Preparation of monoclonal antibody and uses thereof

A monoclonal antibody and immune antigen technology, applied in the field of immunochemical analysis, can solve the problems of time-consuming, laborious, high instrument cost, unfavorable sample detection, etc.

Inactive Publication Date: 2009-03-25
EAST CHINA NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Chromatographic analysis can indeed accurately and quantitatively analyze the residues of Sudan Red in food with high sensitivity, but the cost of the instrument is high, and the experimental sample processing steps are cumbersome, time-consuming and laborious, which is not conducive to the detection of large quantities of samples

Method used

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  • Preparation of monoclonal antibody and uses thereof
  • Preparation of monoclonal antibody and uses thereof
  • Preparation of monoclonal antibody and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Synthesis of Antigens

[0029] The molecular structural formula of carboxy sudan red is as follows:

[0030]

[0031]In a 100-mL beaker, add 140mg of carboxysudan red and dissolve in 3mL of N,N-dimethylformamide (DMF), add 103.2mg of dicyclohexylcarbodiimide (DCC) and 75mg of N-hydroxysuccinate while stirring imide (NHS), stirred overnight at room temperature. The next day, the precipitate was removed by centrifugation, and the supernatant was taken for later use. Take another 100-mL beaker, add 5mL 0.1mol / LPBS (pH 7.2), 340mg BSA, stir on a magnetic stirrer, add 0.5mL N,N-dimethylformamide (DMF) until the BSA dissolves, and then The above supernatant was slowly added dropwise while stirring, the mouth of the beaker was sealed, and the reaction was stirred and reacted at 4°C for 4h. After centrifugation, the precipitate was removed, and the supernatant was loaded into a dialysis membrane and dialyzed with normal saline at 4°C for 3 days, and the medium ...

Embodiment 2

[0034] Embodiment 2: Preparation of monoclonal antibody

[0035] (1) Animal immunity

[0036] Ten female Balb / C mice were differentiated into 2 groups, 5 mice in the immune group and 5 mice in the negative control group.

[0037] The carboxy sudan red I-BSA immunization antigen was diluted with physiological saline, and the immunization dose of each mouse was 50ug / only. The immunization program used 2 basic immunizations and 3 booster immunizations, and the interval between each immunization was 15 days. The first basic immunization was immunized with Freund's complete adjuvant emulsified antigen, and multi-point subcutaneous injection was used on the back of the mouse neck; the second basic immunization and the subsequent two booster immunizations were used to emulsify the antigen with Freund's incomplete adjuvant, using mouse Multi-point subcutaneous injection or intraperitoneal injection on the back of the neck; the last booster immunization uses the antigen without adjuv...

Embodiment 3

[0046] Example 3: Identification of Monoclonal Antibodies - Indirect ELISA Method

[0047] (1) Determination of the optimum antigen coating concentration and the optimum dilution factor (working concentration) of the antibody

[0048] The checkerboard titration test using the indirect ELISA detection method was carried out, and the point where the absorbance value (OD) was about 1.0 was used as a reference point, and factors such as antigen concentration and antibody dilution were comprehensively considered to determine the optimal antigen coating concentration and antibody optimal concentration. Dilution factor (i.e. working concentration).

[0049] The operating procedure of the indirect ELISA detection method: Take a 96-well microtiter plate, add 100ul of the coating antigen carboxy Sudan Red-OVA at a coating concentration diluted with coating buffer to each well, coat overnight at 4°C, wash with washing solution 0.05 Wash the plate 3 times with %PBST, add 300ul of blockin...

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Abstract

The invention discloses the preparation of a monoclonal antibody and application thereof, and in particular relates to the preparation of a monoclonal antibody capable of synchronously detecting residues of Sudan I and para red and application thereof, which belong to the technical field of immunochemistry analysis. A complex formed by crosslinking a Sudan red derivative and bovine serum albumin is taken as immune antigen, the immune antigen is used to immunize a Balb / C mouse, sensitized spleen cells and myeloma cells SP2 / 0 of the mouse are fused to prepare hybridoma cells, a monoclonal hybridoma cell strain is obtained through an indirect ELISA screening method and a limited dilution method, and the hybridoma cell strain is injected into an abdominal cavity of the mouse to produce the monoclonal antibody. The invention provides an antigen prepared based on the method and an enzyme-linked immunoassay (ELISA) method that the characteristics of the monoclonal antibody are suitable to qualitatively and quantificationally detect the Sudan I, the para red and the content of the Sudan I and the para red.

Description

technical field [0001] The invention relates to the preparation and application of a monoclonal antibody, in particular to the preparation and application of a monoclonal antibody suitable for simultaneous detection of Sudan I and parared residues, and belongs to the technical field of immunochemical analysis. Background technique [0002] Sudan red (Sudan) is a lipophilic azo compound. It is a red industrial synthetic dyeing agent. It belongs to chemical dyes. It is mainly used in petroleum, engine oil and some other industrial solvents. , flooring, colored wax, fireworks and other fireworks. Since the chemical structure of Sudan Red contains an azo group, after the human body ingests it, the corresponding amine substances will be generated in the body under the action of reductase, which is carcinogenic to the human body. [0003] Although my country and most countries have already banned Sudan Red as an additive in food, it can make food brighter and longer-lasting and i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/531
Inventor 钱旻王群李恺韩红辉顾虹洁汪磊杜冰
Owner EAST CHINA NORMAL UNIV
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