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Hybridoma cell strain AFM1B7, monoclonal antibody thereof and aflatoxin M1 flow lag immunization time-resolved fluorescence quick test kit

A hybridoma cell line, aflatoxin technology, applied in the directions of antifungal/algae/lichen immunoglobulin, analytical materials, measuring devices, etc., can solve the problems of complicated operation, expensive instruments, complicated processing process, etc., and achieve simple operation. , high accuracy and high sensitivity

Active Publication Date: 2013-07-24
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, thin-layer chromatography is the most commonly used detection method for the detection of aflatoxin earlier. This method does not require special equipment and can be carried out in general laboratories, but the amount of reagents is large, the operation is cumbersome, and other components interfere seriously. , poor accuracy, cannot be accurately quantified, and is more harmful to the experimenters and the surrounding environment, so it is not suitable for on-site rapid detection
Precision instrument analysis mainly includes fluorescence spectrophotometry and high-performance liquid chromatography. These methods have high sensitivity and good accuracy, but they are expensive and require a high degree of purification of aflatoxin samples. The sample pretreatment process is cumbersome and time-consuming. High requirements for the experimental environment, it is difficult to achieve rapid detection

Method used

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  • Hybridoma cell strain AFM1B7, monoclonal antibody thereof and aflatoxin M1 flow lag immunization time-resolved fluorescence quick test kit
  • Hybridoma cell strain AFM1B7, monoclonal antibody thereof and aflatoxin M1 flow lag immunization time-resolved fluorescence quick test kit
  • Hybridoma cell strain AFM1B7, monoclonal antibody thereof and aflatoxin M1 flow lag immunization time-resolved fluorescence quick test kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Screening of hybridoma cell line AFM1B7

[0040] 1. Animal immunization

[0041] Six 6-week-old BALB / c mice were purchased and immunized with commercially available aflatoxin M1 complete antigen AFM1-BSA. For the first immunization, aflatoxin M1 complete antigen was emulsified with an equal volume of Freund's complete adjuvant, and then injected subcutaneously at multiple points on the back of the mouse. The second immunization was carried out 4 weeks later, using Freund's incomplete adjuvant to emulsify with an equal volume of aflatoxin M1 complete antigen, and intraperitoneally injecting it into mice. The interval between the third immunization and the second immunization was 4 weeks, and the immunization method was the same. The fourth immunization was carried out 3 weeks after the third immunization, and the immunization method was the same as the second immunization, which was also intraperitoneal injection. The doses for the four immunizations were th...

Embodiment 2

[0049] Example 2: Determination of the variable region sequence of the hybridoma cell line AFM1B7 antibody

[0050] (1) Total RNA extraction: use the total RNA extraction kit from Tiangen Company and follow the instructions to extract the total RNA that can produce the hybridoma cell line AFM1B7;

[0051] (2) cDNA synthesis: using the total RNA obtained in step 1 as a template, oligo(dT) 15 As a primer, perform reverse transcription according to the SuperScriptTM-2II reverse transcriptase instructions to synthesize the first strand of cDNA; primer oligo(dT)15 was purchased from Invitrogen;

[0052] (3) Cloning of variable region genes by PCR method: Design primers according to the conserved sites of mouse antibody gene sequences in GENEBANK, and use cDNA as a template to amplify antibody light and heavy chain variable region genes. The PCR program was: 94°C for 30s, 58°C for 1min, 72°C for 1min, 30 cycles of amplification, and finally 72°C for 10min. After the PCR product wa...

Embodiment 3

[0054] Example 3: Preparation, purification, subtype and identification of monoclonal antibodies

[0055]The hybridoma cell line AFM1B7 obtained in Example 1 was injected into BALB / c mice treated with Freund's incomplete adjuvant in advance, the ascites of the mice was collected, and the antibody was purified by octanoic acid-ammonium sulfate method. The specific operation was as follows: Filter mouse ascites with double-layer filter paper, centrifuge at 12000r / min for 15min at 4°C, absorb the supernatant, mix the obtained ascites supernatant with 4 times the volume of acetate buffer, slowly add n-octanoic acid under stirring, The required volume of n-octanoic acid is 33μL, mix at room temperature for 30min, stand at 4°C for 2h, then centrifuge at 12000r / min for 30min at 4°C, discard the precipitate, filter the obtained supernatant with double-layer filter paper, and add 1 / 10 of the filtrate volume Phosphate buffer solution with a molar concentration of 0.1mol / L and a pH value...

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Abstract

The invention relates to a hybridoma cell strain AFM1B7, a monoclonal antibody thereof and an aflatoxin M1 flow lag immunization time-resolved fluorescence quick test kit. The hybridoma cell strain AFM1B7 is collected in China Center for Type Culture Collection (CCTCC), and the collection number is CCTCC No.C201020. The monoclonal antibody secreted by the hybridoma cell strain AFM1B7 has high sensitivity and good specificity, the 50% inhibition concentration against aflatoxin M1 is 52pg / mL, and the cross reaction rate with aflatoxins B1, B2, G1 and G2, vomitoxin, zearalenone and fumonisin is less than 0.3%. The aflatoxin M1 flow lag immunization time-resolved fluorescence quick test kit prepared from the monoclonal antibody can be used for quantitatively measuring the content of aflatoxin M1, is simple and quick to operate, and has high accuracy.

Description

technical field [0001] The invention relates to hybridoma cell line AFM1B7, its monoclonal antibody and aflatoxin M1 flow hysteresis immune time-resolved fluorescence rapid test kit. Background technique [0002] Aflatoxins are mainly secondary metabolites secreted by Aspergillus flavus and Aspergillus parasiticus, and are natural toxic compounds that can cause various damages to humans and animals. More than 20 types of aflatoxins have been discovered, mainly including aflatoxin A1 (AFA1), B2 (AFB2), AFG and M1 (AFM1). Aflatoxin M1 (AFM1) is a hydroxylated metabolite of AFB1. After mammals ingest AFB1-contaminated feed, it will be secreted in milk after hydroxylation in the body. Typically, when animals ingest AFB1-contaminated food, the excretion of AFM1 is 1%–3% of the AFB1 intake. A large number of researchers have conducted in-depth studies on the toxicity and carcinogenicity of AFM1, and the results of the research have also prompted the International Agency for Rese...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/14G01N33/577C12R1/91
Inventor 李培武李冉张奇丁小霞张文张兆威
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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