WO1990012871A1

WO1990012871A1 - Cerebrovascular amyloid protein-specific monoclonal antibody sv17-6e10

Info

Publication number: WO1990012871A1
Application number: PCT/US1990/002003
Authority: WO
Inventors: Kwang S. Kim; Henryk M. Wisniewski; Guang Y. Wen; Cheng-Mo James Chen; Victor J. Sapienza
Original assignee: Research Foundation For Mental Hygiene, Inc.
Priority date: 1989-04-14
Filing date: 1990-04-13
Publication date: 1990-11-01
Also published as: AU5439790A

Abstract

Disclosed is monoclonal antibody SV17-6E10 and a specific-binding fragment thereof which is specifically reactive with a peptide whose concentration level is elevated in individuals having Down's syndrome or Alzheimer's disease as compared to individuals of substantially the same age who are not so-afflicted and which does not react with other peptides of human origin. Also disclosed is a hybridoma cell line capable of producing the monoclonal antibody, a reagent composition which incorporates the monoclonal antibody or specific-binding fragments thereof and an immunoassay method for their use.

Description

⁰ Cerebrovascular amyloid protein-specific monoclonal antibody SV17-6E1

BACKGROUND OF THE INVENTION

Field Of The Invention 5 The present invention relates to neurological disorders, and, more particularly, to antibodies that are specific for peptides associated therewith.

0 Brief Description Of The Prior Art

Alzheimer's disease is characterized by three major pathological markers. They are neuritic plaques, neurofibrillary tangles and cerebrovascular aπvyloidosis. Glenner, et al., Biochem. Biophys . Res.

•_|_5 Comm., 120 :885 (1984) and Wisniewski, et al., Int.

Symp. Dementia and Amyloid "Neuropathology" , Suppl. 3_.: 87 (1986). The accumulation of two types of aberrant fibrils, the paired helical filaments and the amyloid are characterized neuropathologically in

2 Alzheimer's disease/senile dementia of the Alzheimer type. Plaque and cerebrovascular amyloid are primarily made up of a 40- to 42- a ino acid residue peptide, called the B-peptide.

Alzheimer's cerebrovascular amyloid protein c has been purified and a 24 amino acid residue sequence has been reported. Glenner, et al., Biochem. Biophys. Res. Comm., 122: 1131 (1984). It has also been observed that the sequenced portion of the cerebrovascular amyloid peptide is part of the sequence of a precursor protein. Robakis, et al., Proc. Nat. Acad. Sci. USA, 81=4190 (1987) and Robakis, at al., The Lancet, 1:384 (1987).

Down's Syndrome is a disability charactei ized by the inheritance of an extra copy of chromosome 21 c in each cell. Older persons afflicted with Down's Syndrome display dementia that resembles Alzheimer's disease. The cerebral tissues of these individuals exhibit the same neuropathological findings of Alzheimer's disease, i.e., amyloid-containing neuritic plaques, neurofibrillary tangles and cerebrovascular amyloidosis. The amyloid deposits of Down's Syndrome contain the same peptide as the amyloid deposits found in the brains of Alzheimer's disease victims. See, Glenner et al., Biochem. Biophys. Res. Comm., 12.2:1131 (1984).

10 As applied to other peptides and antigens, including many associated with various diseases, monoclonal antibody products of hybridoma technology has often proven valuable in studying factors associated with these diseases end in diagnosis of •tc the disease in clinical settings. Kim, et al.,

Neuroscience Research Communications, 2.:121 (1988) discloses production and characterization of monoclonal antibodies specific for the synthetic cerebrovascular amyloid protein described in Glenner

20 et al., Biochem. Biophys. Res. Comm., 122:1131 (1984) , supra.

SUMMARY OF THE INVENTION Hybridoma technology is one of the most

«c important biological tools for the analysis of complex antigens. Using the SV17-6E10 monoclonal antibodies of the present invention, which are specific to a recently acquired amino acid sequence of the beta-amyloid precursor protein, it has become possible

~_Q to study the beta-amyloid precursor protein which leads to significant deposits of vascular and neuritic amyloid plaques in Alzheimer's disease a d Down's syndrome. These monoclonal antibodies provide the potential for non-invasive diagnosis of Alzheimer's

35 ^u disease using body fluids such as spinal fluid, serum or urine.

