Magnetic recognition system

A magnetic and ferromagnetic technology, applied in the field of magnetic recognition labels, can solve the problems of protein orientation not being optimal, deterioration, etc.

Inactive Publication Date: 2014-04-16
ITI SCOTLAND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because when a protein is coupled to a magnetic bead or magnetic particle surface, the required orientation of the protein may not be optimal (see image 3 ), thus exacerbating the problem

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0156] Example 1 - Design and Manufacturing of Fusion Proteins

[0157] To illustrate the present invention, fusion proteins were designed using a commercially available murine anti-fibronectin antibody. Fusion proteins of anti-fibronectin scFv linked genetically to MT2 or ferritin via short flexible linkers were made. This example details the construction, characterization and isolation of the fusion protein.

[0158] The design of the anti-fibronectin ferritin or MT2 fusion protein is based on combining the V from the mouse anti-fibronectin antibody H and V L Genes are cloned into vectors. Both genes are linked by a short, flexible linker consisting of small uncharged amino acids. immediately after V L At the 3' end of the gene, another short flexible linker was ligated to either the ferritin gene or the MT2 gene. Both fusion proteins have hexahistidine regions for purification on nickel columns. Fusion protein translation was terminated at the inserted stop codon at ...

Embodiment 2

[0199] Embodiment 2——SPR analysis

[0200] Anti-fibronectin ferritin and MT2 fusion protein inclusion body preparations were used in surface plasmon resonance (SPR) analysis using a SensiQ instrument (ICX Nomadics).

[0201] For these experiments, fibronectin peptides were coupled to the carboxyl chip surface. The fusion protein preparation was then flowed through the chip and binding (K a ) and dissociation (K d )dynamics.

[0202] Fusion protein analysis samples

[0203] Six samples of each fusion protein at different concentrations of 0.0013 μM to 0.133 μM in running buffer were prepared, as shown in Table 2 and Table 3 below.

[0204] Table 2 - Metallothionein fusion proteins 75kDa:

[0205] 400 μL 10 μg / mL (0.133 μM) was obtained from 40 μL 100 μg / mL 75kDa / 360 μL running buffer, then

[0206] μM FP

μg / mL FP

10μg / mLFP (μL)

Running buffer (μL)

0.0013

0.1

20(of 1μg / mL)

180

0.0065

0.5

10

190

...

Embodiment 3

[0221] Example 3——Magnetized ferritin

[0222] Ferritin usually contains hydrated iron(III) oxide. To generate paramagnetic ferritin, magnetite (Fe 3 o 4 ) to replace these ions. The method used in this experiment involves adding iron ions to apoferritin and oxidizing these ions under controlled conditions.

[0223] Material

[0224] ●Reverse osmosis water (RO water)

[0225] 50mM N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPSO) buffer (pH8.6) (Sigma A6659)

[0226] ●0.1M sodium acetate buffer (pH 4.5)

[0227] ●Phosphate-buffered saline (PBS), 10mM phosphate, 140mM NaCl (pH 7.4)

[0228] ●Trimethylamine-N-oxide (TMA) (Sigma 317594)

[0229] ●0.1M ammonium ferrous (II) sulfate

[0230] ●Horse spleen apoferritin (Sigma A3641)

[0231] method

[0232] Trimethylamine-N-oxide (TMA) was heated to 80 °C (30 min) in an oven to remove Me 3 N, then cooled to room temperature. 114mg TMA was added to 15mL RO water to make a 0.07M solution. Iron ...

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Abstract

Provided is a label for an analyte, which label is attached to a magnetic or magnetisable substance, the label comprising: (a) a recognition moiety for attaching the label to the analyte; and (b) a moiety for binding or encapsulating the magnetic or magnetisable substance; wherein the moiety for binding or encapsulating the magnetic or magnetisable substance comprises a metal-binding protein, polypeptide, or peptide.

Description

technical field [0001] The present invention relates to magnetic recognition labels capable of attaching a small amount of magnetic (or magnetizable) substance to an analyte via a recognition agent for the analyte. A significant advantage of such labels is that they enable the attachment of very small volumes of magnetic substances to analytes, allowing the analytes to be affected by magnetic fields even in confined spaces such as microfluidic systems. The presence of the magnetic species allows more complex spatial manipulation of the analyte, which is particularly beneficial in microfluidic systems. The present invention also relates to products, methods and applications related to said markers. Background technique [0002] It is well known that magnetic beads can be used to manipulate molecules involved in assay methods (see eg US2006 / 084089). Typically, the beads are associated with molecules (such as antibodies) that recognize and bind the analyte. The magnetic prop...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/544G01N33/58
CPCG01N2446/80G01N33/5434
Inventor 普拉把尤特·德哈尔大卫·普里查德克莱尔·吉克
Owner ITI SCOTLAND
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