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A specific anti-ceftiofur monoclonal antibody hybridoma cell line 2e5 and its application

A technology of hybridoma cell lines and cloning antibodies, which is applied in the direction of specific peptides, instruments, peptides, etc., can solve the problems of high instrument cost and complicated operation, and achieve low cross-reaction rate, good detection sensitivity and affinity, and good specificity. Effects of cloning antibody cell lines

Active Publication Date: 2018-10-26
无锡迪腾敏生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the detection methods of ceftiofur are mainly instrument detection methods and immunoassay detection methods. Relatively low requirements for personnel, so it is suitable for rapid screening of a large number of samples

Method used

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  • A specific anti-ceftiofur monoclonal antibody hybridoma cell line 2e5 and its application
  • A specific anti-ceftiofur monoclonal antibody hybridoma cell line 2e5 and its application
  • A specific anti-ceftiofur monoclonal antibody hybridoma cell line 2e5 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Preparation of hybridoma cell line 2E5

[0022] (1) Synthesis of complete antigen: Take 4.5 mg of EFT, add 2.0 mg of EDC and 1.0 mg of NHS, dissolve in DMF, stir at room temperature, and activate for 4 hours; another 5 mg of BSA is dissolved in 2 mL of CB, 0.05 M, pH 9.6 In the solution, the above-mentioned activated ceftiofur solution was slowly added dropwise to the CB solution of BSA, stirred at room temperature overnight, and dialyzed at 4°C for three days to obtain the immunogen, which was subpackaged and stored at -20°C.

[0023] (2) Animal immunization: healthy BALB / c mice aged 6-8 weeks were selected for immunization. After the complete antigen of ceftiofur (1 mg / mL) was emulsified with the same amount of Freund's adjuvant, BALB / c mice were immunized by subcutaneous multi-point injection, 100 μL each. Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for the booster immunization. The immunization...

Embodiment 2

[0029] Example 2 Preparation and Identification of Monoclonal Antibody

[0030] Take 8-10 week-old BALB / c mice, and inject 1 mL of paraffin oil into each mouse; 7 days later, each mouse is injected with 1×10 6 Hybridoma cell 2E5, ascites fluid was collected from the seventh day, the ascites fluid was purified by octanoic acid-saturated ammonium sulfate method, and the obtained monoclonal antibody was stored at -20°C.

[0031] The mouse monoclonal antibody subtype identification kit was used to identify the immunoglobulin subtype of the monoclonal antibody purified from ascitic fluid, as shown in Table 1, the subtype was IgG2b.

[0032] Table 1

[0033]

[0034] Determination of IC of monoclonal antibodies against EFT, CEX and CEF using indirect competition ELISA and indirect ELISA 50 They were 2.4ng / mL, 76.9ng / mL and 45.28ng / mL respectively, and the cross-reactivity rates were all less than 10%. It can be used for specific rapid detection of ceftiofur.

Embodiment 3

[0035] Example 3 Application of Antibody

[0036]The monoclonal antibody prepared by the hybridoma cell line 2E5 through in vivo ascites was applied to the addition and recovery test of ceftiofur ELISA, and the specific steps were as follows:

[0037] a. 0.5μg / mL EFT-EDC-OVA diluted with carbonate buffer solution (CBS) was used as the original coating to coat the 96-well microtiter plate, 100μL per well, after coating at 37°C for 2h, wash with PBST Wash the plate three times, each time with 250 μL per well, each time for 3 min, and pat dry;

[0038] b. Block with CBS containing 0.2% gelatin, 200 μL per well, block for 2 h at 37°C, wash the plate three times with PBST washing solution, 250 μL per well for 3 min each time, and pat dry;

[0039] c. Prepare ceftiofur standard solutions of 0, 0.2, 0.5, 1, 2, 5, 10, and 20 ng / mL with phosphate buffered saline (PBS). Add the standard solution and the extract of the sample to be tested to the sealed microtiter plate, 50 μL per well,...

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Abstract

The invention discloses a specific anti-ceftiofur monoclonal antibody hybridoma cell strain 2E5 and the application thereof and belongs to the technical field of food safety immunodetection. The monoclonal cell strain 2E5 is preserved in the China General Microbiological Culture Collection Center, CGMCC for short, with the preservation number of CGMCC No.10873. The crude drug ceftiofur and BSA are coupled to prepare immunogen by means of the active ester method, and ceftiofur and OVA are coupled to prepare coating antigen. The specific anti-ceftiofur hybridoma cell strain is obtained after a mouse is immunized. The monoclonal antibody secreted by the cell strain only aims at ceftiofur specifically, the crossing-over rates between the monoclonal antibody and cefalexin and between the monoclonal antibody and cefquinome are 3.12% and 5.3% respectively, the crossing-over rates between the monoclonal antibody and other cephalosporin antibiotics are all smaller than 0.1%, and therefore specificity is high. The anti-ceftiofur monoclonal antibody hybridoma cell strain has high detection sensitivity and compatibility, the crossing-over rates with other cephalosporin antibiotics are low, and therefore specific detection of ceftiofur can be achieved. The invention also provides a new idea for preparing a specific antibody aiming at a specific cephalosporin so as to obtain a good specific monoclonal cell strain.

Description

technical field [0001] The invention relates to a specific anti-ceftiofur monoclonal antibody hybridoma cell line 2E5 and application thereof, belonging to the technical field of food safety immunoassay. Background technique [0002] Ceftiofur (EFT), English name is ceftiofur, molecular formula is C 19 h 17 N 5 o 7 S 3 , the molecular weight is 523.56, and the chemical name is (6R,7R)-7-[2-(2-aminothiazol-4-yl)(methoxyimino)acetamido]-3-[(2-furyl Carbonyl)thiomethyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid. The CAS accession number is 80370-57-6. Ceftiofur is an antimicrobial drug and is a third-generation animal-specific cephalosporin semi-synthesized from cephalosporins. [0003] Ceftiofur is the first cephalosporin antibiotic approved for use in animals. Its nucleus is 7-aminocephalosporanic acid (7-ACA) obtained by cleavage of cephalosporin C. It has a broad-spectrum bactericidal effect and is effective against Gram-positive and Gram-negative...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/44G01N33/577
CPCC07K16/44C07K2317/92
Inventor 郭玲玲朱雨婷刘丽强吴晓玲宋珊珊
Owner 无锡迪腾敏生物科技有限公司
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