In one aspect, the invention provides a monoclonal antibody, designated SV17-6E10, and antigen - binding fragments thereof which are 5 specifically reactive with a peptide whose concentration level is elevated in individuals having Alzheimer's disease or Down's syndrome as compared to individuals of substantially the same age who are not so-afflicted and which does not react with other

•JQ peptides of human origin. The monoclonal antibodies of the invention are of subclass IgG, . Papain-digestion produced antigen-binding fragments (e.g. Fab) have also been shown to specifically recognize amyloid plaque. ic The monoclonal antibody is produced by a hybridoma formed by fusion of cells from a myeloma line, usually of mouse origin, and antibody-producing cells, also usually of mouse origin, ^' previously immunized with a peptide whose concentration level is Λ elevated in individuals having Alzheimer's disease or Down's syndrome as compared to individuals of substantially the same age who are not so-afflicted and which do not react with other peptides of human origin, such as one which forms a portion of a

25 cerebrovascular amyloid protein characteristic of

Alzheimer's disease or Down's syndrome. Preferably, a mouse NSO myeloma line is used, particularly with antibody-producing cells from a mouse previously immunized with a peptide comprising the sequence ,_Q comprising asp - ala - glu - phe - arg - his - aπp - ser - gly - tyr - glu - val - his - his - gin - lys - leu. Further in this regard, the invention provides a hybridoma cell line capable of producing the monoclonal antibodies described above. In another aspect, the invention provides a composition for quantitatively determining a peptide whose concentration level characteristic of Alzheimer's disease or Down's syndrome in an individual. The composition comprises a monoclonal antibody or antigen - binding fragment thereof in accordance with the invention and a detectable moiety which is directly or indirectly associated therewith. In one embodiment, the monoclonal antibody is conjugated to an enzyme and the detectable moiety comprises a chromogenic redox substrate for the enzyme. In another embodiment, the monoclonal antibody is conjugated to one partner of a specific binding pair and the other partner of the specific binding pair is conjugated to a detectable moiety or a substance capable of rendering the moiety detectable. One example of this embodiment is where the monoclonal antibody or antigen - binding fragment thereof additionally serves as an antigen of the specific binding pair. The partner for the monoclonal or fragment is an anti-immunoglobulin antibody, usually anti - IgG and usually from a species other than that of its partner, which is labeled, such as with a fluorophore like fluorescein isothiocyanate. Another example is where one partner of the specific binding pair is selected from biotin and its binding analogs and the other partner is selected from avidin and its binding analogs. Usually, the detectable moiety is a chromophore, fluorophore or luminophore and the substance capable of rendering it detectable is an energy donor or catalyst therefor.

Further in this regard, the invention provides an immunoassay method. The method comprises contacting a sample, from the individual suspected of having Alzheimer's disease or Down's syndrome, with the composition of the invention and quantitatively observing any detectable response.

Monoclonal antibody SV17-6E10 is very unique in that no monoclonal antibody reactive to this peptide (17 amino acid residue) has previously been reported. Monoclonal antibody SV17-6E10 has been used, as exemplified below, in immunoassays for characterizing the amyloid peptide and precursor amyloid peptides in connection with biogenesis of B-amyloid peptide plaques found in Alzheimer's

10 disease and Down's syndrome afflicted brains.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1(a) shows amyloid plaques stained with SV17-6E10 monoclonal antibody, •_jc Fig. 1(b) shows a serial section stained with SCVAP-2F9 monoclonal antibody. A serial section stained with SCVAP-4G8 monoclonal antibody produced results identical to those shown in Fig. 1(b).

20 DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Example 1 Monoclonal Antibody SV17-6E10 to Amyloid Peptide

In the experiments reported by this example, 2 a synthetic peptide, an antigenic conjugate thereof and tryptic fragments of the peptide were used to prepare and characterize the monoclonal antibody of the present invention.

30 17 Amino Acid Residue Synthetic B-Amyloid Pe ti_de 1SV17)

Preparation of SV17 was as follows. A 17-residue synthetic cerebrovascular amyloid peptide (SV17), having the sequence asp - ala - glu - phe -

35 arg - his - asp - ser - g]y - tyr - gin - val - his - " his - glu - lys - leu, corresponding to a portion of the published sequence of the Alzheimer's cerebrovascular amyloid peptide was obtained from Biosearch Laboratories, San Rafael, CA. The sequence mentioned was published in Glenner, et al., Biochem.

