Antibodies to A4 amyloid peptide

Monoclonal antibodies to a 28-mer peptide present within A4-amyloid are described. These antibodies exhibit unexpected specificity for amyloid plaque structures previously unrecognized in Alzheimer's disease brains. These monoclonal antibodies are useful as reagents for use in assays and imaging of A4-amyloid in Alzheimer's disease patients.

FIELD OF THE INVENTION 
The present invention relates to antibodies with specificity to A4 amyloid 
polypeptide found in the brain of Alzheimer's Disease (AD) patients, and 
to uses thereof, especially for the neuropathological definition of AD 
senile plaque subtypes, and for the fine, detailed imaging of AD brain 
tissue. 
BRIEF DESCRIPTION OF THE BACKGROUND ART 
Although the presence of amyloid in the plaques of Alzheimer's Disease 
patients was noted over 60 years ago (Divry, 1927), molecular mechanisms 
that produce amyloid in the aged human brain and for the increased 
deposition of this fibrous material in Alzheimer's Disease remain unknown. 
Also undescribed is the contribution, if any, of amyloid to the active 
process of plaque formation vis-a-vis a secondary and more passive role 
that indicates only the terminal stages of parenchymal deterioration. 
Recently, some progress has been made in defining the partial structure of 
amyloid fibrillary protein. Glenner et al. (1984a) purified amyloid from 
meningeal vessels of an AD brain; a 4.2 kD polypeptide was isolated and 
shown to have a unique 24 amino acid sequence (.beta.-polypeptide, FIG. 
1). A polypeptide of similar sequence was subsequently isolated from the 
cerebrovascular amyloid of a Down's syndrome brain (Glenner et al., 
1984b); a single amino acid substitution, of glutamic acid for glutamine 
at position 11, distinguished the two polypeptides. Similar results were 
independently obtained by Masters et al. (1985a) who partly purified and 
analyzed amyloid plaque cores from the AD cerebral cortex; the 28 amino 
acid sequence of the Glu variant was obtained (A4 sequence, FIG. 1). The 
A4 sequence differs from the .beta.-polypeptide disclosed by Glenner et 
al. (U.S. Pat. No. 4,666,829) by the three amino acids at positions 11, 27 
and 28. The significance of this report is that the A4 was derived from 
amyloid plaque cores, a hallmark feature of AD. The amino acid sequence of 
A4 varies from that of the .beta.-polypeptide derived from vascular 
amyloid (see FIG. 1). 
Using polyclonal antisera to a synthetic .beta.-amyloid polypeptide 
containing residues 1 through 10 (FIG. 1), it was shown that neuritic 
plaque amyloid shares antigenic determinants with the similar fibrillary 
lesion of cerebral vessels (Wong et al.. 1985). The same antisera failed 
to detect neurofibrillary tangles (NFTs). By contrast, antiserum raised 
against residues 1 to 11 of the A4 polypeptide failed to detect vascular 
amyloid or neuritic plaques but, rather, exhibited exclusive specificity 
for the NFT; and antisera to the A4 peptide extending from residues 11 to 
23 stained both plaques and vessels (Masters et al., 1985b). Thus, there 
is precedent to believe that antibodies to the .eta.-peptide and the A4 
peptide are not identical with regard to their specificities. 
Glenner et al., U.S. Pat. No. 4,666,829 disclose the preparation of 
antibodies using the first 10 amino acids of the .beta.-amyloid 
polypeptide (FIG. 1). 
SUMMARY OF THE INVENTION 
In order to facilitate studies on the molecular mechanisms involved in 
fibrous protein accumulation in the aged AD demented brain and to provide 
improved neuropathological aids for the diagnosis of AD subtypes, we 
prepared antibodies, both polyclonal and monoclonal antibodies (Mabs), to 
a synthetic amyloid polypeptide with the known 28 amino acid A4 sequence 
(Masters et al., 1985a, FIG. 1). The Mabs were routinely characterized on 
AD cortical and hippocampal sections, and shown to be useful to carry out 
an analysis of individual epitopic sites of Alzheimer--type amyloid. 
Three Mabs were specifically and extensively characterized and used to 
obtain information on the following: (a) detailed morphological features 
of plaque amyloid revealed by individual target epitopes; (b) the 
identification of new subtypes of amyloid deposits in the AD brain; (c) 
the relationship of plaque maturation to the deposition of epitopes. The 
latter study was made possible through computer-assisted imaging and 
microdensitometry. 
