO₃ 2.93 g respectively dissolved in a small amount of ultrapure water was mixed, and then filled up to about 800 mL with ultrapure water. The pH was adjusted to 9.6. CBS was filled to 1000 mL by adding ultrapure water and stored at 4° C. for later use;

Phosphate buffer (PBS): 8.00 g NaCl, 0.2 g KCl, 0.24 g KH₂PO₄, 3.62 g Na2HPO₄12 H₂O were dissolved in 800 mL pure water with pH adjusted to 7.2 to 7.4 by NaOH or HCl, and filled to 1000 mL;

PBST: PBS with 0.05% Tween 20;

TMB: A liquid: Na₂HPO₄12H₂O 18.43 g, citric acid 9.33 g, dissolved in pure water and filled to 1000 mL; B liquid: 60 mg TMB dissolved in 100 mL ethylene glycol. TMB is the mixture of A, B liquid with a volume ratio of 1:5. A, B liquid could be mixed just when needed.

While embodiments of the present invention have been illustrated and described, various modifications and improvements can be made by persons skilled in the art. It is intended that the present invention is not limited to the particular forms as illustrated, and that all the modifications not departing from the spirit and scope of the present invention are Within the scope as defined in the appended claims.

Claims

1. A hybridoma cell strain secreting anti-dinitolmide monoclonal antibodies and deposited at China General Microbiological Culture Collection Center (CGMCC) at No. 1 West Beichen Road, Chaoyang District, Beijing 100101, China on Nov. 28, 2019, with deposit number of CGMCC No. 19165.

2. Anti-dinitolmide monoclonal antibody secreted by the hybridoma cell strain of claim 1.

3. A method of making complete immunogen of dinitolmide comprising the following steps: dissolving 4.5 mg of 3,5-dinitro-2-methylbenzoic acid, as well as 5.0 mg EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and 3.7 mg NHS (N-hydroxyamide) in DMF (N,N-dimethylformamide), stirring and activating at room temperature for 6 hour to obtain an activated solution; dissolving another 15 mg BSA(bovine serum protein) in 3 mL, 0.05M, pH 9.6 carbonate buffer solution; adding the activated solution dropwise to the BSA solution, stirring at room temperature overnight, and then taking out immunogen and dialyzing with PBS for 3 days; and storing the immunogen by aliquoted storage at −20° C. and obtaining the complete immunogen of dinitolmide.

4. A method of detecting dinitolmide residues in food comprising applying the anti-dinitrotoamine monoclonal antibody of claim 2 to food and detecting dinitolmide residues in food based on the interaction of the anti-dinitrotoamine monoclonal antibody with the food.