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Detection of conformationally altered proteins and prions

a technology applied in the field of conformationally altered proteins and detection methods, can solve the problems of difficult diagnosis of prions, complicating detection, late detection, etc., and achieves the effects of convenient prophylactic or remedial treatment, rapid diagnosis, and reliable, affordable and safe methods

Inactive Publication Date: 2008-07-17
ADLYFE INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0020]The invention provides reliable, affordable, and safe methods for the detection of conformationally altered proteins and prions associated with a variety of diseases. Methods of the invention can be applied to obtain rapid diagnoses and facilitate prophylactic or remedial treatments. Significantly, the methods of the invention use small amounts of sample and are therefore less invasive and more readily applied than known diagnostic techniques. Further, methods of the invention can be used to analyze samples from a living subject and are not limited to samples obtained post mortem; and may be utilized in a manner that ensures that infectious material is not propagated during testing.
[0022]Upon interaction of a sample containing conformationally altered protein or prions with a conformational probe as defined hereinafter, the probe undergoes a conformational change and adopts the conformation of, and aggregates with, the conformationally altered protein (which may be soluble or insoluble) or prions. The resulting aggregates which exhibit ββ sheet formation, may be readily detected using standard analytical techniques. As a result, the invention facilitates rapid and cost-effective analysis of small sample sizes and is widely applicable to tissues and body fluids from a variety of sources including, but not limited to, the brain.

Problems solved by technology

Prion diseases are transmissible and insidious.
Diseases caused by prions are hard to diagnose: the disease may be latent or subclinical (abnormal prions are detectable but symptoms are not).
Moreover, normal homologues of a prion-associated protein exist in the brains of uninfected organisms, further complicating detection.
Brain-tissue-based assays can lead to late detection and require slaughtering the animal to be tested.
Although results are obtained in six to seven hours, the test does not account for the six-month lag time between PrPS accumulation in the brain and the onset of clinical symptoms.
Tonsillar biopsy sampling, and blood and cerebrospinal sampling, while accurate, can require surgical intervention and take weeks to obtain results.
Electrospray ionization mass spectrometry (ESI-MS), nuclear magnetic resonance NMR, circular dichroism (CD) and other non-amplified structural techniques require large amounts of sample and expensive equipment that is typically located a substantial distance from the sample source.
Detection methods for conformationally altered proteins associated with the aforementioned disorders such as Alzheimer's disease and CAA are also inadequate in that, like the previously mentioned prion detection techniques, they often require postmortem tissue sampling,

Method used

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  • Detection of conformationally altered proteins and prions
  • Detection of conformationally altered proteins and prions
  • Detection of conformationally altered proteins and prions

Examples

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example 1

Materials and Methods

[0125]A sample may be obtained for testing and diagnosis through use of the instant invention as follows. A sample may be prepared from tissue (e.g. a portion of ground meat, or an amount of tissue obtained by a biopsy procedure) by homogenization in a glass homogenizer or by mortar and pestle in the presence of liquid nitrogen. The amount of material should be between about 1 mg and 1 gm, preferably between 10 mg and 250 mg, ideally between 20 and 100 mg. The material to be sampled may be suspended in a suitable solvent, preferably phosphate-buffered saline at a pH between 7.0 and 7.8. The addition of Rnase inhibitors is preferred. The solvent may contain a detergent (e.g., Triton X-100, SDS, or sarkosyl). Homogenization is performed for a number of excursions of the homogenizer, preferably between 10 and 25 strokes; ideally between 15 and 20 strokes. The suspended sample is preferably centrifuged at between 100 and 1,000 g for 5-10 minutes and the supernatant ...

example 2

[0130]Poly-lysine was used as a model peptide. Experiments were performed using model systems to illustrate the conformational changes involved in the transition from a predominately alpha-helix to a beta-rich form. The model system chosen used non-neurotoxic polyamino acid polylysine. The polyamino acid was chosen because of availability and safety; and normally evidences random coil conformation at pH values between 5 and 9.

[0131]FIG. 3 depicts a CD graph of an experiment in which poly-L-lysine 20 micro Molar (μμM) 52,000 molecular weight (MW) was used as a peptide model.

[0132]As also illustrated in FIG. 3:

Sample 24, which was maintained at pH 7, 25° C., exhibited a minimum at approximately 205 nanometers (nm), indicating a random coil structure;

Sample 26 which was maintained at pH 11 (near the isoelectric point), at 50° C., resulted in a minimum at approximately 216 nanometers (nm) indicating a β-sheet structure (see FIG. 11 for exemplary CD spectra of protein conformations);

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example 3

[0133]FIG. 4 illustrates general CD results of experiments that were conducted: (1) using poly-L-lysine; and (2) at varying temperatures and pH, to observe the effect of random coil to beta-sheet conformational changes under varying environmental conditions. The results indicate that both temperature and pH play an important role in the transition. The results also indicate that the addition of a relatively small amount of β-sheet peptide to a random coil sample can result in a shift towards a β-rich conformation and that such changes can be accelerated depending on the temperature and pH environment of the samples.

[0134]More specifically, FIG. 4 illustrates an absorbance graph generated using a poly-L-lysine of 52,000 molecular weight (MW) at 70 micromolar (μM) as a peptide probe in accordance with the experimental technique described in Examples 1-3. FIG. 4 illustrates that:

Sample 32 (pH 11, 25° C.) evidenced a plateau at approximately 0.12, indicating a pre-dominantly α-helical s...

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Abstract

The invention provides methods and kits for detecting conformationally altered proteins and prions in a sample. In one embodiment, the conformationally altered proteins and prions are associated with amyloidogenic diseases.

Description

RELATED APPLICATIONS[0001]This application is a continuation of U.S. patent application Ser. No. 10 / 728,246 filed Dec. 4, 2003, which is a continuation-in-part of U.S. patent application Ser. No. 10 / 161,061, filed May 30, 2002, which claims priority from U.S. Provisional Patent Application Ser. No. 60 / 295,456, filed May 31, 2001. This application is also a continuation-in-part of U.S. application Ser. No. 10 / 494,906 filed Sep. 7, 2004, which is a National Stage of Application Serial No. PCT / US02 / 17212 filed May 30, 2002, which claims priority from U.S. Provisional Application Ser. No. 60 / 295,456 filed May 31, 2001. The entire contents of the aforementioned applications are incorporated herein by reference.FIELD OF THE INVENTION[0002]The invention provides methods and kits for detecting conformationally altered proteins and prions in a sample.[0003]In one embodiment, the conformationally altered proteins and prions are associated with amyloidogenic diseases.BACKGROUND OF THE INVENTIO...

Claims

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Application Information

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IPC IPC(8): G01N33/53C07K16/00C07K14/47G01NG01N33/483G01N33/68
CPCC07K14/4711G01N2800/2828G01N33/6896G01N33/542G01N33/582
Inventor ORSER, CINDYGROSSET, ANNEDAVIDSON, EUGENE A.
Owner ADLYFE INC
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