The antibodies can be used in in vitro immunoassay procedures for 
AD-amyloid. They can also be used in imaging (e.g. cytochemical or in 
vivo) neurons for evidence of AD-amyloid. For immunoassays and/or imaging, 
the antibodies can be detectably labelled with, e.g., radio, enzyme or 
fluorescent labels. They can also be immobilized on insoluble carriers. 
The striking aspect of Mabs prepared to the A4 28-mer peptide is that they 
define previously undescribed amyloid formations in the AD brain. These 
Mabs represent, therefore, a unique class of Mabs.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The antibodies of the present invention have specificity to one or more 
epitopes present on the A4 28-mer peptide shown in FIG. 1. The antibodies 
of the invention can be polyclonal or monoclonal, provided that they are 
made with the A4 28-mer polypeptide as an immunogen. Both of these types 
of antibodies can be utilized in the multiple applications described 
herein below. 
The term "epitope" as used in this invention is meant to include any 
determinant responsible for specific interaction with an antibody 
molecule. Epitopic determinants usually consist of chemically active 
surface groupings of molecules such as amino acids or sugar side chains 
and have specific three dimensional structural characteristics as well as 
specific charge characteristics. 
Polyclonal antibodies can be generated in any suitable animal such as, for 
example, mice, rabbits or goats. The A4-amyloid 28-peptide can be injected 
by itself or linked to appropriate immunoactivating carriers, such as KLH. 
Further detailed descriptions of immunization protocols can be found in 
the Examples. 
Monoclonal antibodies can be produced in various ways using techniques well 
understood by those having ordinary skill in the art and will not be 
repeated here. Details of these techniques are described in such books as 
Monoclonal Antibodies-Hybridomas: A New Dimension in Biological Analysis, 
edited by Roger H. Kennett et al., published by Plenum Press (1980). 
For example, additional hybridomas to those specifically disclosed in the 
invention, which produce monoclonal antibodies which enable the detection 
of A4-amyloid can be easily produced and isolated with minimal screening. 
Hybridomas producing monoclonal antibodies specific for epitopes which are 
found on the A4 28-mer peptide are most effectively produced by first 
immunizing an animal from which hybridomas can be produced such as, for 
example, a Balb/c mouse, with initial subcutaneous injections of the 
28-mer peptide in Freund's adjuvant, followed by booster injections within 
a few days. The fusion can be carried out using any of the techniques 
commonly known to those of ordinary skill in the art. The screening of the 
hybridomas to determine which ones are producing monoclonal antibodies 
specific for the 28-mer peptide is straightforward and can be done either 
in a standard ELISA or RIA format. For example, in an RIA screening format 
the culture supernatant, or ascites fluid from a hybridoma producing 
monoclonal antibody is reacted with .sup.125 I-28-mer peptide. 
The antibodies of the present invention can be utilized in immunoassays for 
the detection of A4-amyloid polypeptide wherever it may occur, including 
fluid or semi-fluid human samples. The immunoassays can be competitive or 
sandwich, as is otherwise well known they all depend on the formation of 
antibody-antigen immune complex. These assays are not described herein in 
any further detail, as they are well known to those of skill in the art. 
For purposes of the assays, the antibodies can be immobilized or labeled. 
There are many carriers to which the antibodies can be bound for 
immobilization and which can be used in the present invention. Well-known 
carriers include glass, polystyrene, polypropylene, polyethylene, dextran, 
nylon, amylases, natural and modified celluloses, polyacrylamides, 
agaroses, and magnetite. The nature of the carrier can be either soluble 
to some extent or insoluble for purposes of the invention. Those skilled 
in the art will know many other suitable carriers for binding the 
antibodies, or will be able to ascertain such, using routine 
experimentation. 
Depending on the particular embodiment of the invention, one or more of the 
antibodies will be coupled with a detectable label such as an enzyme, 
radioactive isotope, fluorescent compound, chemiluminescent compound, or 
bioluminescent compound. 
Those of ordinary skill in the art will know of other suitable labels for 
binding to the antibodies or will be able to ascertain such using routine 
experimentation. Furthermore, the binding of these labels to the 
antibodies can be done using standard techniques commonly known to those 
of ordinary skill in the art. 
