Assay for disease related conformation of a protein and isolating same

a protein and conformation technology, applied in the field of disease related conformation of a protein and isolating same, can solve the problems of not being able to reliably compare glycosylation and peptide mapping patterns in diagnostics, which are still debated, and achieve the effects of enhancing sensitivity, quick and accurate determination of protein presence, and enhancing signal

Inactive Publication Date: 2001-08-16
RGT UNIV OF CALIFORNIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0020] An advantage of the present invention is that the immunoassay can quickly and accurately determine the presence of proteins in the disease related conformation (e.g., PrP.sup.Sc, .beta.A4 and transthyretin) even though the antibody used in the assay does not bind or has a very low degree of binding affinity for the protein in the disease related conformation and the disease related conformation is present in a lower concentration than the non-disease conformation.
[0021] A feature of the invention is that the signal obtained can be enhanced by the use of transgenic animals, e.g., mice which are used to detect the presence of a protein in a sample.
[0022] Another feature is that time-resolved, dissociation-enhanced fluorescence or a dual wavelength, laser driven fluorometer can be used to enhance sensitivity.
[0023] Another advantage is that the assay can detect levels of the disease causing conformation of a protein at a concentration of 1.times.10.sup.3 particles / ml or less.
[0024] A specific object is to provide a diagnostic assay for determining the presence of infectious prion protein in variable sample materials obtained or derived from human, primate, monkey, pig, bovine, sheep, goat, deer, elk, cat, dog, mouse, chicken, and turkey tissues and / or body fluids.
[0025] Another specific object is to provide a diagnostic assay for determining the presence of .beta.A4 protein in variable sample materials obtained or derived from human, primate, monkey, pig, bovine, sheep, goat, deer, elk, cat, dog, mouse, chicken, and turkey tissues and / or body fluids.

Problems solved by technology

However, the reliability of both glycosylation and peptide mapping patterns in diagnostics of multiple prion strains is currently still debated [Collings, Hill et al.
Unfortunately, such does not appear to be possible with current PrP.sup.Sc assays--it is estimated that the current sensitivity limit of proteinase-K and Western blot-based PrP.sup.Sc detection is in a range of 1 .mu.g / ml which corresponds to 10.sup.4-10.sup.5 prion infectious units.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0129] Detection of PrP.sup.Sc in Hamster Brain

[0130] To determine the levels of PrP.sup.Sc in affected hamsters, a prion infected hamster and a normal hamster were each sacrificed and their brains removed. A 10% (w / v) homogenate of each of the brains was prepared by dispersing the brain tissue in PBS. The brain homogenate was then subjected to a low speed centrifugation of 500.times.g for 15 minutes to separate the suspended proteins from unwanted cellular debris. The total protein concentration of the supernatant (S1) was measured using a BCA Protein Assay (Pierce) and the concentration of each brain homogenate was adjusted with PBS to 3.5 mg / ml. A portion of the homogenate was saved to serve as a control of total brain proteins.

[0131] The metalloendopeptidase dispase (Worthington) was added to the remainder of each sample at an enzyme to protein ratio of 1:35. The homogenates were digested with 100 .mu.g / ml dispase for 60 min at 37.degree. C. in the presence of either 0.15% Zwitt...

example 2

Detection of PrP.sup.Sc in Mouse Brain

[0139] To determine the levels of PrP.sup.Sc in affected mice, a prion infected mouse and a normal mouse are each sacrificed and their brains removed. A 10% (w / v) brain homogenate from normal and prion infected mice is prepared in PBS. After a low speed centrifugation at 500.times.g for 15 min, the total protein in the supernatant (S1) is measured using spectrophotometric assays, and the concentration is adjusted to 2.5 mg / ml with PBS.

[0140] The samples are digested with 500 U / ml Leucolysin for 45 min at 37.degree. C. in the presence of 2% Sarkosyl. The digestion is stopped by the addition of 50 mM EDTA. An aliquot of the proteins obtained in S1 both before and after the leucolysin digestion are electrophoresed at 4.degree. C. on an 8% polyacrylamide slab gel as described in the Laemmli reference but in the absence of SDS and 2-mercaptoethanol. This allows the nondenatured proteins to migrate through the polyacrylamide while preserving the nativ...

example 3

[0145] Detection of PrP.sup.Sc in Cow Brain

[0146] A 10% (w / v) brain homogenate from normal and prion infected cows is resuspended in 1 L of 25 mM Tris-HCl, pH 8.0, 5 mM EDTA (buffer A). This is centrifuged at 10,000.times.g for 20 min, and the supernatant containing soluble periplasmic proteins is discarded. The pellet is resuspended in 1 L of buffer A, passed through a cell disrupter twice (Microfluidics International, model MF110), and centrifuged at 30,000.times.g for 1 h, after which the supernatant is discarded and the pellet is washed once in buffer A and centrifuged again at 30,000.times.g for 1 hour. At this stage the pellet could be stored at -20.degree. C. prior to hydrolysis.

[0147] The .beta.-Lytic Metalloendopeptidase digestion is done at an enzyme to protein ratio of 1:40. The protein is digested with 100 .mu.g / ml .beta.-Lytic Metalloendopeptidase (Sigma) for 75 min at 40.degree. C. in a buffered pH 8.0 solution containing 0.2% Sarkosyl. The digestion is stopped by the ...

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Abstract

An assay method is disclosed which isolates and detects the presence of a disease related conformation of a protein (e.g., PrPSc) present in a sample also containing the non-disease related conformation of the protein (e.g., PrPC). The sample is treated (e.g., contacted with protease) in a manner which hydrolyzes the disease related conformation and not the non-disease related conformation. The treated sample is contacted with a binding partner (e.g., a labeled antibody which binds PrPSc) and the occurrence of binding provides and indication that PrPSc is present. Alternatively the PrPSc of the treated sample is denatured (e.g., contacted with guanadine) or unfolded. The unfolded PrPSC is contacted with a binding partner and the occurrence of binding indicates the presence of PrPSc in the sample. In another embodiment, PrPSc and PrPC are reacted with a labeled antibody that binds both conformations and a conformation that binds only the disease related conformation, and the presence of the disease related conformation is determined by comparing the two.

Description

CROSS-REFERENCE[0001] This application is a continuation of U.S. application Ser. No. 09 / 169,574, filed Oct. 9, 1998, which is incorporated herein by reference in its entirety and to which application we claim priority under 35 USC .sctn.120.[0003] This invention relates to the field of bioassays and more particularly to an assay which makes it possible to isolate and detect a disease conformation of a protein present in a native sample also containing a non-disease conformation of the protein.[0004] Prions are infectious pathogens that cause invariably fatal prion diseases (spongiform encephalopathies) of the central nervous system in humans and animals. Prions differ significantly from bacteria, viruses and viroids. The dominating hypothesis is that no nucleic acid is necessary to allow for the infectivity of a prion protein to proceed.[0005] A major step in the study of prions and the diseases they cause was the discovery and purification of a protein designated prion protein [Bo...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61L2/00C07K14/47G01N33/53C12Q1/37G01N33/569G01N33/68
CPCA61L2/0088C07K14/47G01N33/56983G01N33/6896G01N2333/964G01N2800/2828G01N33/68
Inventor PRUSINER, STANLEY B.SAFAR, JIRI G.
Owner RGT UNIV OF CALIFORNIA
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