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Conformationally abnormal forms of tau proteins and specific antibodies thereto

a technology of tau protein and specific antibodies, applied in the field of alzheimer's disease, can solve problems such as cell degeneration and retraction of neuronal processes

Inactive Publication Date: 2009-05-14
NOVAK MICHAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0012]“Antibody” as used herein is meant to include intact molecules and fragments thereof as well as synthetic and biological derivatives thereof, such as for example Fab, F(ab′)2 an FV fragments-free or expressed e.g. on the surface of filamentous phage on pIII or pVIII or other surface proteins, or on the surface of bacteria, which are capable of binding an antigen. Fab, F(ab′)2 and FV fragments lack the FC fragments of intact antibody, clear more rapidly from the circulation and may have less non-specific tissue binding of antibody. Furthermore FV antibody (often called as minibody) can be easily engineered to carry on its C-terminus specific tracer and used for early intravital presymtomatic diagnosis of AD, since stage I, II and III of AD that is recognized by the antibodies according to the present invention is not associated with intellectual decline.

Problems solved by technology

Moreover, tauons cause upon microinjection to differentiated human neurons significant displacement of endogenous tau from microtubule bound tau fraction, retraction of neuronal processes and degeneration of the cells.

Method used

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  • Conformationally abnormal forms of tau proteins and specific antibodies thereto
  • Conformationally abnormal forms of tau proteins and specific antibodies thereto
  • Conformationally abnormal forms of tau proteins and specific antibodies thereto

Examples

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example 1

Preparation of the Monoclonal Antibodies of DC 11 Family Specific for Tauons

Preparation of Soluble and Insoluble Tauons as Antigens for Immunization (FIG. 1)

[0046]For isolation of tauons from human AD brains, a new approach was developed which is partially based on the methods described by Kopke et al., (1993) and Greenberg and Davies (1990). Human brains, showing changes characteristic for I.-III. Braak's stage of AD with short post mortem delay (PMD) were selected. Blocks of the temporal lobe including the enthorinal and transenthorinal regions, amygdala and hippocampal region were selected. The tissue was dissected and immediately immersed into minimal essential medium (Gibco). Tissue was finely minced and pushed through a 150 μm mesh wire screen. At this stage the brain sample was divided into two aliquots: sample A and sample B.

[0047]Sample A was further processed in 20 mM TRIS, pH 8, 0.32 M sucrose, 10 mM mercaptoethanol, 5 mM EGTA, 1 mM EDTA, 5 mM MgSO4, 5 mM benzamidine, 10 ...

example 2

Quantitative Determination of the Abnormally Truncated Tau Proteins (Tauons) Using Family of DC-11 Monoclonal Antibodies

[0055]Tauons were isolated as described above. The combination of monoclonal antibody DC 30 (recognizing both normal and pathological tau) and family of DC-11 monoclonal antibodies (specific for abnormally truncated tau) allows quantification of tauons in the tested samples prepared from AD-brains. Antibodies were purified from serum-free medium by protein A column chromatography. The wells of high-binding microtiter plate (Nunc) were coated with mixture of DC-11 monoclonal antibodies at a concentration of 10 μg / ml (50 μl / well) in PBS overnight at 4° C. Non-specific binding in wells was saturated by adding 200 μl of 1% nonfat dried milk in phosphate buffered saline (PBS) for 60 min at room temperature. The plates were washed 3 times with PBS-0.05% Tween 20 (v / v). The serially diluted standards containing recombinant tauons at concentrations ranging between 100-1000...

example 3

Detection of Tauons by Western Blotting Using Monoclonal Antibody DC-11

[0057]Purified recombinant full-length human tau and abnormally truncated tau proteins—tauons, were loaded on 5-20% gradient SDS-polyacrylamide gels and run under denaturated conditions according to Laemmli (1970). After SDS-PAGE, the transfer on polyvinyl difluoride membrane (Milipore) was carried out in 10 mM CAPS buffer pH 12 for 1 hr at 350 mA with cooling. After blotting, the membrane was washed in PBS and blocked with 1% dried nonfat milk in PBS for 1 hr at room temperature. Transferred proteins were incubated overnight at 4° C. with monoclonal antibody DC-11. After washing with PBS-0.05% Tween 20 (v / v), rat anti-mouse immunoglobulin labeled with horse radish peroxidase was used at a dilution 1 / 1000 and incubated 1 hr at room temperature. The membrane was then washed four times in PBS-Tween 20, developed with substrate solution (12 mg 4-chloro-1-naphtol, 4 ml methanol, 16 ml PB, 0.03% v / v H2O2) and the reac...

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Abstract

The invention relates to antibodies with a specificity to an abnormally truncated form of tau protein, which is conformationally different from normal tau, and does not bind to normal tau protein, conformationally different tau proteins (“tauons”) and diagnostic and therapeutical aspects in relation to Alzheimer's disease and related tauopathies.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of U.S. application Ser. No. 10 / 470,928, filed on Nov. 25, 2003, which is a U.S. national phase application under 35 U.S.C. § 371 of PCT Application No. PCT / EP02 / 00897 filed 29 Jan. 2002, which claims priority to Austrian Application No. A 175 / 2001 filed 2 Feb. 2001. The entire text of each of the above-referenced disclosures is specifically incorporated by reference herein without disclaimer.BACKGROUND[0002]The invention relates to Alzheimer's disease and to other tauopathies. Alzheimer's disease (AD) is the most common chronic neurodegenerative disorder which is characterized clinically by a progressive and irreversible loss of cognitive and behavioral function. The disease can persevere for over 10 years, advancing from mild symptoms to extremely severe manifestations. AD afflicts approximately 10% of the population over the age of 65 and 20% of the population over the age of 80. As a result of growin...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/53C07K16/18C12N5/06A61K39/395A61P25/14A61P25/16A61P25/28A61P43/00C07K14/47C12N5/10C12N5/12C12N15/09G01N33/563
CPCA61K2039/505C07K16/18C07K14/4711A61P25/14A61P25/16A61P25/28A61P43/00
Inventor NOVAK, MICHAL
Owner NOVAK MICHAL
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