The antibodies can be bound to an enzyme. This enzyme, in turn, when later 
exposed to its substrate will react to the substrate in such a manner as 
to produce a chemical moiety which can be detected, as, for example, 
spectrophotometric or fluorometric means. Examples of enzymes that can be 
used to detectably label are malate dehydrogenase, staphylococcal 
nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, 
alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, alkaline 
phosphatase, asparaginase, glucose oxidase, beta-galactosidase, 
ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, 
glucoamylase, and acetylcholine esterase. 
The presence of an antibody can also be detected by labeling it with a 
radioactive isotope. The presence of the radioactive isotope could then be 
determined by such means as the use of a gamma counter or a scintillation 
counter. Isotopes which are particularly useful are .sup.3 H, .sup.125 I, 
.sup.32 P, .sup.35 S, .sup.14 C, .sup.51 Cr, .sup.36 CI, .sup.57 Co, 
.sup.58 Co, .sup.59 Fe, .sup.75 Se, and .sup.152 Eu. 
It is also possible to detect the presence of the antibody by labeling it 
with a fluorescent compound. When the fluorescently labeled antibody is 
exposed to light of the proper wavelength, its presence can then be 
detected due to fluorescence of the dye. Among the most important 
fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, 
phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and 
fluorescamine. 
Another way in which the antibody can be detectably labeled is by coupling 
it to a chemiluminescent compound. The presence of the 
chemiluminescent-tagged antibody is then determined by detecting the 
presence of luminescence that arises during the course of a chemical 
reaction. Examples of particularly useful chemiluminescent labeling 
compounds are luminol, isoluminol, aromatic-acridinium ester, imidazole, 
acridinium salt, and oxalate ester. 
Likewise, a bioluminescent compound may also be used to label the antibody. 
Bioluminescence is a special type of chemiluminescence which is found in 
biological systems and in which a catalytic protein increases the 
efficiency of the chemiluminescent reaction. The presence of a 
bioluminescent binding partner would be determined by detecting the 
presence of luminescence. Important bioluminescent compounds for purposes 
of labeling are luciferin, luciferase, and aequorin. 
The antibodies for use in the assay of the invention are ideally suited for 
the preparation of a kit. Such a kit may comprise a carrier means being 
compartmentalized to receive in close confinement one or more container 
means such as vials, tubes, and the like, each of said container means 
comprising one of the separate elements to be used in the method. 
For example, one of the container means may comprise a first antibody bound 
to an insoluble or partly soluble carrier. A second container may comprise 
soluble, detectably-labeled second antibody, in lyophilized form or in 
solution. The carrier means may also contain a third container means 
comprising a detectably-labeled third antibody in lyophilized form or in 
solution. Such a kit can be used for sandwich assays. See, e.g., David et 
al. U.S. Pat. No. 4,376,110 herein incorporated by reference. 
In addition, the carrier means may also contain a plurality of containers 
each of which comprises different, predetermined amounts of known 
A4-amyloid antigen. These latter containers can then be used to prepare a 
standard curve into which can be interpolated the results obtained from 
the sample containing the unknown amount of A4-amyloid antigen. 
Imaging can be carried out in vitro or in vivo. In vitro imaging can be 
done with the labels mentioned previously. In vivo imaging is done with 
diagnostically effective labeled antibodies. The term "diagnostically 
effective" means that the amount of detectably labeled antibody 
administered is sufficient to enable detection of the site of amyloid 
presence when compared to a background signal. 
Generally, the dosage of detectably labeled antibody for diagnosis will 
vary depending on considerations such as age, condition, sex, and extent 
of disease in the patient, counterindications, if any, and other 
variables, to be adjusted by the individual physician. Dosage can vary 
from 0.01 mg/kg to 2,000 mg/kg, preferably 0.1 mg/kg to 1,000 mg/kg. 
The term "diagnostically labeled" means that the immunoglobulin has 
attached to it a diagnostically detectable label. 
There are many different imaging labels and methods of labeling known to 
those of ordinary skill in the art. Examples of the types of labels which 
can be used in the present invention include radioactive isotopes and 
paramagnetic isotopes. 
For diagnostic in vivo imaging, the type of detection instrument available 
is a major factor in selecting a given radionuclide. The radionuclide 
chosen must have a type of decay which is detectable for a given type of 
instrument. In general, any conventional method for visualizing diagnostic 
imaging can be utilized in accordance with this invention. 
