WO2018054394A1 - Hybridoma cell line nan-1 which secretes sulfonamide antibiotic-resistant monoclonal antibody, and application of hybridoma cell line - Google Patents

Abstract

A hybridoma cell line NaN-1 which secretes a sulfonamide antibiotic-resistant monoclonal antibody, and an application of the hybridoma cell line, belonging to the field of food safety immunoassays. The antibody secreted by the hybridoma cell line NaN-1 is capable of identifying twenty-five sulfonamide antibiotics. The hybridoma cell line has been stored in the China General Microbiological Culture Collection Center (CGMCC), the accession number being CGMCC No. 12028. The monoclonal antibody secreted by the cell line NaN-1 is capable of identifying twenty-five sulfonamide antibiotics and has good detection sensitivity. The IC50 for sulfadiazine (SD), sulfamerazine (SMR), sulfadimethoxine (SDM) and sulfathiazole (STZ) is respectively: 4.51, 1.80, 1.98 and 0.53 μg/L. The present invention can be used for sulfonamide antibiotic multi-residue detection within the field of food safety.

Description

Hybridoma cell line NaN-1 secreting monoclonal antibody against sulfa antibiotics and application thereof Technical field

The present invention relates to a hybridoma cell line NaN-1 secreting a monoclonal antibody against sulfa antibiotics and the use thereof, and relates to a hybridoma cell line NaN-1 and a monoclonal antibody against sulfa antibiotics thereof, belonging to food safety immunology Testing area.

Background technique

Sulfonamide antibiotics (SAs) are synthetic antibacterial drugs that have been used in clinical practice for nearly 50 years. They have the advantages of wide antibacterial spectrum, stable nature and easy to use. Such antibiotics are also a class of antibacterial drugs widely used in aquaculture, aquaculture and other aquaculture production, which are often used for the prevention and treatment of livestock diseases, and as feed additives to promote animal growth. However, due to its potential harm to human body and the emergence of resistance to pathogenic bacteria, its residue in food has caused widespread concern. In recent years, various countries have strengthened the monitoring of SAs and formulated the maximum residue of SAs in animal-derived foods. The Codex Alimentarius Commission, the European Union and some countries in Europe and the United States clearly stipulate that the total content of SAs in food should not exceed 0.1 mg/kg.

At present, the detection methods of SAs mainly include instrumental detection methods (such as high performance liquid chromatography, high performance liquid chromatography tandem mass spectrometry), enzyme-linked immunosorbent assay (ELISA, Enzyme-Linked Immunosorbent Assay) and colloidal gold immunochromatography. Because the sample preparation method of the instrument analysis method is cumbersome, time-consuming, expensive, and requires professional operation, simultaneous detection of a large number of samples cannot be achieved. ELISA is an extremely efficient and sensitive detection method. The purity of the sample is not high and the operation is simple. It is suitable for rapid on-site detection of a large number of samples. However, in order to achieve simultaneous detection of sulfa antibiotics, it is a prerequisite to prepare a monoclonal antibody.

Summary of the invention

The object of the present invention is to provide a group-selective monoclonal antibody hybridoma cell line NaN-1 resistant to sulfa antibiotics, and the accession number is CGMCC No.12028, and the antibody prepared from the cell line can recognize twenty-five kinds of SAs, and It has good detection sensitivity and can be used to establish immunological detection methods for SAs.

The basic steps for preparing the NaN-1 cell strain provided by the present invention are as follows:

1) Synthesis of immunogen: 500 mg of aminothiazoleacetic acid was dissolved in 7.5 mL of anhydrous methanol, and 400 mg of thionyl chloride was added to the solution. After the reaction mixture was refluxed for 2 h, the volatiles were evaporated, evaporated, evaporated, evaporated, evaporated, evaporated. The acid chloride was dissolved in 15 mL of pyridine, and reacted at room temperature for 3 hours. The reaction mixture was concentrated to give Intermediate II. 134 mg of Intermediate II was dissolved in 5 mL of 1M sodium hydroxide solution, heated to 80 ° C, and reacted under nitrogen for 2 h. After the end of the reaction, the volatiles were evaporated, and the residue was crystallised from hydrochloric acid Dry the obtained target liquid and evaporate the organic solvent The reagent is finally concentrated under vacuum to obtain the target hapten.

The hapten is a product having a carboxyl group and is coupled by a carbodiimide method. The specific steps are as follows: 20 mg of hapten is weighed and dissolved in 5 mL of N,N-dimethylformamide, and 15.1 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is added with 12.8. Mg N-hydroxysuccinimide was activated at room temperature for 4 h; 50 mg of bovine serum albumin was weighed and dissolved in 6 mL of carbonate buffer solution, and the activated droplets were added to the protein solution and allowed to react at room temperature overnight. The unconjugated hapten was removed by dialysis against a 0.01 M phosphate buffer solution for 3 days to obtain a complete antigen.

