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Detection kit and detection method using antibody to modify immuno-PCR reaction

A detection kit and antibody modification technology, which is applied in the field of immunological detection, can solve the problems of varying degrees of improvement, difficulty in breaking through 1000 times of sensitivity, and decline in antibody specificity and affinity, achieving a wide dynamic range, wide detection sensitivity, and simplified The effectiveness of the detection process

Active Publication Date: 2016-04-20
程永升 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This coupling technology not only requires the antibody to be coupled in an inactive buffer, but also the coupling site is full of randomness, often due to the shielding of the coupled DNA near the antigen-antibody binding region, resulting in a lack of antibody specificity and drop in affinity
This is also the reason why the sensitivity of Immuno-PCR has been difficult to break through 1000 times in examples over the years and the degree of improvement varies.

Method used

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  • Detection kit and detection method using antibody to modify immuno-PCR reaction
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  • Detection kit and detection method using antibody to modify immuno-PCR reaction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1: Biotin Modification of Insulin Antibody 3A6

[0069] The Insulin monoclonal antibody 3A6 (ab1965) from Abcam needs to be modified with biotin before it can be specifically coated into a streptavidin PCR96well microplate (Pierce, CatNo.15500) for Immuno-PCR detection . The antibody was modified using the SiteClick series labeling kit (S10467) and DIBO-labeled biotin (C10412) from LifeTechnologies.

[0070] (1) Purification and modification of antibodies are carried out first.

[0071] The first round of purification: first add 0.5mg / ml 250μL 3A6 antibody solution to the antibody concentrator (an ultrafiltration centrifuge tube with unknown molecular weight cut-off) attached to the kit, which can concentrate the antibody and replace the buffer; Then add 500 μL of antibody replacement buffer (bufferA) to the concentrator and centrifuge at 6000×g at 4°C for 5 minutes. After the centrifugation is completed, use a pipette to suck up the remaining solution to wash...

Embodiment 2

[0076] Example 2: ssDNA modification of Insulin antibody

[0077] (1) Synthesis of ssDNA with DIBO marker (dibenzocyclooctyne):

[0078] a. Generate a random sequence with a length of 100bp through the DNA sequence random synthesis website (http: / / www.facμLty.ucr.edu / ~mmaduro / random.htm)

[0079] 5'-ATGGGGCTGGATAAAACTGCCCTGGTGACCGCCATCAACAACCCGAATACGTGGCATTTCAGGAGGCGGCCGGAGGGGGATGTTTTCTACTATTCGAGG-3' (SEQ ID No. 1).

[0080] Through the blastn search program (http: / / www.ncbi.nlm.nih.gov / BLAST / Blast.cgi), it was confirmed that the sequence does not have any known sequences homologous to it, so as to ensure that the subsequent PCR detection signal is specific information from antibody binding .

[0081] After determining the sequence of the ssDNA, the 5' terminal DIBO marked ssDNA (SEQ ID No.1) and two PCR primers for amplifying the fragment were synthesized by IDT (www.idtdna.com):

[0082] 5'-ATGGGGCTGGATAAAACTGC-3' (SEQ ID No.2) and

[0083] 5'-CCTCGAATAGTAGAAAACATCCCC-3'...

Embodiment 3

[0091] Embodiment 3: the making of Insulin immune PCR standard curve

[0092] (1), Preparation of Immuno-PCR microwell plate:

[0093] The DIBO-labeled biotin-modified Insulin antibody 3A6 solution obtained in Example 1 was coated on a streptavidin PCR microwell plate (Pierce Company, CatNo. 15500): after overnight incubation for 12 hours at 4°C, Antibody-coated microwell plates were washed three times with PBST buffer (1×PBS, 0.1% Tween20), and then incubated with PBST-BSA buffer (1×PBS, 0.1% Tween20, 5% BSA) at 4°C The plate was left overnight, and then the microplate was washed with PBST buffer three times for use to obtain an Immuno-PCR microplate.

[0094] (2), InsuLin Immuno-PCR standard curve formulation:

[0095] a. First prepare the human source Insulin standard substance (mother solution, 10mg / mL, Sigma Company), the concentration of the standard substance is from 33ng / ml to 3.3x10E-5ng / ml10-fold equal dilution, and there are 7 different concentrations in total; th...

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Abstract

The present invention belongs to the field of immunological detection methods. A purpose of the present invention is to provide a detection kit and a detection method using antibody to modify an immuno-PCR reaction. The detection method comprises synthesis of DIBO-labeled ssDNA, biotin modification of Insulin monoclonal antibody, preparation of ssDNA-modified immuno-PCR micro-pore plate of Insulin monoclonal antibody, establishment of Immuno-PCR standard curve of Insulin, and other steps. According to the present invention, with the application of the antibody modification technology in the immuno-PCR reaction, the detection on the ultra low concentration biotin marker can be performed, the sensitivity of the detection method of the present invention can exceed 1000 times the sensitivity of the conventional ELISA method, and the extremely-low concentration biomarker can be detected.

Description

technical field [0001] The invention belongs to the field of immunological detection methods, and in particular relates to a detection kit and a detection method utilizing antibody modification for immuno-PCR reaction. Background technique [0002] ELISA detection technology has been more and more widely used in clinical and research markets since its invention (Lequin, R.M. (2005). Enzyme Immunoassay (EIA) / Enzyme-Linked Immunosorbent Assay (ELISA). Clinical Chemistry 51, 2415–2418.) . With increasingly sophisticated detection requirements, end users have higher and higher requirements for in vitro diagnostic sensitivity, dynamic range, and multi-channel detection capabilities. Antibody-based detection technologies are becoming more and more diverse, and many new technologies have emerged. According to different technical routes and requirements, they are divided into the following categories: [0003] 1. Increased sensitivity. After years of technical upgrading and matu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/577
Inventor 程永升吴雪萍
Owner 程永升
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