Another important factor in selecting a radionuclide for in vivo diagnosis 
is that the half-life of a radionuclide be long enough so that it is still 
detectable at the time of maximum uptake by the target, but short enough 
so that deleterious radiation upon the host is minimized. Ideally, a 
radionuclide used for in vivo imaging will lack a particulate emission, 
but produce a large number of photons in a 140-200 keV range, which may be 
readily detected by conventional gamma cameras. 
For in vivo diagnosis, radionuclides may be bound to antibody either 
directly or indirectly by using an intermediary functional group. 
Intermediary functional groups which are often used to bind radioisotopes 
which exist as metallic ions to antibody are diethylenetriaminepentaacetic 
acid (DTPA) and ethylenediaminetetracetic acid (EDTA). Typical examples of 
metallic ions which can be bound to immunoglobulins are .sup.99m Tc, 
.sup.123 I, .sup.111 In, .sup.131 I, .sup.97 Ru, .sup.67 Cu, .sup.67 Ga, 
.sup.125 I, .sup.68 Ga, .sup.72 As, .sup.89 Zr, and .sup.201 Tl. 
The antibodies used in the method of the invention can also be labeled with 
paramagnetic isotopes for purposes of in vivo diagnosis. Elements which 
are particularly useful (as in Magnetic Resonance Imaging (MRI) 
techniques) in this manner include .sup.157 Gd, .sup.55 Mn, .sup.162 Dy, 
.sup.52 Cr, and .sup.56 Fe. 
Preparations of the imaging antibodies for parenteral administration 
include sterile aqueous or nonaqueous solutions, suspensions, and 
emulsions. Examples of non-aqueous solvents are propyleneglycol, 
polyethyleneglycol, vegetable oil such as olive oil, and injectable 
organic esters such as ethyloleate. Aqueous carriers include water, 
alcoholic/aqueous solutions, emulsions or suspensions, including saline 
and buffered media, parenteral vehicles including sodium chloride 
solution, Ringer's dextrose, dextrose and sodium chloride, lactated 
Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient 
replenishers, electrolyte replenishers, such as those based on Ringer's 
dextrose, and the like. Preservatives and other additives may also be 
present, such as, for example, antimicrobials, anti-oxidants, chelating 
agents, and inert gases and the like. See, generally, Remington's 
Pharmaceutical Science. 16th ed., Mac Eds, 1980. 
EXPERIMENTAL 
MATERIALS AND METHODS 
Postmortem Brain Tissues 
All formalin-fixed postmortem brains were obtained from the McLean Hospital 
Brain Tissue Resource Center. Prefrontal cortex (PC) or hippocampal 
sections were used. 
Preparation and Characterization of the Synthetic Amyloid Polypeptide 
The A4 amyloid polypeptide of 28 residues (FIG. 1), corresponding to the 
previously reported sequence of Masters et al. (1985a), was synthesized on 
a Biosearch SAM2 synthesizer using the general procedure of Merrifield 
(1963). Purification was achieved with a 3 X 65 cm column of Sephadex G50 
(10-40 .mu.). Aliquots were removed, spotted onto TLC plates and sprayed 
with fluorescamine to locate protein. Material was pooled and loaded onto 
an analytical HPLC column (Vydac C.sub.18, catalog No. 218TP54). Elution 
was carried out at a flow rate of 1.7 ml/min. with 0.05% trifluoroacetic 
acid/H.sub.2 O for 5 min followed by a 5-100% linear gradient of 0.05% 
TFA/CH.sub.3 CH for 17 min. The optical density profile at 230 nm revealed 
a single major peak that was further analyzed. Amino acid analysis was 
carried out in 6M HCl containing 1% phenol for 18 hours at 110.degree. C. 
The sample was dried under N.sub.2, dissolved in citrate buffer and 
analyzed with an LKB 4151 Alpha Plus amino acid analyzer. Amino acid 
analyses were consistent with the published sequence and indicated an 
approximate yield of 95-98%. Peptide samples were stored as a dry powder 
at -20.degree. C. until used. For some experiments, the peptide was linked 
to Kehole Limpet Hemocyamin (BABCO, Berkeley, Calif.) prior to use. 
