Magnetic recognition system

A magnetic and labeling technology, which is applied in the direction of material inspection products, measuring devices, instruments, etc., can solve the problems of deterioration, protein orientation is not optimal, etc.

Inactive Publication Date: 2009-09-23
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 manufacture of fusion proteins

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

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

Embodiment 2

[0199] Example 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 were prepared at various concentrations ranging from 0.0013 μM to 0.133 μM in running buffer, as shown in Tables 2 and 3 below.

[0204] Table 2 - Metallothionein Fusion Protein 75kDa:

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

[0206] μM FP

μg / mL FP

10μg / mL FP (μL)

Running buffer (μL)

0.0013

0.1

20(of 1μg / mL)

180

0.0065

0.5

10

190

0.013

...

Embodiment 3

[0221] Example 3 - Magnetoferritin

[0222] Ferritin usually contains hydrated iron (III) oxide. To generate paramagnetic ferritin, magnetite (Fe 3 O 4 ) to replace these ions. The method used for this experiment involved 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 (pH 8.6) (Sigma A6659)

[0226] ●0.1M sodium acetate buffer (pH4.5)

[0227] Phosphate-buffered saline (PBS), 10 mM phosphate, 140 mM 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] Heat trimethylamine-N-oxide (TMA) to 80 °C (30 min) in an oven to remove Me 3 N, then cooled to room temperature. 114 mg of TMA was added to 15 mL of RO water to produce a 0.07M solution. Iro...

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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 identification labels capable of attaching small amounts of magnetic (or magnetizable) substances to the analyte by means of an analyte using an identification agent. A significant advantage of the labels is their ability to attach small volumes of magnetic material to the analyte, allowing the analyte to be affected by magnetic fields even in confined spaces such as microfluidic systems. The presence of the magnetic species allows for 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 control the molecules involved in the assay method (see eg US2006 / 084089). Typically, the magnetic beads are linked to molecules (eg, antibodies) capable of recognizing and binding the analyte. ...

Claims

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Application Information

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IPC IPC(8): G01N33/544G01N33/58
CPCG01N33/5434G01N2446/80
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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