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Biotin-labeled polymer nanoparticle simulated antibody and application thereof in Bt protein enzyme-linked immunosorbent assay

A technology of biotin-labeling and nano-particles, which is applied in the field of biotin-labeling polymer nano-particle mimic antibodies and their preparation, can solve the problems of harsh storage and reaction conditions, difficulties in the preparation of natural antibodies, and poor environmental tolerance, and achieve improved Effect of selective affinity, good specificity, and increased workload

Active Publication Date: 2019-03-26
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Then, biotin-labeled polymer nanoparticles were used instead of natural biological antibodies to establish an enzyme-linked immunoassay method for Bt protein, which retained the advantages of good specificity, high accuracy, and easy operation of the enzyme-linked immunosorbent assay, and overcome the difficulties in the preparation of natural antibodies , high cost, poor environmental tolerance, easy inactivation, harsh storage and reaction conditions

Method used

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  • Biotin-labeled polymer nanoparticle simulated antibody and application thereof in Bt protein enzyme-linked immunosorbent assay
  • Biotin-labeled polymer nanoparticle simulated antibody and application thereof in Bt protein enzyme-linked immunosorbent assay
  • Biotin-labeled polymer nanoparticle simulated antibody and application thereof in Bt protein enzyme-linked immunosorbent assay

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Synthesis of biotin-labeled polymer nanoparticles mimic antibody NP1

[0046] Weigh 10% molar percentage of acrylamide-NITIHITDTNNK-amide, 88% isopropylacrylamide and 2% N,N-methylenebisacrylamide into a 50mL round bottom flask, add 5mL ultrapure water, Ultrasound for 5min to fully dissolve. Add 10 mg of sodium dodecyl sulfonate to the dissolved functional monomer solution (the total substance is 0.2 mmol), add double distilled water to 20 mL, continue to sonicate for 5 minutes to make it fully dissolved, and then add a magnetic stir bar. The rubber stopper seals the round bottom flask. The round bottom flask was placed on a magnetic stirrer and stirred at a speed of 50r / min. Insert a long needle connected to a nitrogen cylinder through the rubber stopper and insert it to the bottom of the solution. Insert another short needle into the rubber stopper and place it on the top of the round-bottomed flask. Keep nitrogen gas flowing for 30 minutes to completely rem...

Embodiment 2

[0047] Example 2: Synthesis of biotin-labeled polymer nanoparticles to mimic antibody NP2

[0048] Weigh 10% acrylamide-NITIHITDTNNK-amide, 2% N-(3-methacrylamidopropyl)-D-biotinamine, 86% isopropylacrylamide and 2% N,N-methylenebisacrylamide was put into a 50mL round-bottomed flask, 8mL ultrapure water was added, and it was fully dissolved by ultrasound for 8min. Add 10 mg of sodium dodecyl sulfonate to the dissolved functional monomer solution (the amount of the total substance is 0.2 mmol), add double distilled water to 20 mL, continue ultrasonication for 3 minutes to make it fully dissolved, and then add a magnetic stir bar. The rubber stopper seals the round bottom flask. Place the round bottom flask on a magnetic stirrer and stir at a speed of 160r / min. Insert a long needle connected to a nitrogen cylinder through the rubber stopper and insert it to the bottom of the solution. Insert another short needle into the rubber stopper and place it on the top of the round-bottome...

Embodiment 3

[0049] Example 3: Synthesis of biotin-labeled polymer nanoparticles mimic antibody NP3

[0050] Weigh 10% acrylamide-NITIHITDTNNK-amide, 4% N-(3-methacrylamidopropyl)-D-biotinamine, 84% isopropylacrylamide and 2% N,N-methylenebisacrylamide was put into a 50mL round-bottomed flask, 5mL ultrapure water was added, and it was fully dissolved by ultrasonic for 5min. Add 10 mg of sodium dodecyl sulfonate to the dissolved functional monomer solution (the total substance is 0.2 mmol), add double distilled water to 20 mL, continue to sonicate for 5 minutes to make it fully dissolved, and then add a magnetic stir bar. The rubber stopper seals the round bottom flask. Place the round bottom flask on a magnetic stirrer and stir at a speed of 300 r / min. Insert a long needle connected to a nitrogen cylinder through the rubber stopper and insert it to the bottom of the solution. Insert another short needle into the rubber stopper and place it on the top of the round-bottomed flask. Keep nitrog...

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Abstract

The invention discloses a biotin-labeled polymer nanoparticle simulated antibody. The antibody refers to polymer nanoparticles prepared by simulating a molecular recognition mechanism between a Bt Cry1Ac protein and a brush border membrane receptor cadherin Bt-R1 in tobacco hornworms, and reacting a synthetic acrylamide-polypeptide-amide monomer and other coexistence monomers and biotin-labeled monomers. The polypeptide comprises an amino acid sequence NITIHITDTNNK, and the coexistence monomers refer to acrylamide and / or acrylate compounds. The invention further discloses a Bt enzyme-linked immunosorbent assay kit and a detection method established based on the biotin-labeled polymer nanoparticle simulated antibody. The Bt protein detected by the method is diversified in type, wide in application range, low in cost and small in workload. Meanwhile, the advantages of the traditional biological antibody based enzyme linked immunosorbent assay such as excellent specificity, high accuracyand simplicity and convenience in operation are remained, and the defects that the natural antibody is difficult to prepare, high in cost, poor in environmental tolerance, easy to inactivate and harshin preservation and reaction conditions and the like are overcome.

Description

Technical field [0001] The invention belongs to the field of biological analysis, and relates to a biotin-labeled polymer nanoparticle mimic antibody and a preparation method thereof, and a Bt protease-linked immunoassay method based on the biotin-labeled polymer nanoparticle mimic antibody. Background technique [0002] Bt is the abbreviation of Bacillus thuringiensis, and its Chinese name is Bacillus thuringiensis. Bt is a kind of aerobic Gram-positive bacteria, rod-shaped, endophytic spores, is an important insecticidal bacteria. Bt bacteria enter the stable growth phase, forming a diamond-shaped or biconical parasporal crystal protein (Parasporal crystal protein) around the spores, usually called Insecticidal crystal protein (ICP) or delta-endotoxin or thuringikin Bt toxic protein. Bt protein has high specific insecticidal activity against many important agricultural pests, including Lepidoptera, Coleoptera, Diptera, Hymenoptera, Pseudomonas and nematodes, and against human...

Claims

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Application Information

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IPC IPC(8): C08F289/00C08F220/56C08F222/38C08F220/54C08F220/14G01N33/569G01N33/58G01N33/68
CPCC08F289/00G01N33/56911G01N33/581G01N33/68C08F220/56C08F222/385C08F220/54C08F220/14
Inventor 刘名茗胡森华陈雅露禹云莉李雨欣程巧连陈秀华刘凡
Owner HUAZHONG AGRI UNIV
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