Preparation of Polyclonal Antibodies (Pabs) 
New Zealand white female rabbits were used for the production of polyclonal 
antibodies to the synthetic amyloid peptide. In some cases the 28-mer 
A4-amyloid polypeptide (AP) as shown in FIG. 1 linked to KLH was used as 
the antigen. Subdermal injection was carried out using 1 mg of AP that had 
been emulsified in Freund's complete adjuvant. After 3 weeks the animals 
were bled and tested for reactivity. Animals were injected again after 3 
weeks using 1 mg of AP in Freund's incomplete adjuvant. Two weeks later 
the serum was tested and was observed to give a positive reaction at 
1/1,000 dilution. Rabbits were then injected with 1 mg AP for 2 monthly 
intervals after which the serum was positive at a dilution of greater than 
1/10,000 when assayed by immunoblotting. 
Preparation of Monoclonal Antibodies (Mabs) 
Balb/c mice were injected subdermally with 1 mg each of AP in Freund's 
complete adjuvant. After 3 weeks sera were positive at a dilution of 
1/1,000 using the assays described. At 5 and 4 days prior to fusion 100 
.mu.g of AP was injected both subcutaneously and intraperitoneally in 
phosphate buffered saline. Spleen cells were isolated and fused with 
plasmacytoma P3 NS1/1-4 Ag-1 cells (Galfre et al., 1977). Supernatants 
were tested for antibody activity after 10-14 days using the assay 
procedures described below. Positive colonies were subcloned by limiting 
dilution and used in further experimentation. One hybridoma, designated 
10H3, has been deposited before the filing date of the present application 
at the American Type Culture Collection, Rockville, Md., under the terms 
of the Budapest Treaty and given accession number HB9542. 
Dot Blot Assay 
Antibodies were tested for reactivity to AP using the BioRad dot blot 
apparatus according to the manufacturer's directions. For initial 
screening, 1 mg of AP was sonicated in 0.5 ml of 1% sodium dodecylsulfate 
in H.sub.2 O and added to an equal volume of 2.5% Triton X-100, 0.3M NaCl, 
40 mM Tris HCl, pH 7.4. One .mu.g AP was added to each well followed by 50 
.mu.l of 10% BSA. For Mab assays, 150 .mu.l of culture supernatant was 
added per well. Pab assays used serum diluted 1/500, 1/1,000 and 1/10,000 
in 150 .mu.l Tris buffered saline (TBS) containing 0.15M sodium chloride, 
20 mM Tris, pH 7.4. After filtration TBS was used for intermediate washes 
between antibody additions and prior to adding substrate. 50 .mu.l of 
horseradish peroxidase-conjugated affinity purified, goat anti-mouse or 
anti-rabbit IgC (Cappel), diluted 1/2,000 in 5% BSA, 0.5% Triton X-100, 
0.15M NaCl, 20 mM Tris hydrochloride, pH 7.4 was added to each well. The 
reaction product was visualized using diaminobenzidine, 0.5 mg/ml, 
imidazole, mg/ml, and H.sub.2 O.sub.2, 0.015%. Negative controls consisted 
of tissue culture media or pre-immune sera, omission of AP, and addition 
of a monoclonal antibody supernatant specific for a protein other than AP. 
For subsequent assays, the amount of antigen per well was varied over the 
range 0.001-1 .mu.g and antibodies were tested at different dilutions. For 
these assays, individual strips of nitrocellulose containing various 
amounts of antigen were immunostained as described by Brown et al. (1983). 
PAGE Procedures 
PAGE procedures were carried out as described previously (Brown et al., 
1981, 1982). Variations from the published procedures are described in the 
text. 
Immunoblot Assay 
Electrophoretic transfer of proteins to nitrocellulose membranes and 
immunostaining procedures were carried out as previously described (Brown 
et al., 1983). 
Immunohistology 
Formalin fixed human postmortem brain tissue was cut on a vibrotome at 50 
.mu.m. All sectioned material were pre-treated prior to staining for 10 
min by incubation in 1% H.sub.2 O.sub.2. The reaction was stopped after 7 
min. by placing the sections in water. Tissues were mounted on gelatinized 
glass slides, air dried and coverslipped using Permount (Fisher). Immune 
serum was applied at a dilution of 1/1000, pre-immune serum at a dilution 
of 1/200 and Mab culture supernatants were undiluted. 
Thioflavin S Staining 
Formalin-fixed sections were placed in a 0.1% solution of Thioflavin S 
(Sigma) in TBS for 10 min. Excess stain was removed by placing the tissue 
sections in 70% ethanol for to 2 min and then in water. Sections were 
coverslipped using a solution containing 20% polyvinyl alcohol (Sigma), 
10% glycerol and 50 mM Tris HCl, pH 8.5. Where indicated in the text, 
double staining was carried out on the same tissue: a section stained with 
Thioflavin S was subsequently stained by means of the immunocytochemical 
procedure described above. 
