Binding of pathological forms of prion proteins

A protein and form technology, applied in disease diagnosis, peptide preparation method, peptide source, etc.

Inactive Publication Date: 2005-07-20
MICROSENS BIOPHAGE LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0016] It has been reported that the PrP c In combination, throug

Method used

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  • Binding of pathological forms of prion proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1: Application of biotinylated pentosan polysulfate and subsequent affinity capture from inferior Normal Prion

[0071] introduce

[0072] Biotin was conjugated to pentosan polysulfate using standard chemistry. This biotinylated pentosan polysulfate was bound to the inferior prion protein in the brain homogenate, after binding, the pentosan polysulfate / prion was captured using streptavidin-derivatized superparamagnetic beads Complex. The captured inferior prions were then eluted from these beads and treated with an immune-based Bio-RadPlatelia TM Measured with BSE detection kit; this kit cannot distinguish between normal and inferior prion protein, and can give signals of both proteins. A study was carried out on a set of two BSE-infected and two non-infected bovine brains to illustrate that under the specific conditions described, pentosan polysulfate-specific capture of prion proteins in brain homogenates can be specific .

[0073] method

[007...

Embodiment 2

[0101] Embodiment 2: Use immobilized biotinylated pentosan polysulfate to separate normal prion and bad prion protein

[0102] introduce

[0103] Biotin was conjugated to pentosan polysulfate using standard chemistry. Streptavidin-derivatized superparamagnetic beads were coated with this biotinylated pentosan polysulfate. The coated beads were then used to specifically capture the prion protein in the brain homogenate. Subsequently, the captured inferior prion protein was eluted from these beads and immuno-based Bio-Rad Platelia TM It is detected by the BSE detection kit; this kit cannot distinguish between normal and inferior prion proteins, and can give signals of both proteins. A group of 3 BSE-infected and 3 non-infected bovine brains was studied and used to demonstrate that under the special conditions described, pentosan polysulfate can specifically capture the prion protein in these brain homogenates.

[0104] method

[0105] Preparation of magnetic beads ...

Embodiment 3

[0133] Example 3. Biotinylation of PPS

[0134] Rationale for this method

[0135] Substitution of approximately one-tenth of the sugar residues in the xylan backbone of pentosan sulfate with uronic acid residues, followed by substitution of methyl esters at some of the carboxyl groups, thus present in this molecule A large number of free carboxyl groups can be derivatized with carbodiimide to form active esters. These can then be substituted with amino groups to create an amide bond. In this particular case, EDC and NHS were chosen to form the active ester and biotin hydrazide was chosen as the amino group. Two reactions were performed, in the first reaction, biotin hydrazide was initially present without any NHS addition, and in the second reaction, NHS / EDC was reacted with PS and biotin hydrazide simultaneously.

[0136] Material

[0137] Pentosan sulfate (Norton Healthcare) was a gift from Stephen Dealer.

[0138] Biotin Hydrazide 100mg, Pierce #21339 mw 258.33 ...

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Abstract

Infective aggregating forms of proteins such as PrP, amyloid, and tau are bound selectively in the presence of the normal form protein using a polyionic binding agent such as dextran sulphate or pentosan (anionic), or polyamine compounds such as pDADMAC (cationic) under selective binding conditions including the use of n-lauroylsarcosine at mildly alkaline pH, and may then be assayed.

Description

field of invention [0001] The present invention relates to the use of anti-protease binding agents, typically polyionic substances, such as polyanionic substances or polycationic substances, polyanionic substances including pentosan polysulfate, dextran sulfate or other polyanionic polyglycosides, polyanionic substances Cationic species include hexadimethylamine bromide, polyamidoamine dendrimers, or poly(dipropylenedimethylammonium chloride), optionally under selective conditions to capture pathological or dysmorphic forms of the prion protein PrP c , and proteins that resemble aggregated abnormal forms of proteins that are not aggregated in their normal form. Background of the invention [0002] Prion diseases, also known as transmissible spongiform encephalopathy or TSEs, have only recently been recognized. Bovine spongiform encephalopathy (BSE) was first reported in 1985. The first case of variant Creutzfeldt Jakob disease (vCJD) was reported in 1996. VCJD is a fatal ...

Claims

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Application Information

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IPC IPC(8): G01N33/68C07K1/22C07K14/47G01N33/53
CPCG01N2800/2828G01N2333/968G01N33/68G01N33/54326G01N33/6896G01N33/6803
Inventor A·R·莱恩C·J·斯坦利S·M·威尔森
Owner MICROSENS BIOPHAGE LTD
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