Chemistry: analytical and immunological testing – Peptide – protein or amino acid
Reexamination Certificate
1994-08-26
2002-04-02
Ludlow, Jan (Department: 1743)
Chemistry: analytical and immunological testing
Peptide, protein or amino acid
C436S164000, C436S177000, C436S811000
Reexamination Certificate
active
06365414
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is directed to various assays for detection of A&bgr; amyloid, screening candidate agents for their ability to prevent or reverse the formation of A&bgr; amyloid in vitro, as well as kits which are used in the present methods.
2. Related Art
Aggregation of A&bgr; in the brain is believed to contribute to dementia, characteristic of Alzheimer's disease (AD) and Down's syndrome, a condition characterized by premature AD. A&bgr;, a 4.3-kDa peptide, is the principal constituent of the cerebral amyloid deposits, a pathological hallmark of Alzheimer's disease (AD) (Masters et al.,
Proc. Natl. Acad. Sci. USA
82:4245-4249 (1985); Glenner & Wong, Biochem.
Biophys. Rev. Commun.
120:885-890 (1984)). A&bgr; is derived from the much larger amyloid protein precursor (APP) (Kang et al.,
Nature
325:733-736 (1987); Tanzi et al.,
Science
235:880-884 (1987); Robakis et al.,
Proc. Natl. Acad. Sci. USA
84:4190-4194 (1987); Goldgaber et al.,
Science
235:877-880 (1987)), whose physiological function remains unclear. The cause of Alzheimer's disease remains elusive; however, the discovery of mutations of APP close to or within the A&bgr; domain (Goate et al.,
Nature
349:704-706 (1991); Levy et al.,
Science
248:1124-1126 (1990); Murrell et al.,
Science
254:97-99 (1991); Hendricks et al.,
Nature Genet.
1:218-221 (1992), linked to familial AD (E. Levy et al.,
Science
248:1124 (1990); A&bgr; Goate et al.,
Nature
349:704 (1991); M. Chartier-Harlin et al.,
Nature
353:844 (1991); J. Murrell, M. Farlow, B. Ghetti, M. D. Benson,
Science
254:97 (1991); L. Hendricks et al.,
Nature Genet.
1:218 (1992); M. Mullan et al.,
Nature Genet.
1:345 (1992)), indicates that the metabolism of A&bgr; and APP is likely to be intimately involved with the pathophysiology of this disorder.
Soluble A&bgr; is secreted in cell cultures and is found as a 40-residue peptide (A&bgr;
1-40
) in the cerebrospinal fluid (CSF) (Shoji et al.,
Science
258 126-129 (1992); Seubert et al.,
Nature
359:325-327 (1992); Haass et al.,
Nature
359:322-325 (1992)), but is not found at elevated levels in sporadic AD cases (M. Shoji et al.,
Science
258:126 (1992); P. Seubert et al.,
Nature
359:325 (1992)). Physiological factors which can induce the aggregation of soluble A&bgr; are of interest in determining the cause of A&bgr; amyloid formation. Synthetic A&bgr;
1-40
remains soluble at concentrations up to 16 mg/ml in neutral phosphate buffer (Tomski & Murphy,
Arch. Biochem. Biophys.
294:630-638 (1992)), indicating that overproduction of soluble A&bgr; cannot sufficiently explain A&bgr; precipitation. Hence, biochemical mechanisms which promote A&bgr; amyloid formation in sporadic cases would appear to be relevant to the pathogenesis of AD. Furthermore, soluble A&bgr; in cerebrospinal fluid is not increased in AD cases (Shoji et al.,
Science
258: 126-129 (1992)), indicating that other pathogenetic mechanisms are likely to be involved.
In recent years, the study of A&bgr; peptide has led to making cell lines that express or overexpress A&bgr; or its precursor protein, APP or increased amounts of its more amyloidogenic A&bgr;
1-42
form. See N. Suzuki et al.,
Science
264:1336-1340 (1994); X-D Cai et al.,
Science
259:514-516 (1993); F. S. Esch et al.,
Science
248:1122-1124 (1990). Moreover, monoclonal antibodies to A&bgr; peptide have been generated (see, e.g. U.S. patent Ser. No. 5,231,000, issued Jul. 27, 1993). These monoclonal antibodies are useful as reagents for use in detecting presence of A&bgr; amyloid.