Computer-Assisted Image Enhancement 
Specimens of prefrontal cortex immunostained for amyloid protein were 
visualized through a Leitz Laborlux 12 light microscope equipped with a 
MCI 65 televideo camera that interfaced with a computer. The transmitted 
images of immunostained amyloid deposits were processed by the computer 
using software equipped with pseudocolor optical density coding. The 
transmitted image was reconstructed on a separate monitor according to the 
individual colors assigned to each level of gray in the image. Thus, 
internal density variations within amyloid deposits could be assessed. 
RESULTS 
Peptide Preparation and Characterization 
A synthetic amyloid peptide (AP) of 28 amino acids (FIG. 1), with a 
calculated molecular weight of 3.2 kD, was synthesized and then analyzed 
by PAGE procedures prior to immunological studies. The AP was dissolved in 
PAGE sample buffer containing 2% SDS, 5% mercaptoethanol, and 9.5 M urea 
and was electrophoresed on a 10% gel containing 0.1% SDS. After staining 
with Coomassie blue the peptide appeared as a broad band at approximately 
23-25 kD and a narrow band that migrated at the gel front during 
electrophoresis. The higher molecular weight species appeared to be an 
aggregate since it was PG,18 eliminated by adding urea to the separating 
gel: the AP was dissolved in sample buffer containing 9.5M urea and 
electrophoresed on a 10% or 15% gel that contained 6M urea and 2% SDS. 
After staining with Coomassie blue, the predominant species appeared as a 
3-4 kD band which is consistent with the mass of a denatured polypeptide. 
Thus, the synthetic AP has aggregational properties not unlike the 
naturally occurring amyloid protein of 4 kD (Masters et al., 1985b). In 
experiments that follow, antibodies to AP were characterized with respect 
to both the aggregated and denatured forms. 
Characterization of Polyclonal Antibodies (Pabs) 
Sera was collected from rabbits immunized against AP and tested by the 
various immunoassay procedures. Using a rapid dot blot screening 
procedure, it was observed that antiserum Scarlet-1 prepared to the 
unconjugated polypeptide was reactive at the 0.01 .mu.g level and produced 
staining above background with 0.001 .mu.g of antigen. At this antigen 
level, the reaction product from pre-immune serum was barely observable. 
Scarlet-1 was more potent than serum from a rabbit immunized with the 
KLH-linked derivative of AP. 
Scarlet-1 was characterized with respect to the state of aggregation of AP. 
Although the dot blot assay may have contained primarily aggregated 
complexes of the polypeptide, this possibility was examined further by 
PAGE. AP was electrophoresed in a gel containing 2% SDS under conditions 
that allowed aggregates to form. An electroblot was prepared from the same 
gel and immunostained with Scarlet-1. The antiserum detected the lower 
molecular weight form as well as aggregated species. Thus, antiserum 
produced in response to the amyloid polypeptide was extremely potent 
irrespective of the state of aggregation of AP. 
Characterization of Monoclonal Antibodies (Mabs) 
The supernatants of 24 hybridomas were positive for AP as indicated by the 
dot blot assay. Of these, three Mabs with particularly strong binding 
properties were further characterized. Dot blots demonstrated that the 
three Mabs, designated 4E12, 5E2 and 10H3, were at least as reactive as 
the polyclonal antisera. In some immunocytochemical experiments (see 
below), a mixture of the three Mabs in equal parts was used since the 
mixed preparation was also intensively reactive towards the synthetic 
peptide. As with polyclonal sera, each of the Mabs reacted strongly with 
the AP that had been previously treated with SDS prior to electrophoresis. 
Immunodetection of Amyloid with Polyclonal Antisera 
Initial immunostaining studies employed polyclonal antisera to AP; the data 
obtained served as a basis for comparison with later experiments using 
Mabs. Prefrontal cortex (PC) of an AD case contained numerous plaques and 
interneuronal NFTs that were readily visualized by staining with 
Thioflavin S (Kelenyi, 1967). Immunostaining the same tissues with 
Scarlet-1 clearly revealed amyloid plaques that were typical of those seen 
in the PC and hippocampus of four AD cases. 
