Nanometer antibody 2018AFB-N11 having high specific recognition of Aflatoxins B1 and application of nanometer antibody 2018AFB-N11

A 2018AFB-N11, aflatoxin technology, applied in the biological field, can solve the problems of low sensitivity and specificity, achieve good specificity, high sensitivity, and reduce production costs

Active Publication Date: 2019-05-03
颐思特(武汉)生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology provides an effective way for reducing harmful chemicals called flutaxes that may occur naturally or intentionally during processing food products such as fava bean curdings (FBC). By recognizing this compound from natural sources like Fusarium oxysporum nivea, researchers have found ways to develop better methods to detect these substances without causing damage on crops being processed.

Problems solved by technology

This patented technical problem addressed in this patent relates to developing efficient and accurate ways to test or determine levels of exposure to certain types of pesticides including aflatictoxic compounds called fulvic acid residue(FAR). Current testing techniques involve expensive laboratory tests like gaschrometry and liquid scintigraphy, making them difficult to implement at home due to limitations associated with current assay technologies. There exists a new technique called Quantum Layer Chemistry Analysis (QLC), where enzyme activity plays key roles during bacterial growth process.

Method used

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  • Nanometer antibody 2018AFB-N11 having high specific recognition of Aflatoxins B1 and application of nanometer antibody 2018AFB-N11
  • Nanometer antibody 2018AFB-N11 having high specific recognition of Aflatoxins B1 and application of nanometer antibody 2018AFB-N11
  • Nanometer antibody 2018AFB-N11 having high specific recognition of Aflatoxins B1 and application of nanometer antibody 2018AFB-N11

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Aflatoxin B 1 Construction of Nanobody Gene Library

[0023] 1. Animal immunity

[0024] Buy a 2-year-old male alpaca, immune to aflatoxin B 1 complete antigen (AFB 1 -BSA, Sigma). 200 μg aflatoxin B 1 After the complete antigen was emulsified with Freund's incomplete adjuvant, the alpaca was injected subcutaneously at multiple points. Immunize once every 2 weeks, 7-10 days after each immunization, blood was collected from the alpaca, and the serum titer was determined by indirect ELISA method. After the first immunization with the highest titer, 10 mL of blood was taken to extract total RNA.

[0025] 2. Aflatoxin B 1 Construction of Nanobody Gene Library

[0026](1) Extraction of total RNA: select the first immunization with the highest titer of alpaca serum, and 7-10 days after immunization, take 10 mL of blood from the alpaca vein, and extract total RNA: use the LeukoLOCK Total RNA Isolation Kit of Life Technology Company to extract Total RNA in al...

Embodiment 2

[0055] Example 2: Aflatoxin B 1 Nanobody Screening and Sequence Determination

[0056] 1. Aflatoxin B 1 Panning of Nanobodies

[0057] use AFB 1 -BSA (1μg / well) and 3% BSA-PBS solution (used as a negative control) were respectively coated on the ELISA plate, overnight at 4°C; the next day, after pouring off the coating solution, the plate was washed 3 times with PBST, and then washed with 3% Block with skimmed milk powder for 1 hour; wash the plate 3 times with PBST, and coat with AFB 1 - Add 100 μL of the above-mentioned rescued aflatoxin nanobody gene library to the wells of BSA, and incubate at 37°C for 1 h; after washing the plate 10 times with PBST, add 100 μL, 500 ng / mL AFB to each well 1 (Aflatoxin) solution, shake at room temperature (20°C-30°C) for 30min to elute. Transfer the eluate to wells coated with 3% BSA-PBS solution, and incubate at 37°C for 1 h (to remove non-specific adsorption); after incubation, take the supernatant and infect 2 mL of ER2738 bacterial...

Embodiment 3

[0064] Example 3: Aflatoxin B 1 Preparation of Nanobody 2018AFB-N11

[0065] (1) Obtain the ability to secrete aflatoxin B 1 The ER2738 bacterial solution of Nanobody 2018AFB-N11 was extracted with Qiagen’s DNA mini-extraction kit, transformed into Top10F’competent cells, and spread on LB-Ampicillin plate;

[0066] (2) Pick a product containing aflatoxin B 1 The Top10F' colony of Nanobody 2018AFB-N11 plasmid was cultured in 100mL SB ampicillin liquid medium, 250rpm, 37℃ to OD 600 =0.5-0.8, add 100 μL of 1.0M IPTG solution to induce overnight.

[0067] (3) Centrifuge at 10,000 rpm for 15 min at 4°C, carefully remove the supernatant in a sterile operating bench, and use a bacterial protein extraction kit (Clontech Technology) for periplasmic protein extraction to obtain a crude protein extract. The crude protein extract was dialyzed overnight against equilibration buffer (50 mM phosphate, 300 mM sodium chloride, 20 mM imidazole; pH 7.4).

[0068] (4) Purify the antibody usi...

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Abstract

The invention discloses a nanometer antibody 2018AFB-N11 having high specific recognition of Aflatoxins B1 and an application of the nanometer antibody 2018AFB-N11. The Aflatoxins B1 are used for complete antigen immunity of alpacas, RNA in blood of the alpacas after immunity is extracted, a one-step method RT-PCR system is used for amplification of alpaca heavy chain antibody variable region VHHgenes, and after connection with a pComb3X carrier, colon bacillus is transformed to obtain a high-quality nanometer antibody gene pool, the Aflatoxins B1 complete antigen is coated to coated wells, aphage display nanometer antibody gene pool is screened, a nanometer antibody gene capable of specifically recognizing Aflatoxins B1is obtained, then the gene is transformed to colon bacillus, and a nanometer antibody strain which can be efficiently expressed in the colon bacillus is established. The Aflatoxin B1 nanometer antibody 2018AFB-N11 is high in sensitivity and good in specificity, and the 50% inhibition concentration IC 50 of the Aflatoxins B1 is 1.26ng/mL. The cross reaction rate with Aflatoxins B2, G1, G2 and M1 is smaller than 0.1%. The nanometer antibody 2018AFB-N11 can be used for research and development of a detection reagent for high specific recognition of Aflatoxins B1.

Description

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Claims

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Application Information

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Owner 颐思特(武汉)生物科技股份有限公司
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