Characterization of new polyclonal antibodies specific for 40 and 42 amino acid-long amyloid beta peptides: their use to examine the cell biology of presenilins and the immunohistochemistry of sporadic Alzheimer's disease and cerebral amyloid angiopathy cases.
BACKGROUND
In Alzheimer's disease (AD), the main histological lesion is a proteinaceous deposit, the senile plaque, which is mainly composed of a peptide called A beta. The aggregation process is thought to occur through enhanced concentration of A beta 40 or increased production of the more readily aggregating 42 amino acid-long A beta 42 species.


METHODS
Specificity of the antibodies was assessed by dot blot, Western blot, ELISA, and immunoprecipitation procedures on synthetic and endogenous A beta produced by secreted HK293 cells. A beta and p3 production by wild-type and mutated presenilin 1-expressing cells transiently transfected with beta APP751 was monitored after metabolic labeling and immunoprecipitation procedures. Immunohistochemical analysis was performed on brains of sporadic and typical cerebrovascular amyloid angiopathy (CAA) cases.


RESULTS
Dot and Western blot analyses indicate that IgG-purified fractions of antisera recognize native and denaturated A beta s. FCA3340 and FCA 3542 display full specificity for A beta 40 and A beta 42, respectively. Antibodies immunoprecipitate their respective synthetic A beta species but also A beta s and their related p3 counterparts endogenously secreted by transfected human kidney 293 cells. This allowed us to show that mutations on presenilin 1 triggered similar increased ratios of A beta 42 and its p 342 counterpart over total A beta and p3. ELISA assays allow detection of about 25-50 pg/ml of A beta s and remain linear up to 750 to 1500 pg/ml without any cross-reactivity. FCA18 and FCA3542 label diffuse and mature plaques of a sporadic AD case whereas FCA3340 only reveals the mature lesions and particularly labels their central dense core. In a CAA case, FCA18 and FCA3340 reveal leptomeningeal and cortical arterioles whereas FCA3542 only faintly labels such structures.


CONCLUSIONS
Polyclonal antibodies exclusively recognizing A beta 40 (FCA 3340) or A beta 42 (FCA3542) were obtained. These demonstrated that FAD-linked presenilins similarly affect both p342 and A beta 42, suggesting that these mutations misroute the beta APP to a compartment where gamma-secretase, but not alpha-secretase, cleavages are modified. Overall, these antibodies should prove useful for fundamental and diagnostic approaches, as suggested by their usefulness for biochemical, cell biological, and immunohistochemical techniques.
Barelli
H
H
IPMC du CNRS, UPR411, Valbonne, France.
Lebeau
A
A
Vizzavona
J
J
Delaere
P
P
Chevallier
N
N
Drouot
C
C
Marambaud
P
P
Ancolio
K
K
Buxbaum
J D
JD
Khorkova
O
O
Heroux
J
J
Sahasrabudhe
S
S
Martinez
J
J
Warter
J M
JM
Mohr
M
M
Checler
F
F
D000544:Alzheimer Disease / Q000473:pathology*; D016229:Amyloid beta-Peptides / Q000276:immunology / Q000302:isolation & purification*; D000918:Antibody Specificity; D016657:Cerebral Amyloid Angiopathy / Q000473:pathology*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D015151:Immunoblotting; D008565:Membrane Proteins / Q000276:immunology / Q000302:isolation & purification*; D010446:Peptide Fragments / Q000276:immunology / Q000302:isolation & purification*; D011233:Precipitin Tests; D053764:Presenilin-1
D016229:Amyloid beta-Peptides; D008565:Membrane Proteins; C508853:PSEN1 protein, human; D010446:Peptide Fragments; D053764:Presenilin-1; C078592:amyloid beta-protein (1-40); C075222:amyloid beta-protein (1-42)