The same results were obtained with antiserum prepared against the 
synthetic amyloid polypeptide that had been linked to KLH prior to 
immunization. The preimmune serum of Scarlet-1 failed to stain AD brain 
tissue, and the immune serum was infrequently able to detect neuritic 
plaques in normal controls. 
Scarlet-1 antisera strongly bound to the vasculature of AD brains. A 
longitudinal section of a vessel, stained with Thioflavin S and with 
immune serum, demonstrated that anti-AP serum detected the most intensely 
stained features observable with the fluorescent dye. Immunostaining 
further revealed the close association of amyloid material in the 
parenchyma with the vascular amyloid. The double label technique applied 
to blood vessels cut in cross-section confirmed that all vascular layers 
that bound Thioflavin S were detectable with Scarlet-1 immune serum. 
Monoclonal antibodies were prepared to the A4 28 amino acid polypeptide 
derived from AD brain amyloid (Masters et al., 1985a, FIG. 1). The 
supernatants of 24 hybridomas were positive; of these, three Mabs with 
particularly strong binding properties were further characterized. Dot 
blots demonstrated that the three Mabs, designated 4E12, 5E2 and 10H3, 
were strongly reactive towards the polypeptide even at extremely high 
dilutions. In some immunocytochemical experiments (see below) a mixture of 
the three Mabs in equal parts was used. 
Initial studies were aimed at establishing the specificity of the Mabs by 
immunostaining AD brain sections using an avidin-biotin horseradish 
peroxidase procedure and analyzing the epitopic distribution by 
conventional imaging methods. Sections of prefrontal cortex were used. A 
section stained with thioflavin S, which is known to react with brain 
amyloid, was counterstained with Mab 5E2. The antibody bound to the 
amyloid deposit with a distribution that overlapped the fluorescent dye. 
In other studies, observations on numerous plaques stained with the three 
Mabs indicated that these antibodies provided more detailed architectonic 
information than previously reported for AD brain amyloid deposits. The 
Mabs demonstrated that target epitopes occurred in deposits of different 
sizes and different morphologies. For example, it was observed that the 
10H3 epitope was localized both within a core and in a peripheral ring 
whereas in other instances the same epitope was more randomly distributed. 
Immunodetection of an amyloid core surrounded by a ring during the course 
of these studies was an original finding as were other morphologies 
described below. 
Although it is not known whether or not the three Mabs under consideration 
are specific for the same or different epitopic sites of the A4 28-mer 
amyloid polypeptide sequence, when mixed together they provided 
particularly intense staining of tissues; however, the dark reaction 
product remained in sharp contrast to low background staining. In control 
studies, there was no staining with the Mab mixture applied to 
neurologically normal controls beyond light background staining. The 
specific and intense staining of amyloid with the mixed Mabs appeared to 
be the method of choice for more detailed studies using computer-enhanced 
imaging methods in order to analyze the epitopic distribution without 
interference from other components of the senile plaque. 
As expected, the Mabs that reacted with parenchymal deposits of amyloid 
also detected the amyloid of blood vessels, as indicated by double 
staining experiments (thioflavin and Mabs). 
In order to improve the visualization of immunostained patterns and gather 
more detailed information on the distribution of epitopes in the various 
amyloid conformations, we used computer-enhanced imaging procedures that 
provided increased resolution of structural features. Sections of 
prefrontal cortex immunostained with the Mab mixture were viewed by means 
of a light microscope equipped with a televideo camera that interfaced 
with a computer for the production of processed images. The images were 
digitized, size estimates were made, and pseudocolor gray scaling was used 
to display different levels of density. The major types of amyloid 
deposits were distinguished in terms of size, internal organization, and 
internal density. 
Four classes of amyloid deposits of different sizes were identified. Small 
punctate amyloid deposits (9.06.+-.0.24 .mu.m diameter) were the most 
commonly observed immunostained configuration. Often seen adjacent to 
punctate deposits were minute amounts of material that may represent 
precursor forms. Amyloid accumulations referred to as macular amyloid 
deposits (30.87.+-.1.28 .mu.m diameter) were considered together as a 
class since they are distinguished by a larger diameter. In one example, 
multiple foci of dense deposits were present throughout the field; a 
second macular structure of similar size contained only diffuse reaction 
product. Another example appeared as a darker staining accumulation of 
amyloid. 