SUMMARY OF THE INVENTION
The process described in this invention involves the rapid induction of A&bgr; amyloid by a heavy metal cation such as zinc to form amyloid. In a preferred embodiment of the invention, the proportion of an A&bgr;
1-40
solution which remains filtrable after incubation with zinc is assayed and the effects of candidate pharmacological agents on the filtrate are measured to determine their ability to maintain the solubility of A&bgr; in physiological solution and thus prevent A&bgr; amyloid formation.
A method for the in vitro induction of A&bgr; amyloid has been previously described (J. T. Jarrett et al., Biochem. 32:4693-4697 (1993)). However, this method has many disadvantages, such as a requirement for high concentrations of peptide and prolonged incubation periods (days) with results that are qualitative rather than quantitative. In contrast, some of the major advantages of the present invention are that the technique is reliable, rapid (can be carried out in minutes), is easily quantifiable, and is achieved with low micromolar concentrations of peptide.
Hence, the present invention relates to an in vitro method for the rapid screening of candidate reagents which are likely to be effective in preventing or reversing the formation of amyloid deposits in vivo which are characteristic of Alzheimer's disease and related pathological conditions. Promising candidate reagents which are selected through one of the in vitro methods of the present invention may then be tested for their effectiveness in vivo in patients which are suffering from Alzheimer's disease or who are at risk for developing Alzheimer's disease.
One aspect of the invention relates to a rapid analytical method for detection of A&bgr; amyloid formation in a biological fluid which comprises:
(a) preparing a first set of reaction mixtures comprising neat biological fluid from a control human subject, and serial dilutions of the same made in aqueous buffer or physiological solution;
(b) preparing a second set of reaction mixtures comprising neat biological fluid from a human patient suspected of amyloidosis, and serial dilutions of the same made in aqueous buffer or physiological solution;
(c) adding an equal amount of AB peptide comprising at least amino acids 6 to 28 of A&bgr; to each serial dilution sample;
(d) contacting each of the first and the second set of reaction mixtures with an amount greater than 300 nM of a heavy metal cation capable of binding to an A&bgr; peptide comprising at least amino acids 6 to 28 of A&bgr;;
(e) centrifuging each of the first and the second sets of reaction mixtures to give a first and a second set of pellets, respectively; and
(f) comparing the amount of amyloid in the first and the second set of pellets and thereby detecting excessive A&bgr; amyloid formation in the biological fluid from the human patient suspected of amyloidosis.
A second aspect of the invention relates to a method for determining whether a compound inhibits the formation of A&bgr; amyloid which comprises:
(a) pre-filtering an aqueous buffer solution of A&bgr; peptide, which comprises at least the region in the A&bgr; peptide from amino acid number 6 to 28 to give a first filtrate;
(b) measuring the amount of A&bgr; peptide in the first filtrate obtained in step (a);
(c) contacting the first filtrate obtained in step (a) with a heavy metal cation capable of binding to the peptide comprising at least amino acids 6 to 28 of A&bgr; to give a reaction mixture;
(d) contacting the reaction mixture obtained in step (c) with a candidate anti-amyloidotic agent;
(e) filtering the reaction mixture obtained in step (d) to give a second filtrate; and
(f) comparing the amount of A&bgr; peptide in the second filtrate with the amount of A&bgr; peptide in the first filtrate, thereby determining whether the candidate compound inhibits formation of A&bgr; amyloid.
A third aspect of the invention relates to a method for determining whether a compound inhibits formation of A&bgr; amyloid which comprises:
(a) assembling a first and a second reaction mixture, wherein each reaction mixture comprises an equal amount of a pre-filtered A&bgr; peptide solution, which comprises at least the region in the A&bgr; peptide from amino acid number 6 to 28, and an aqueous buffer or physiological solution;
(b) contacting each of the first and the second reaction mixtures with an equal amount of a candidate anti-amyloidotic agent;
(c
Bush Ashley I.
Tanzi Rudolph E.
Ludlow Jan
Sterne Kessler Goldstein & Fox
The General Hospital Corporation
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