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Method for enriching and tracking pathologic modified prions-proteins (PrP)

A prion, pathology technology, applied in chemical instruments and methods, biochemical equipment and methods, disease diagnosis, etc., can solve the problems of limited protein binding ability, high cost, incompatibility, etc.

Inactive Publication Date: 2005-09-07
PRIONTYPE +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method is only suitable for the detection of PrP in body fluids and tissues (excluding plasminogen) Sc , but not suitable, for example, for the detection of PrP in serum Sc
Other disadvantages of this approach are its high cost and the limited protein binding capacity of plasminogen-loaded magnetic particles

Method used

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  • Method for enriching and tracking pathologic modified prions-proteins (PrP)
  • Method for enriching and tracking pathologic modified prions-proteins (PrP)
  • Method for enriching and tracking pathologic modified prions-proteins (PrP)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Isolation of PrP from brain homogenate using different peptides in the form of MTP Sc

[0045] Microtiter plate (MTP) (Nunc-Immuno TM Plate Maxisorp TM Surface, F96 (Nunc, Roskilde, Denmark)), was coated with the peptides listed in Table 1 (see figure 2 ). The coating was performed by co-incubating each well with 100 μl of peptide solution (10 μg / ml in 0.1 M carbonate buffer, pH 9.6) at 4° C. for 16 hours. The liquid was aspirated by vacuum and the wells were washed three times with 300 μl of wash buffer (PBS (10 mM phosphate buffer, 0.15 M NaCl, pH 7.2); 0.05% Tween 20)). Free binding sites were blocked by incubation with 0.5% casein in wash buffer for 1 hour at room temperature.

[0046] After a wash phase (300 μl wash buffer / well), the coated MTP was covered with foil and mixed with 100 μl PrP-containing Sc Samples / well (brain homogenates from BSE positive animals, which were prepared on Platelia  OD > 4.0; a positive finding confirmed by immu...

Embodiment 2

[0050] Example 2: PrP Binding to KLVEF in the MTP Model Sc Elution of

[0051] PrP Sc The binding to the capture molecule is very strong and requires relatively strong conditions to elute PrP from the solid support Sc . Elution conditions were detected using the MTP model.

[0052] As in Example 1, MTP was coated with peptide 2 (KLVEF) and filled with PrP-containing Sc brain homogenate (Platelia  Medium OD > 3.0). After washing 3 times with 300 μl of washing buffer (PBS; 0.05% Tween 20) per well, 100 μl of potent elution buffer (see Table 2) was added per well and incubated at room temperature for 5 minutes. Subsequently, the liquid is removed and the Platelia  BSE Detection Kit (Bio-RadLaboratories, Hercules, USA) measures the PrP eluted in the elution buffer Sc The amount of and PrP left on the MTP Sc amount. Comparing the elution efficiencies (Table 2) shows that only the wash-containing buffer (containing 5% SDS) is suitable for the complete elution of PrP...

Embodiment 3

[0056] Example 3: Covalent binding of peptide KLVFF to EAH Sepharose

[0057] If bound via the amino group, the peptide will be fixed at the N-terminus and side chain (Lys), which disrupts the three-dimensional structure. To this end, the specific binding of the β-sheet binding molecule KLVFF to the solid support can be achieved through the carboxyl group at the C-terminus of the peptide. EAH-Sepharose 4B (Amersham Pharmacia Biotech, Uppsala, Sweden) was used as carrier material.

[0058] To prepare for the binding reaction, the EAH-Sepharose gel was washed with 0.5M NaCl and any excess liquid was completely removed. The ligand was a pentapeptide with the sequence KLVEF, which was dissolved in water at a final concentration of 5 mg / ml and the pH was adjusted to 4.5 with HCl. Resuspend the gel in ligand solution (1 part gel + 2 parts ligand solution) and add EDC (N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide ) to make the final concentration 0.1M. The binding reaction w...

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Abstract

The invention relates to a method for enriching and tracking pathologic modified prions-proteins (PrP) of living organisms.

Description

technical field [0001] The present invention relates to methods for detecting pathologically altered prion proteins in living organisms. Background technique [0002] Transmissible Spongiform Encephalopathies (TSE) are contagious, often fatal, degenerative disorders of the central nervous system. Histopathological changes in the brain and pathologically altered prion protein (PrP) in these diseases Sc ) (which is the naturally occurring cellular prion protein (PrP C ) is related to the accumulation of conformers). Replication of prions that occur during the course of the disease, is PrP Sc and PrP C The result of a direct interaction between the pathological PrP Sc The conformation is imposed on PrP C superior. By working with PrP c For comparison, PrP Sc is characterized by an increased content of beta sheets and high resistance to proteases such as proteinase K. [0003] PrP Sc This resistance is currently used in in vitro diagnostics to detect bovine spongiform...

Claims

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Application Information

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IPC IPC(8): G01N33/53C07K7/02C07K7/06C12Q1/37G01N33/531G01N33/543G01N33/545G01N33/68
CPCG01N2800/2828G01N33/54313G01N2333/968G01N33/6896
Inventor 克劳迪娅·恩格曼卡特加·霍施勒乔尔格·莱曼乔尔格·加伯特乌尔里克·克鲁姆莱
Owner PRIONTYPE
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