5 Biophys. Res. Comm., 120: 885 (1984). The preparation as received was purified by reverse phase liquid chromatography. The purified SV17 peptide (1 mg) was conjugated to keyhole-limpet hemocyanin (6 mg) with glutaraldehyde (3 ul) in sodium phosphate (1.2 ml, 50 •JO mM, pH 7.5) at 4°C for 2 hours to provide the SV17-KLH antigen.

Preparation of Hybrid Cell Lines

Procedures for producing antibody-secreting

15 hybrid cell lines as described in Togashi, et al., Arch. Virol., £7.:149 (1981) and modified as described in Kisi, et al., J. Clin. Microbiol., 18:331 (1983) were applied as follows.

BALB/CJ female mice (Jackson Laboratories,

20 Bar Harbor, ME) were immunized via the back foot pads with Ribi adjuvant (200 ul) (Ribi Immunochem Research, Inc., Hamilton, MT) containing SV17-KLH antigen (25-50 ug) . At 21 days after the initial injection, these mice were immunized

25 intraperit^'oneally with SV17-KLH antigen (25-50 ug) in Ribi adjuvant (200 ul) four times at about four week intervals. Four days prior to fusion (at six weeks after the last immunization) the mice received intraperitoneal booster injections of SV17 (200 ug) n without adjuvant. Immune spleen cells were harvested as described in Galfre, et al., Methods Enzymol., 2:73 (1981). A mouse with the highest titer after SV17-KLH antigen immunization was selected for hybrid clone production.

35 ⁰ NSO myeloma cells were obtained from Dr.

Choi, Kyung Hee of Albert Einstein Medical School, New York, NY. The immune spleen cells were incubated with the NSO myeloma cells and fused at a ratio of 5:1 in the presence of polyethyleneglycol 1500

5 (Boehringer Mannheim, Indianapolis, IN) as the fusing agent. The cells were maintained in Iscoves modified

Dulbecco minimum essential medium containing 15% fetal bovine serum, 10 -4 M hypoxanthme, 1.6 x

-5 . -7

10 M thyrnidme and 4.0 x 10 M aminopterin for

•_j_0 14 days. Subsequent feedings with medium containing only hypoxanthine and thymidine were performed on days 18, 22 and 25. After day 25, cells were fed with medium that did not include hypoxanthine, thymidine or aminopterin. Normal BALB/CJ mouse macrophages

•-c (6,000 cells per well in 96 wells of a microtiter plate) as a feeder layer were used for the initial hybridoma production and also for subcloning the selected hybrids by limiting dilutions.

20 Establishment of Hybrid Clones Secreting Anti-SV17 ABs

After the initial hybridoma production, as 4 described above, 5 x 10 hybridoma cells in 1.0 ml were added to each well of 96 well tissue culture

25 plates. Hybrid cell growth was detected by the ELISA technique using the synthetic peptide antigen (1 ug/ml) coated on the wells. Supernatant fluid in several wells were found to contain SV17 - specific antibody.

₃₀ The hybrid that produced SV17-6E10 antibody, as determined by the ELISA procedure, was established and cloned three times by limiting dilutions to obtain a pure clone. Normal BALB/CJ mouse macrophages (6,000 cells per well in 96 wells of a 5 microtiter plate) as a feeder layer were used here also. Stable cloned hybrid cell lines were established and are referred to here as purified antibodies .

Example 2 Monoclonal Antibody SV17-6E10 Immunocytochemistry Reagent Monoclonal antibody SV17-6E10 was used in the experiments reported by this example as an immunocytochemistry reagent. Paraffin -embedded, formalin - fixed Alzheimer and normal control cortex sections (6 urn) were stained using the avidin-biotin complex technique (Bethesda Research Laboratories, Bethesda, MD) . Amyloid present in neuritic plaques and cortical and meningeal vessels in brain sections of Alzheimer's pat-ients were stained and those of non-a flicted controls were not. No staining of Alzheimer neurofibrillary tangles or axons/neuroEilaments either in tissue sections or in isolated neurons were observed with monoclonal antibody SV17-6E10.

