Immunoaffinity column based on bispecific monoclonal antibody for aflatoxin B1 and ochratoxin A and application of immunoaffinity column

A monoclonal antibody, aflatoxin technology, applied in the field of immunoaffinity column, can solve the problems of complicated operation, time-consuming and high cost

Pending Publication Date: 2021-09-10
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to provide an anti-aflatoxin B in order to overcome the problems of complex, time-consuming and costly operations when traditional IAC detects multiple biological toxins 1 (AFB 1 ) and ochratoxin A (OTA) bispecific monoclonal antibody preparation method, and an immunoaffinity column based on the bispecific monoclonal antibody

Method used

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  • Immunoaffinity column based on bispecific monoclonal antibody for aflatoxin B1 and ochratoxin A and application of immunoaffinity column
  • Immunoaffinity column based on bispecific monoclonal antibody for aflatoxin B1 and ochratoxin A and application of immunoaffinity column
  • Immunoaffinity column based on bispecific monoclonal antibody for aflatoxin B1 and ochratoxin A and application of immunoaffinity column

Examples

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Effect test

Embodiment 1

[0045] Embodiment 1 Aflatoxin B 1 (AFB 1 ) and the preparation of ochratoxin A (OTA) hybridoma cells

[0046] 1. Experimental method

[0047] 1. Aflatoxin B 1 Preparation of Artificial Antigen and Ochratoxin A Artificial Antigen

[0048] AFB 1 artificial antigen (AFB 1 -KLH and AFB 1 -OVA) is prepared as follows: 10mg AFB 1 and 20mg of carboxymethylhydroxylamine hemihydrochloride (CMO) were dissolved in a mixed solution of methanol-pyridine-water (volume ratio V:V:V=4:1:1), and dried to obtain a brown oil. Add an equal amount of chloroform and primary water to wash, collect the organic phase, and obtain the hapten AFB 1 -O. Take 2.5mg AFB 1 -O was dissolved in 200 μL DMF, then added equimolar 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) , stirred overnight at room temperature to obtain an activated solution. Add the activation solution dropwise to 2 mL of 5 mg / mL keyhole limpet hemocyanin (KLH) or 2.5 mg / mL ovalbu...

Embodiment 2

[0055] Example 2 HGPRT gene-deficient AFB 1 Mutation and Identification of Mutants in Hybridoma Cell Lines

[0056] 1. Experimental method

[0057] 1. AFB 1 HGPRT gene mutation mutagenesis treatment of hybridoma cell line

[0058] Before carrying out the induction treatment of cells, the AFB obtained in Example 1 1 The hybridoma cell line E4 was cultured in HAT medium for 2 days, and the culture condition was 5% CO 2 , 37°C; then cultured in HAT medium for 2 days to restore the cells to normal growth; take 5×10 5 AFB 1 The hybridoma cells were inoculated in a 10 cm culture dish containing HAT medium and cultured for 24 hours, then subjected to mutagenesis treatment, and added complete culture containing different concentrations of N-methyl-N'-nitro-nitrosoguanidine (MNNG) mutagen The concentration of MNNG in the culture medium was set to 5.0 μg / mL, 10.0 μg / mL, 20.0 μg / mL and 40.0 μg / mL respectively, and the culture time was set to 2 h, 4 h, 6 h and 10 h, respectively. A...

Embodiment 3

[0077] 1. Experimental method

[0078] The experimental method of this embodiment is basically the same as that of Example 2, the difference is that the cell lines that undergo HGPRT mutagenesis treatment are other AFBs obtained in Example 1 1 For the hybridoma cell lines E1-E3, the mutagenesis conditions were the optimal mutagenesis conditions determined in Example 2: the concentration of MNNG was 10 μg / mL, and the treatment time was 6 h.

[0079] 2. Experimental results

[0080] AFB 1 After the hybridoma cell lines E1-E3 were treated with the mutagenic conditions, the survival rates of the hybridoma cell lines were calculated by trypan blue staining, and the survival rates of E1-E3 were 68.45%, 70.20%, and 69.03%, respectively. It shows that the optimized mutagenesis conditions in Example 2 can obtain HGPRT-deficient cells with a relatively suitable and stable survival rate, which can be used in cell fusion experiments.

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Abstract

The invention discloses an immunoaffinity column based on a bispecific monoclonal antibody for aflatoxin B1 and ochratoxin A and application of the immunoaffinity column, and specifically discloses a preparation method of the bispecific monoclonal antibody for aflatoxin B1 (AFB1) and ochratoxin A (OTA). According to the invention, a hybridoma cell capable of stably secreting an antibody capable of simultaneously recognizing both AFB1 and OTA is obtained, and the prepared bispecific monoclonal antibody can be used in the preparation of an immunoaffinity column. The affinity column can simultaneously extract two objects AFB1 and OTA only by coupling one antibody, coupling rate is easy to adjust and measure, number of binding sites of the two objects is equal, structure is uniform, and interference is less. The immunoaffinity column disclosed by the invention can be used in combination with ELISA, has characteristics of simplicity, convenience, rapidness, strong specificity and relatively high sensitivity, and has important application value in detecting and monitoring AFB1 and OTA multi-residues in food and agricultural products.

Description

technical field [0001] The invention relates to the field of biotechnology, more specifically, to an immunoaffinity column based on aflatoxin B1 and ochratoxin A bispecific monoclonal antibody and its application. Background technique [0002] Aflatoxin B 1 (aflatoxin B 1 ,AFB 1 ) and ochratoxin A (OTA) are common mycotoxin contaminants in cereals. AFB 1 It is classified as a first-class carcinogen by the International Agency for Research on Cancer, and OTA is classified as a 2B carcinogen. due to AFB 1 OTA and OTA are both mandatory inspection items for cereal products, and both of them often contaminate cereal foods at the same time, so it is necessary to provide a fast and simple detection method for simultaneous detection of both. Immunoassay has the advantages of specificity, sensitivity, simplicity and speed, and has become a research hotspot in recent years. At present, there have been researches on AFB at home and abroad 1 and OTA immunoassay research reports...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/14C12N15/01C12N15/06G01N33/577G01N33/531B01D15/22B01D15/20
CPCC07K16/14C12N15/01C12N5/16G01N33/577G01N33/5308G01N33/531B01D15/22B01D15/206C07K2317/31
Inventor 肖治理卢迪莎谢波王序杨金易王弘罗林
Owner SOUTH CHINA AGRI UNIV
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