2) Immunization of mice: After complete emulsification of complete antigen and an equal amount of Freund's adjuvant, BALB/c mice were immunized by subcutaneous injection into the back. The first immunization was performed with complete Freund's adjuvant, followed by incomplete Freund's adjuvant. There was a one-month interval between the first immunization and booster immunization, and the interval between booster immunizations was 21 days. The last time was directly immunized with complete antigen (without adjuvant), and serum titer and inhibition were detected by indirect ELISA;

3) Cell fusion and cell line establishment: Mouse spleen cells and mouse myeloma cells were fused by polyethylene glycol (PEG 4000) method, cultured in HAT medium, positive cell wells were detected by indirect ELISA, and further utilized. The inhibitory effect of positive cell wells was determined by indirect competitive ELISA. The positive cells with the best inhibition were subcloned three times by limiting dilution method, and finally the hybridoma cell line NaN-1 was obtained.

The present invention also provides a monoclonal antibody against sulfa antibiotics which is secreted by the anti-sulfa antibiotic monoclonal antibody hybridoma cell line NaN-1 of the accession number CGMCC No. 12028. The anti-SAs monoclonal antibody has good detection sensitivity to twenty-five sulfa antibiotics, among which, sulfadiazine (SD), Sulfamerazine (SMR), sulfamethoxine (Sulfamerazine, SMR) The IC _{50 of} Sulfadimidine, SDM) and Sulfathizzole (STZ) were 4.51, 1.80, 1.98 and 0.53 μg/L, respectively.

The anti-sulfonamide antibiotic monoclonal antibody can be used in the field of food safety to detect the content of sulfa antibiotics in food.

The invention provides an anti-sulfa antibiotic monoclonal antibody hybridoma cell line NaN-1, and the monoclonal antibody secreted by the cell line can recognize twenty-five sulfa antibiotics, and has good detection sensitivity, wherein The IC _{50 of} sulfadiazine, sulfamethazine, sulfadimethoxine and sulfathiazole are: 4.51, 1.80, 1.98 and 0.53 μg/L, respectively, which can be used for multi-residue detection of sulfonamides in food safety.

Biomaterial preservation

The monoclonal antibody hybridoma cell line NaN-1 has been deposited with the General Microbiology Center of the China Microbial Culture Collection Management Committee on January 20, 2016, referred to as CGMCC. Address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, China. The Institute of Microbiology, Chinese Academy of Sciences, is classified as a monoclonal cell strain with the accession number CGMCC No.12028.

DRAWINGS

Figure 1: Standard curve for inhibition of sulfadiazine (SD) by monoclonal antibodies secreted by NaN-1.

detailed description

The invention screens cell supernatant by cell fusion, HAT selective medium culture, indirect ELISA and indirect competition ELISA by immunizing mice with complete antigen of SAs, and finally obtains wide recognition and good sensitivity to SAs. Monoclonal antibody hybridoma cell line NaN-1.

Example 1: Preparation of hybridoma cell line NaN-1

Solution configuration:

Carbonate buffer (CBS): Weigh 1.59g of Na ₂ CO _{3 and} 2.93g of NaHCO ₃ , dissolve in a small amount of double distilled water, mix with double distilled water to about 800mL, adjust the pH to 9.6, add Double distilled water to a volume of 1000mL, stored at 4 ° C for use;

Phosphate buffer (PBS): 8.00 g NaCl, 0.2 g KCl, 0.2 g KH ₂ PO ₄ , 2.9 g Na ₂ HPO ₄ · 12H ₂ O, dissolved in 800 mL of pure water, adjusted to pH 7.2 with NaOH or HCl 7.4, constant volume to 1000mL;

PBST: PBS containing 0.05% Tween 20;

TMB color developing solution: liquid A: Na ₂ HPO ₄ · 12H ₂ O 18.43 g, citric acid 9.33 g, pure water to 100 mL; B liquid: 60 mg TMB dissolved in 100 mL of ethylene glycol. A and B liquids are mixed with a volume ratio of 1..5 to be a TMB coloring liquid, which is currently used.

1. Synthesis of antigen

500 mg of aminothiazoleacetic acid was dissolved in 7.5 mL of anhydrous methanol, and 400 mg of thionyl chloride was added to the solution. After the reaction mixture was refluxed for 2 h, the volatiles were evaporated, evaporated, evaporated, evaporated, evaporated, evaporated. The acid chloride was dissolved in 15 mL of pyridine, and reacted at room temperature for 3 hours. The reaction mixture was concentrated to give Intermediate II. 134 mg of Intermediate II was dissolved in 5 mL of 1M sodium hydroxide solution, heated to 80 ° C, and reacted under nitrogen for 2 h. After the end of the reaction, the volatiles were evaporated, and the residue was crystallised from hydrochloric acid The obtained target liquid was dried and evaporated to an organic solvent, and finally concentrated under vacuum to obtain a target hapten.