The immunoreactivity of monoclonal antibody SV17-6E10 was then compared with that of SVCAP-2F9 (or SVCAP-4G8) on the amyloid plaques. Comparison of their immunoreactivities with amyloid plaques using serial sections (6 urn thickness) revealed that there were two types of amyloid plaques present. Regardless of high concentration of SV17-6E10 used, one group of plaques was lightly stained while the other group of plaques was darkly stained (Fig. la). When SCVAP-2F9 or SCVAP-4G8 monoclonal antibody was used, these two groups of plaques all reacted with equal staining intensity (Fig. lb). Monoclonal antibody SV17-6E10 was raised against the amino acid residue 1-17 of the synthetic amyloid B-protein while SCVAP-2F9 and SCVAP-4G8 are monoclonal antibodies ^ϋ specific to an epitope in the 17-24 amino acid segment of the synthetic amyloid B-protein. Judging from these observations, the amount of the peptide with amino acids 1-17 was not equally present in these plaques. However, the amount of B amyloid 5 protein having amino acids 17-24 was approximately the same in all the plaques as evidenced by equal staining density. The availability of SV17-6E10 and SCVAP-2F9 or SCVAP-4G8 makes it possible to reveal for the first time that the plaques are composed of

^■ _j^O more peptides with amino acid residues 17-24 and less of the peptide having amino acid residues 1-17 in some plaques.

The immunohistochemical staining procedure used was as follows. The deparaffinized sections (6

]_ urn thickness) were washed in 0.05M Tris-based buffer (TBS) (pH 7.6) and 0.15M NaCl (TBS) for 5-10 minutes and immersed in undiluted 99% formic acid (Sigma Chemical Co., St. Louis, MO) for 3-4 hours. They were then washed with tap water (3-4 times) and then

2o washed with TBS (5-6 times) . They were further immersed in 0.3% hydrogen peroxide for 20 minutes and washed with TBS (5-6 times) . After treatment with normal horse serum (Vector stain Elite ABC Kit, anti-mouse IgG made in horse) for 30 minutes, sections

25 were incubated with primary mouse monoclonal antibody (SCVAP-2F9, SCVAP-4G8 or SV17-6E10) to amyloid B-protein (diluted 1:50-4,000) overnight in a moist chamber at room temperature. All slides were washed with 0.05 M TBS after each incubation. Secondary 0 antibody (biotinylated anti-mouse IgG, Vector

Laboratories, Inc., Burlingame, CA) was applied for 30 minutes. Avidin-biotinylated peroxidase (Vector-Stain Elite ABC Kit) was used for 45 minutes. They were then stained with 3,3'

25 -diaminobenzidine tetrahydrochloride (DAB) for 5-10 10

° minutes. Sections were lightly stained with

Hematoxylin (RICCA Chemical Co., Arlington, TX) for 30 seconds, dehydrated and covered with permount and cover slips.

5 Example 3

Fab Fragments Of Monoclonal Antibody SV17-6E10 Fab fragments of monoclonal antibody SV17-6E10 were prepared and tested as follows. Immunoglobulin IgG purified by protein A column was

•_j_0 concentrated to 20 mg/ml in 20 mM phosphate/10 mM EDTA. A portion (0.5 ml) was mixed with digestion buffer (0.5 ml; 42 mg cysteine - HC1 in 12 ml phosphate buffer; pH 7.0). Then, a portion (0.5 ml) of 50% slurry of immobilized papain, which had been

•^ rinsed twice with digestion buffer, was incubated (5 hours at 37°C) with constant shaking. The solubilized Fab and Fc fragments and undigested IgG from the immobilized papain gel were recovered. The recovered supernatant was applied to a protein A

2 column which had been equilibrated with binding buffer. Eluate contained Fab fragments.

The Fab fragments were dialyzed against PBS and used for immunostaining of brain sections. When avidin/biotin complex staining method or FITC

25 immunofluorescent staining procedures were used, the Fab fragments of SV17-6E10 selectively stained only amyloid plaques.

Example 4 0 Double Antibody Sandwich Immunoassay

In the experiments reported by this example, B-amyloid protein was detected using the double antibody sandwich procedure described in Kim, et al., J. Clin. Microbiol., 1£:331 (1983).

35 u Falcon ELISA plates with 96 wells were coated with purified SV17-6E10 monoclonal antibody (50 ul; 5 ug/ml in carbonate buffer; pH 9.6) overnight at 4°C. After the plates were washed 10 times with distilled water, they were coated with PBS 5 containing 10% goat serum, 3% bovine serum albumin, and 1% Triton X-100 (PBSTGB) for one hour at room temperature; washed lOx again with distilled water; were then coated with 50 ul of the 24 amino acid residue synthetic B-protein (referred to as "SCVAP2";

₁₀ sequence = DAEFRHDSGYEVHHQKLFAEDV) ; diluted in

PBSTGB; and were thereafter incubated for 2 hours at room temperature. Note that amino acid residue 11 is gluta ine here.