Also observed with ring-like amyloid deposits (40.51.+-.4.65 .mu.m 
diameter) in which the central region contained little or no 
immunodetectable amyloid. The rarest configuration was a ring+core amyloid 
deposit (a ring of amyloid that contained a distinct and separate amyloid 
core, the ring measured 48.73.+-.7.36 .mu.m diameter and the core measured 
12.85.+-.2.20 .mu.m diameter). Previous immunostaining of amyloid deposits 
with polyclonal sera to a synthetic polypeptide did not detect the ring or 
ring with core patterns (Master et al., 1985b; Wong et al., 1985). 
With respect to neocortex, punctate deposits were predominantly located in 
layer I and their frequency of occurrence was inversely related to their 
depth within the cortical mantle. By contrast, macular deposits had a 
different distribution. When compared with punctate deposits, the macular 
types were represented to a lesser extent in layer I, but to a greater 
degree in layers II through VI. Ring shapes occurred in all layers. Ring 
with core structures were less frequently encountered than the other 
morphologies. This laminar distribution of the different amyloid classes 
has not been previously described. 
The immunoperoxidase-stained amyloid was subjected to pattern analysis by 
computer-enhanced imaging methods that allowed visualization of the 
various epitopic sites according to their density distribution. Using this 
approach, we observed a greater degree of heterogeneity than was 
previously appreciated for amyloid in the AD brain. Punctate deposits, in 
spite of their small diameter, nevertheless exhibited internal gradients 
of reaction product density. All morphologic types showed a similar 
gradient of amyloid immunoreactivity. These gradients of reaction product 
were not attributable to diffusion during the peroxidase step since both 
short (3-4 min.) and long (7-8 min.) incubations showed deposits of 
similar size and internal heterogeneity. Irrespective of the overall 
morphologic variations among the four groups of amyloid deposits, a common 
feature was the presence of multiple foci of high density. 
It is to be emphasized that the Mabs used in the present study exhibited 
high specificity for amyloid and not necessarily for the senile plaque 
detectable by silver staining methods. Due to this immunologic 
specificity, observations were not made with regard to senile plaques per 
se, which include a variety of cellular and subcellular elements in 
addition to amyloid (Wisniewski and Terry, 1973); instead, our attention 
was focused upon morphologic entities identifiable by Mabs with high 
specificity towards amyloid. In other studies, we have observed that 
Bielchowsky staining demonstrates that layers II and III contain the 
greatest number of senile plaques while not revealing the punctate lesions 
of layer I or other layers in similar tissue sections. For this reason, it 
is important to emphasize that there is only partial overlap between the 
immunodectectable amyloid deposit and the classically defined senile 
plaque. 
While the macular amyloid deposits may correspond to the classical 
descriptions of amyloid within senile plaques (Wisniewski and Terry, 
1973), the remaining forms we described appear to be unique. Specifically, 
Mabs to the A4 28-mer polypeptide visualized a series of amyloid deposits 
in the AD brain that do not appear to have been previously described: 
punctate, ring and ring+core amyloid deposits that each have internal 
density gradients are original findings. The unique aspect of these Mabs 
suggests their use as powerful reagents for the detaield investigation of 
subtypes of AD; such reagents have not been previously available. 
The techniques discussed above appear well-suited to the production of Mabs 
that are unique and which provide new tools to examine the molecular 
pathogenesis of AD. Therefore, we have used the same methods to generate 
two additional peptides for the preparation of antibodies. Kang et al. 
(1987) have reported an A4 amyloid cDNA precursor derived from fetal 
brain. The sequence predicts two polypeptide regions that appear to be 
unique to this molecule. The uniqueness was determined after searches by 
means of the Bionet data bases. The two peptides are as follows: 
I. 
Ala-Glu-Glu-Pro-Tyr-Glu-Glu-Ala-Thr-Glu-Arg-Thr-Thr-Ser-Ile-Ala-Thr-Thr-Th 
r 
II. Arg-His-Val-Phe-Asn-Met-Leu-Lys-Lys-Tyr-Val-Arg-Ala-Glu-Gln-Lys-Asp 
The peptides were synthesized and purified. Injection into mice and rabbits 
produces polyclonal and monoclonal antibodies using procedures previously 
applied to the A4 peptide. 
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Having now fully described this invention, it will be readily apparent that 
the same can be performed within a wide and equivalent range of 
parameters, conditions and the like, without affecting the spirit or scope 
of the invention or any embodiment thereof.