The hapten is a product having a carboxyl group and is coupled by a carbodiimide method. The specific steps are as follows: 20 mg of hapten is weighed and dissolved in 5 mL of N,N-dimethylformamide, and 15.1 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is added with 12.8. Mg N-hydroxysuccinimide was activated at room temperature for 4 h, and 50 mg of bovine serum albumin was weighed and dissolved in 6 mL of carbonate buffer solution, and the activated droplets were added to the protein solution, and reacted at room temperature overnight. The unconjugated hapten was removed by dialysis against a 0.01 M phosphate buffer solution for 3 days to obtain a complete antigen and characterized by UV.

The original synthesis of the coating is similar to the synthesis of the immunogen, and the carrier protein is simply replaced with chicken egg albumin.

2, animal immunity

Healthy 6-8 week old BALB/c mice were selected for immunization. After the complete antigen was mixed and emulsified with an equal amount of Freund's adjuvant, BALB/c mice were immunized by subcutaneous injection into the back. The first immunization was performed with complete Freund's adjuvant, followed by incomplete Freund's adjuvant. There was a one-month interval between the first immunization and booster immunization, and the interval between booster immunizations was 21 days. Blood was collected 7 days after the third immunization, and the serum titer and inhibition of the mice were determined by indirect competitive ELISA. The mice with high titer and more SAs were selected, and the immunization was performed 18 days after the four immunizations without adjuvant. injection.

3, cell fusion

After 3 days of shock immunization, cell fusion was carried out according to the conventional PEG (polyethylene glycol, molecular weight 4000) method. The specific steps are as follows: (1) The mouse spleen was aseptically taken, ground and passed through a 200 mesh cell sieve to obtain spleen cell suspension. Liquid, and perform cell counting; (2) collect SP2/0 cells, suspend in RPMI-1640 basal medium for cell counting; (3) mix spleen cells and SP2/0 cells according to a ratio of 1..10 After centrifugation, the cells were fused with 50% PEG for 1 min, then RPMI-1640 basal medium was added from slow to fast, centrifuged and suspended in RPMI-1640 containing 20% fetal bovine serum and 2% 50×HAT. The solution was applied to a 96-well cell culture plate and cultured in an incubator at 37 ° C in a 5% CO _{2 solution} .

4. Cell selection and cell line establishment

The RPMI-1640 screening medium was semi-exchanged on the third day of cell fusion, and the whole day was changed with RPMI-1640 transition medium containing 20% fetal bovine serum and 1% 100×HT on the 5th day. The cell supernatant was taken for screening on day 7. The screening is divided into two steps: the first step is to select positive cell wells by indirect ELISA, and the second step is to select SD, SMR, SDM and STZ as standard products, and the inhibition effect of positive cells is determined by indirect competition ELISA. Cell wells with good inhibition of these four standards were selected, subcloned by limiting dilution method, and detected by the same method. This was repeated three times to obtain a cell strain NaN-1.

5. Preparation and identification of monoclonal antibodies

8-8 weeks old BALB/c mice were injected with 1 mL of paraffin oil per mouse. After 7 days, each mouse was intraperitoneally injected with 1×10 ⁶ hybridoma cell NaN-1, and ascites was collected from the 7th day. Ascites was purified by caprylic acid-ammonium sulfate method, and the obtained monoclonal antibody was stored at -20 °C.

Using an indirect competition ELISA, the IC ₅₀ of monoclonal antibodies against twenty-five SAs is shown in Table 1:

Table 1 Cross-reaction rate of monoclonal antibodies secreted by NaN-1

Drug name	IC 50 (μg/L)	Cross-reaction rate (%)
Sulfadiazine (SD)	4.51	100.00
Sulfamethazine (SMR)	1.8	250.56
Sulfamethoxine	1.09	413.76
Sulfadimethoxypyrimidine (SDM)	1.98	227.78
Sulfamethoxazole	0.15	3006.67
Sulfamethoxazole	0.21	2147.62

Sulfathiazole (STZ)	0.52	850.94
Sulfaquinoxaline	50.12	9.00
Sulfadimethylpyrimidine	20.32	22.19
Sulfadiazine	20.2	22.33
Sulfachloropyridazine	0.51	884.31
Sulfamethoxazine	1.12	402.68
Sulfadoxine	50.3	8.97
Sulfapyridine	1.2	375.83
Phenyl sulfonamide	124.29	3.63
Sulfaacetamide	115.95	3.89
Sulfaoxazole	138.21	3.26
Sulfonamide	109.19	4.13
Sulfanitrobenzene	149.2	3.02
Sulfaphenazole	80.27	5.62
Sulfaisoxazole	70.65	6.38
Sulfasalazine	90.45	4.99
Sulfonamide	85.69	5.26
Sulfaguanidine	149.13	3.01
Sulfonamide	150.03	3.63

