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A hybridoma cell line secreting anti-total aflatoxin monoclonal antibody and its application

A hybridoma cell line, aflatoxin technology, applied in the biological field, can solve the problems of inability to quantify well, high detection limit, low detection limit, etc., and achieve the effects of high sensitivity, good specificity and good anti-interference.

Active Publication Date: 2020-01-31
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thin-layer chromatography is convenient for detection, but the detection limit is high and cannot be quantified well; high-performance liquid chromatography, liquid chromatography-mass spectrometry, etc. are accurate in detection and low in detection limit, but require complicated sample pretreatment processes, specialized Instruments and professional personnel training
These congenital deficiencies of instrumental analysis methods have greatly limited their practical application.

Method used

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  • A hybridoma cell line secreting anti-total aflatoxin monoclonal antibody and its application
  • A hybridoma cell line secreting anti-total aflatoxin monoclonal antibody and its application
  • A hybridoma cell line secreting anti-total aflatoxin monoclonal antibody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Screening of hybridoma cell line F-3-1

[0028] 1. Hapten synthesis and identification

[0029] The synthetic route of AFs hapten is attached figure 1 shown.

[0030] Add 100 mg of aflatoxin B to a 25 mL round bottom flask 1and 5mL of N,N-dimethylformamide, stirred mechanically to dissolve, slowly added 147.3mg of phosphorus oxychloride dropwise at 0°C, raised the temperature to 40°C, and reacted for 2h under magnetic stirring, then raised the temperature to 80°C and continued to stir React for 3 hours. After the reaction is over, cool naturally, slowly pour into ice water, add w(NaOH)=10% aqueous solution to adjust the pH of the solution to 7~8, use ethyl acetate 30mL, extract three times, combine the organic phases, anhydrous sulfuric acid Sodium was dried and evaporated to dryness. Recrystallized from absolute ethanol to obtain yellow solid aldehyde aflatoxin B 1 103 mg is the AFs hapten, with a yield of 95.37%.

[0031] The above haptens were ident...

Embodiment 2

[0056] Example 2: Preparation, purification and identification of anti-total aflatoxin monoclonal antibodies

[0057] 1. Ascites Preparation

[0058] Inject liquid paraffin into 6-8 week old Balb / c mice, 500 μL / mouse. After 10 days, the hybridoma cells CGMCC No.13806 in the logarithmic growth phase were collected with RPMI-1640 basal medium, counted with a hemocytometer and a microscope, and the cell concentration was 1.0×10 6 ~1.5×10 6 within the range of cells / mL. 0.5 mL of hybridoma cell CGMCC No.13806 was injected into the peritoneal cavity of each mouse. Pay attention to the swelling of the abdomen of the mice after one week, collect ascites in the abdominal cavity of the mice with a sterile syringe, and collect once every one to two days, and collect repeatedly until the mice die naturally. Centrifuge at 5000r / min for 5min at 4°C, collect the supernatant, and remove the floating fat and protein film in the upper layer of ascites.

[0059] 2. Antibody Purification

...

Embodiment 3

[0106] Embodiment 3: the application of anti-total aflatoxin monoclonal antibody

[0107] The anti-total aflatoxin monoclonal antibody secreted by hybridoma cell line F-3-1 is used to prepare aflatoxin ELISA kit for aflatoxin B in traditional Chinese medicine 1 , B 2 , G 1 , G 2 Total analytical testing.

[0108] 1. Composition of ELISA Kit

[0109] (1) ELISA plate coated with anti-total aflatoxin monoclonal antibody;

[0110] (2) Enzyme-labeled aflatoxin antigen: prepared by coupling the coated antigen described in Example 1 with horseradish peroxidase (HRP), and diluted to the optimal working concentration with enzyme-labeled antigen diluent;

[0111] (3) Aflatoxin B 1 Standard: the concentration of the standard solution is 0μg / L, 0.03μg / L, 0.1μg / L, 0.2μg / L, 0.6μg / L;

[0112] (4) Substrate chromogenic solution: made up of A liquid and B liquid, A liquid is the aqueous solution of 2% carbamide peroxide, and B liquid is the aqueous solution of 1% tetramethylbenzidine; ...

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Abstract

The invention provides a hybridoma cell strain F-3-1 for secreting monoclonal antibodies for resisting total aflatoxins. The hybridoma cell strain is preserved in China General Microbiological Culture Collection Center, and the preservation number is CGMCC No.13806. The monoclonal antibodies for resisting total aflatoxins secreted by the cell strain has the advantages of high sensitivity, good specificity, and good anti-interference performance; the 50% inhibition concentration IC50 of aflatoxin B1 is 0.046[mu]g / L, cross reaction rate of the monoclonal antibodies and AFB2 is 110%, cross reaction rate of the monoclonal antibodies and AFG1 is 96%, and cross reaction rate of the monoclonal antibodies and AFG2 is 78%; the cell strain has good anti-interference performance for mycotoxin, pesticides, and heavy metals which may exist in traditional Chinese medicines, and accessories, contaminant microorganisms and the like used in processing, and provides a condition for immunodetection of total amount of aflatoxins B1, B2, G1 and G2 in traditional medicine with real application values.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a hybridoma cell strain secreting anti-total aflatoxin monoclonal antibody and application thereof. Background technique [0002] During the process of production, harvesting, processing, transportation, storage, etc., Chinese herbal medicines may contaminate fungi due to their own properties and the influence of external factors, and then mildew and contaminate mycotoxins. Mildew will not only affect the quality of Chinese medicinal materials, make the medicinal materials lose their original medicinal value, and cause huge economic losses, but also in the process of mildew, a variety of mycotoxins produced will affect the patient's liver, kidney, nerves and hematopoiesis. System, etc. cause serious damage, and may even induce cancer. At the same time, with the popularization of the concept of traditional Chinese medicine health care and health preservation, more and mor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/14G01N33/577
CPCC07K16/14C07K2317/33G01N33/577G01N2333/38
Inventor 何方洋崔海峰洪小栩万宇平吴小胜袁媛鲁亚辉杨昌松杨春燕
Owner BEIJING KWINBON BIOTECH