After the plates were washed, the optimum

•1 dilution of horseradish peroxidase conjugated monoclonal antibody 4G8 (4G8-HRP) at 1/1,600 dilution was added and plates were incubated an additional 2 hours at room temperature. The plates were then washed for 20 minutes with PBST and a final wash with distilled water, and orthophenylene diamine

(OPD) substrate solution (100 ul) was added. After 10 minutes, 75 ul of 1 M H-SO. was added and the color change was determined with a Dynatech Microelisa Auto Reader MC640 with a 490 nm filter. A

2 well that received only PBSTGB was used as a negative control. The cutoff point between positive and negative was the average optical density value of the negative controls plus 2 standard deviations. As reported below, SV17-6E10 : 4G8HRP refers to the 0 reagent wherein SV17-6E10 is used as the capture antibody and 4G8HRP is the detection antibody. Alternatively, in the other embodiment reported, SCVAP-4G8 is the capture antibody and SV17-6E10HRP is the detecting antibody. The results are reported in

2 Table 1. Table 1 Double Antibody ELISA Test SCVAP2 6E10:4G8HRP 4G8:6E10HRP

(picog/ml) Relative OD Reading

1. 500, ,000 658 1533

2. 250, ,000 662 1365

3. 125, ,000 786 1284

4. 62, ,500 694 1268

5. 31, ,250 756 1275 _0 6 6.. 1 155,, ,662255 6 61100 681

7. 7, ,812.50 400 553

8. 3, ,906.25 222 274

9. 1, ,953.12 174 173

10. 976.56 125 82 ₅ 1 111.. 4 48888..2288 9 944 40

12. 244.14 9 23

13. 122.07 21 6

14. 61.04 28 9

15. 30.52 19 1 0 16. 15.25 31 7

17. 7.63 49 27

18. 3.81 43 35

19. 1.91 52 8

20. 0.95 51 0 5

6E10:4G8HRP ELISA Sensitivity 4G8:6E10HRP ELISA

Sensitivity Mean = 33.7 Mean = 11

Sbd. Dev. = 15.7 Std. Dev. = 13 0 Mean + Two Std. Dev. = 65.1 Mean + Two Std. Dev

37.0

5 Positive results were defined as the highest dilution of B-protein giving an optical density value higher than the cut-off optical density value. Both assay systems are capable of detecting as little as 488.28 picograms of B-amyloid synthetic peptide per ml or 24.4 picogram of B-amyloid synthetic peptide present in 50 ul of sample. Therefore, as long as 6E10 and 4G8 were used in combination of either detecting or capture antibody the double antibody sandwich ELISA was a very sensitive test. This successful double antibody sandwich ELISA system for detecting B-protein has never been reported before.

Claims

0 What Is Claimed Is:

1. Monoclonal antibody SV17-6E10 and antigen - binding fragments thereof.

2. The monoclonal antibody or binding fragment of 5 claim 1 which is specifically reactive with a cerebrovascular amyloid protein whose elevated levels are characteristic of Alzheimer's disease.

3. The monoclonal antibody or binding fragment of 0 claim 1 which is of subclass IgGl.

4. The monoclonal antibody or binding fragment of claim 1 which is specifically bindable with a peptide comprising the sequence asp - ala - glu - phe - arg

]_ - his - asp - ser — gly - tyr - gin - val - his - his - gin - lys - leu.

5. The monoclonal antibody or binding fragment of claim 1 which is produced by a hybridoma formed by

2o fusion of cells from a mouse myeloma line and antibody-producing cells from a mouse previously immunized with a peptide whose concentration level is elevated in individuals having Down's syndrome or Alzheimer's disease as compared to individuals of

25 substantially the same age who are not so-afflicted and which does not react with other peptides of human origin.

6. The monoclonal antibody or binding fragment of 0 claim 1 which is produced by a hybridoma formed by fusion of cells from a mouse myeloma line and antibody-producing cells from a mouse previously immunized with a cerebrovascular amyloid protein whose elevated levels are characteristic of 25 Alzheimer's disease or Down's syndrome. ° 7. The monoclonal antibody or binding fragment of claim 1 which is produced by a hybridoma formed by fusion of cells from a mouse NSO myeloma line and antibody-producing cells from a mouse previously immunized with a peptide whose concentration level is

5 elevated in individuals having Down's syndrome or Alzheimer's disease as compared to individuals of substantially the same age who are not so-afflicted, and which monoclonal antibody or binding fragment thereof does not react with other peptides of human 0 origin.