6. Antibody application

The monoclonal antibody prepared by the hybridoma cell line NaN-1 by in vivo ascites was applied to the ELISA addition recovery test of several sulfa antibiotics (SD, SMR, SDM, STZ). The specific steps are as follows:

(1) coating: the original was diluted with 0.05M pH 9.6 carbonate buffer from 2μg / mL, 100μL / well, 37 ° C reaction for 2h;

(2) washing: the solution in the plate was decanted, dried, and washed 3 times with the washing liquid for 3 min each time;

(3) Closure: After pat dry, add 200 μL/well blocking solution and react at 37 ° C for 2 h. Dry after washing;

(4) Loading: The antiserum was diluted from 1..1000 and added to the coated wells of each dilution, 100 μL/well, and reacted at 37 ° C for 1 h; after washing, add 1..3000 diluted HRP. - goat anti-mouse IgG, 100 μL / well, reacted at 37 ° C for 1 h;

(5) Color development: the enzyme plate was taken out, and after thoroughly washing, 100 μL of TMB color developing solution was added to each well, and the reaction was carried out at 37 ° C for 15 minutes in the dark;

(6) Termination and measurement: 50 μL of the stop solution was added to each well to terminate the reaction, and then the OD 450 value of each well was measured with a microplate reader.

(7) Results interpretation: The ELISA titer of serum is the highest dilution factor of serum corresponding to 2.1 times of OD ₄₅₀ value (ie P/N ≥ 2.1).

(8) Adding recovery and sample pretreatment: Take the addition and recovery test of adding SD as an example, take 1 mL of milk into a 15 mL centrifuge tube, add 20 ng, 50 ng, 100 ng SD to the milk, and dilute 10 times with 0.01 M PBS. An indirect ELISA was used for the addition recovery test. The addition amount of SMR was 1 ng, 2 ng, 5 ng; the amount of SDM added was: 10 ng, 20 ng, 50 ng; the amount of STZ added was: 1 ng, 2 ng, 5 ng; NaN-1 monoclonal antibody against SD, SMR The results of the addition and recovery of four drugs, SDM and STZ, the recovery rate is shown in Table 2.

Table 2:

Although the present invention has been disclosed in the above preferred embodiments, the present invention is not limited thereto, and various modifications and changes can be made thereto without departing from the spirit and scope of the invention. The scope of the invention should be determined by the scope of the claims.

Claims (8)

1. An anti-sulfa antibiotic monoclonal antibody hybridoma cell line NaN-1 was deposited with the General Microbiology Center of the China Microbial Culture Collection Management Committee on January 20, 2016, with the preservation number CGMCC No.12028, and the deposit address is Beijing. No. 3, Beichen West Road, Chaoyang District, No. 3, Institute of Microbiology, Chinese Academy of Sciences.
2. A monoclonal antibody against sulfa antibiotics, which is produced by the anti-sulfa antibiotic monoclonal antibody hybridoma cell line NaN-1 of claim 1 having the accession number CGMCC No. 12028.
3. Use of the anti-sulfa antibiotic monoclonal antibody of claim 2, which is useful for preparing a tool for detecting a sulfa antibiotic or for directly detecting a sulfa antibiotic.
4. The use according to claim 3, characterized in that it is used for multi-residue detection of sulfonamides in food safety.
5. The anti-sulfa antibiotic monoclonal antibody according to claim 3, wherein the sulfa antibiotic comprises sulfadiazine, sulfamethazine, sulfamethoxine, sulfamethoxine, sulfamethoxine Azole, sulfamethoxazole, sulfathiazole, sulfaquinoxaline, sulfamethazine, sulfamethazine, sulfachloropyridazine, sulfamethoxazine, sulfadoxine, sulfapyridine, phenyl sulfonamide, sulfacetamide Sulfaoxazole, sulfonamide, sulfanitrobenzene, sulfaphenazole, sulfisoxazole, sulfasalazine, sulfamethamine, sulfaguanidine, sulfamethoxazole.
6. A tool for detecting a sulfa antibiotic, which comprises the anti-sulfa antibiotic monoclonal antibody according to claim 2 or the anti-sulfa antibiotic monoclonal antibody hybridoma cell strain NaN-1 according to claim 1.
7. A tool for detecting a sulfa antibiotic according to claim 6, wherein the tool is based on a kit of enzyme-linked immunosorbent technology or a test strip based on immunochromatography.
8. Use according to claim 6 or 7 in accordance with the detection of the content of sulfa antibiotics in foods.

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