Enzyme-linked immunosorbent assay kit for analyzing chloramphenicol residues based on rabbit monoclonal antibody

An enzyme-linked immunosorbent assay and monoclonal antibody technology, applied in the direction of analytical materials, measuring devices, instruments, etc., can solve problems that have not been reported before, and achieve the effect of reducing detection costs

Inactive Publication Date: 2010-12-15
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This kit uses hybridoma technology and recombinant antibody technology to obtain chloramphenicol rabbit monoclonal antibody, and develops a chloramphenicol indirect ELISA kit based on rabbit monoclonal antibody, which has not been reported at home and abroad.

Method used

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  • Enzyme-linked immunosorbent assay kit for analyzing chloramphenicol residues based on rabbit monoclonal antibody
  • Enzyme-linked immunosorbent assay kit for analyzing chloramphenicol residues based on rabbit monoclonal antibody
  • Enzyme-linked immunosorbent assay kit for analyzing chloramphenicol residues based on rabbit monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1: the synthesis of chloramphenicol immune antigen and coating antigen

[0028] The specific operation steps are as follows: Take 80 mg of small CAP molecules, dissolve them in 1.5 mL of absolute ethanol, and add 12 mL of 1mol / L hydrochloric acid. Add 65mg of zinc powder and heat in a water bath at 70°C for 50min. After cooling, add 0.5mol / L NaNO dropwise at 4°C and pH 1-2 2 Add a few drops, and stir in the dark until the starch potassium iodide test paper turns grayish blue (solution I); weigh 150 mg of BSA, dissolve it in 12 mL of phosphate buffer (PBS, pH 7.4), and cool. (solution II). Then slowly add liquid I into liquid II while stirring, and at the same time add 0.2mol / L NaOH solution to keep the pH at around 7-8. Stirring at 4°C for 12h, the reaction product was dialyzed against PBS for 5d. The coating antigen OVA-CPFX was prepared in the same way and used to coat the microtiter plate.

[0029] The coupling principle of chloramphenicol and carrier...

Embodiment 2

[0031] Example 2: Production of Chloramphenicol Rabbit Monoclonal Antibody

[0032] 1. Immunization of rabbits

[0033] For primary immunization, mix 500 μg of immune antigen with an equal amount of Freund’s complete adjuvant, and after complete emulsification, inject white rabbits (1 mL / only) subcutaneously at 5 points on the back; three weeks later, use 200 μg of immune antigen with Freund’s incomplete adjuvant After mixing, carry out the second immunization in the same way; after that, immunize once every two weeks. After a total of 3 to 7 immunizations, when the serum titer reaches 64,000 or more, inject 500 μg of immune antigen into the ear vein, and collect whole blood from the carotid artery 4 days later. , take the spleen aseptically and prepare for cell fusion.

[0034] 2. Preparation of rabbit monoclonal antibody

[0035] The rabbit splenic lymphocytes were fused with 240E cells in the logarithmic growth phase, and the fusion agent was 50% PEG. The fused cells use...

Embodiment 3

[0036] Embodiment 3: the coating of microtiter plate

[0037] Chloramphenicol coated antigen was diluted to 0.2 μg / mL with pH9.6, 0.05mol / L carbonate buffer (containing 2.93g sodium bicarbonate and 1.59g sodium carbonate, dissolved in double distilled water or ultrapure water 1L) , add 50 μL to each well of the ELISA plate, coat at 4°C for 12 hours or 37°C for 2 hours, pour off the coating liquid, wash with PBST for 3 to 5 times, pat dry, and then add 250 μL of 3% degreasing to each well Milk, sealed at 37°C for 1 hour, washed 3 to 5 times after taking out, dried and sealed for storage.

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Abstract

The invention discloses an enzyme-linked immunosorbent assay (ELISA) kit for analyzing chloramphenicol residues based on a rabbit monoclonal antibody. The kit comprises a body and a 96-well ELISA plate and reagents arranged in the body, wherein a chloramphenicol envelop antigen is enveloped in the plate; and the reagents include (1) washing concentrate; (2) diluting concentrate; (3) chloramphenicol standard solution; (4) a chloramphenicol antibody; (5) a horseradish peroxidase labeled goat anti-rabbit antibody; (6) substrate diluent; (7) a substrate; (8) substrate development solution; and (9) reaction termination solution. The kit is applicable to detection of chloramphenicol residues in such samples as milk, swine urine, etc. The kit can simultaneously detect mass samples, is convenient and quick, is of great practical significance to field monitoring trace analysis, simultaneously greatly lowers the detection cost and has potential economic value.

Description

technical field [0001] The invention relates to a kit, in particular to an enzyme-linked immunosorbent assay kit for chloramphenicol residue analysis based on a rabbit monoclonal antibody. Background technique [0002] Chloramphenicol (Chloramphenicol, CAP), chemical name D-threo-(-)-N-[α-(hydroxymethyl)-β-hydroxy-p-nitrophenylethyl]-2,2-dichloroethyl amides. It belongs to broad-spectrum antibiotics and has a good inhibitory effect on Gram-positive bacteria and negative bacteria, so it is widely used in animal husbandry and aquaculture. However, CAP residues in animal foods are potentially harmful to human health. For this reason, many countries in the world have all formulated the highest residual standard of chloramphenicol, strictly restricting its use. In order to adapt to the international competition and challenges after joining the WTO, it is necessary to establish a rapid detection method with independent intellectual property rights. Therefore, it is of great pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577
Inventor 郑晓冬陆蕾刘娜倪庚
Owner ZHEJIANG UNIV
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