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Aptamer screening method and immunoassay method using aptamer

A screening method and immunoassay technology, applied in the field of aptamer screening and immunoassay using aptamers, can solve the problems of slowing down the speed of new immunoassays, limiting sensitivity enhancement, low signal-to-noise ratio, etc., to achieve stability and sensitivity Excellent, time-saving, bond-enhancing results

Pending Publication Date: 2022-03-01
SB生物科学株式会社
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Accordingly, low signal-to-noise ratios limit the sensitivity enhancement facilitated by probe amplification
In addition, this method still requires Antibody screening to select the best antibody pair, which can slow down the development of new immunoassays
Finally, the synthesis of detection antibodies that bind to DNA probes results in antibody degradation due to the use of many chemical reagents and also requires complex steps including amino modification of DNA probes

Method used

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  • Aptamer screening method and immunoassay method using aptamer
  • Aptamer screening method and immunoassay method using aptamer
  • Aptamer screening method and immunoassay method using aptamer

Examples

Experimental program
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Effect test

Embodiment 1

[0123] Aptamer screening method

[0124] 1. Single-stranded DNA adapter (ssDNA) library design

[0125] In the aptamer selection method, in the first round, a ssDNA oligonucleotide library comprising 40 nucleotide sequences (SEQ ID NO: 2: 5'-ATC CAG AGT GAC GCA GCA-[core Sequence: (NX 40)]-TG GAC ACGGTG GCT TAG T-3').

[0126] All oligonucleotides used in this study were purchased from Integrated DNA Technologies, Inc. (Coralville, Iowa, USA). At this time, after placing the ssDNA library in binding buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 5 mM KCl, 1 mM MgCl 2 ) and denaturing it by heating at 90°C for 5 minutes, it was washed with 0.01% After washing with Tween 20, it was used after cooling on ice for 10 minutes.

[0127] 2. Expression and purification of severe febrile thrombocytopenia syndrome (Server fever with thrombocytopenia syndrome; SFTS) virus nucleoprotein

[0128] The PCR product was obtained by amplifying the nucleotide sequence encoding the protein comp...

Embodiment 2

[0169] Immunoassay using selected aptamers

[0170] 1. Target-specific aptamer screening

[0171] After the inventor commercially purchased Type A influenza virus NP, Type B influenza virus NP, HIV-1p24 protein, Ebola NP and SARS-Cov-2 NP, the method for each An aptamer with binding specificity for a protein. The sequences of the selected aptamers are shown in [Table 2]. The sequences in bold in Table 2 below are the sequences to which the primers bind in the aptamer amplification step, and were made different for each aptamer.

[0172] 【Table 2】

[0173]

[0174] 2. Heterogeneous sandwich immunoassay using screening aptamers

[0175] The inventors investigated the detection efficiency of H-sandwich RPA by using the screened aptamers to detect various concentration ranges of five model target proteins ( Figure 6 ).

[0176] Anti-target antibodies (1 ng / mL) in 0.1 M carbonate buffer (pH 9.6) were immobilized in 96 microwell plates overnight at 4°C. Coated wells were...

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Abstract

Since the aptamer screened by the aptamer screening method of the present invention binds to a site different from the portion where the antibody binds to the target substance, it can be applied to various fields such as sandwich-type biosensors, and since a separate pairing screening process is not required, time is greatly saved. Furthermore, compared with a preparation containing the existing antibody, the aptamer not only has excellent stability and sensitivity, but also can be massively produced in a short time at low cost by a chemical synthesis method, and various modifications can be easily performed to improve the binding force. Furthermore, the immunoassay method using the aptamer screened by the aptamer screening method according to the present invention detects a relative fluorescence signal by selectively amplifying only the aptamer bound to the target substance in the heterogeneous sandwich structure, thereby having the advantage of being capable of sensitively and quickly detecting the target substance.

Description

technical field [0001] The present invention relates to an aptamer screening method and an immunoassay method using the aptamer, more specifically, the immunoassay method relates to a heterogeneous sandwich immunoassay method using nucleic acid amplification technology. Background technique [0002] Aptamers are nucleic acid molecules characterized by exhibiting a high degree of specific binding affinity for molecules through interactions other than classical Watson-Crick base pairing. Aptamers have the property of being able to specifically bind to screening targets, so not only can the presence or absence of targets be determined through aptamer binding, but also the function of targets can be inhibited or activated. Aptamers capable of specifically binding to proteins, including growth factors, transcription factors, enzymes, immunoglobulins, and receptors, have been generated from random sequence oligonucleotide libraries (Library) through an in vitro screening process. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12N15/115C12Q1/6806G01N33/543
CPCC12N15/1048C12N15/115C12Q1/6806G01N33/543C12N2310/16C12Q2541/101C12Q2525/205C12Q2531/113C12N2320/13C12Q1/68C12N15/1086C12Q1/6844
Inventor 金珉坤姜珠映
Owner SB生物科学株式会社