8. The monoclonal antibody or binding fragment of claim 1 which is produced by a hybridoma formed by fusion of cells from a mouse NSO myeloma line and •_j_ antibody-producing cells from a mouse previously immunized with a cerebrovascular amyloid protein whose elevated levels are characteristic of Alzheimer's disease or Down's syndrome^'.

2o

9. A monoclonal antibody which is produced by a hybridoma formed by fusion of cells from a mouse NSO myeloma line and antibody-producing cells from a mouse previously immunized with a peptide comprising the sequence asp - ala - glu - phe - arg - his - asp

25 - ser - gly - tyr - gin - val - his - his - gin - lys

- leu or an antigen - binding fragment of said monoclonal antibody.

10. Monoclonal antibody SV17-6E10 or an antigen -

30 binding fragment of said monoclonal antibody derived from hybridoma cells formed from NSO myeloma cells and spleen cells of a Balb/CJ mouse immunized with a peptide having the sequence asp - ala - glu - phe - arg - his - asp - ser - gly - tyr - gin - val - his - 25 his - gin - lys - leu which is conjugated directly or through a linkage group to keyhole - limpet hemocyanin.

11. A hybridoma cell line capable of producing the monoclonal antibody of claim 1.

12. The hybridoma cell line of claim 11 which is formed by fusion of cells from a myeloma line and antibody-producing cells previously immunized with a peptide whose concentration level is elevated in individuals having Down's syndrome or Alzheimer's disease as compared to individuals of substantially the same age who are not so-afflicted.

13. The hybridoma cell line of claim 11 which is formed by fusion of cells from a mouse myeloma line and antibody-producing cells from a mouse previously immunized with a peptide whose concentration level is elevated in individuals having Down's syndrome or Alzheimer's disease as compcired to individuals of substantially the same age who are not so-afflicted.

14. The hybridoma cell line of claim II which is formed by fusion of cells from a mouse NSO myeloma line and antibody-producing cells from a mouse previously immunized with a peptide whose concentration level is elevated in individuals having Down's syndrome or Alzheimer's disease as compared to individuals of substantially the same age who are not so-afflicted.

15. The hybridoma cell line of claim 11 which is formed by fusion of cells from a mouse NSO myeloma line and antibody-producing cells from a mouse previously immunized with a peptide comprising the sequence asp - ala - glu - phe - arg - his - asp - ser - gly - tyr - gin - val - his - his - gin - lys - leu.

16. A composition for quantitatively determining a peptide whose elevated levels are characteristic of Alzheimer's disease or Down's syndrome in an individual, which composition comprises the monoclonal antibody or specific - binding fragment of claim 1 and a detectable moiety which is directly or indirectly associated therewith. 0

17. The composition of claim 16 wherein said monoclonal antibody or specific - binding fragment is conjugated to an enzyme and said detectable moiety comprises a chromogenic redox substrate for said enzyme. 5

18. The composition of claim 16 wherein said monoclonal antibody or specific - binding fragment is specifically bindable with a substance attached to said detectable moiety. 0

19. The composition of claim 18 wherein the substance attached to said detectable moiety is an antibody specifically bindable with an immunoglobulin. 5

20. The composition of claim 16 wherein said monoclonal antibody is conjugated to one partner of a specific binding pair and which further comprises the other partner of said specific binding pair conjugated to said detectable moiety or a substance

30 capable of rendering said moiety detectable.

21. The composition of claim 20 wherein one partner of said specific binding pair is selected from the _₅ group of biotiii and its binding analogs and the other partner of said binding pair is selected from the group of avidin and its binding analogs.

22. The composition of either of claims 20 or 21 wherein said detectable moiety is a chrσmophore, fluorophore or luminophore and said substance capable of rendering said moiety detectable is an energy donor or catalyst therefor.

23. A method for quantitatively determining a peptide whose elevated levels are characteristic of Alzheimer's disease or Down's syndrome in an individual, which method comprises contacting a sample from said individual with the composition of any of claims 16-20 and quantitatively observing any detectable response.