Quantification of Alzheimer amyloid beta peptides ending at residues 40 and 42 by novel ELISA systems.
BACKGROUND
The amyloid beta (Abeta) peptide is a key molecule in the pathogenesis of Alzheimer's disease. Reliable methods to detect and quantify soluble forms of this peptide in human biological fluids and in model systems, such as cell cultures and transgenic animals, are of great importance for further understanding the disease mechanisms. In this study, the application of new and highly specific ELISA systems for quantification of Abeta40 and Abeta42 (Abeta peptides ending at residues 40 or 42, respectively) in human cerebrospinal fluid (CSF) are presented.


METHODS
Monoclonal antibodies WO-2, G2-10 and G2-11 were thoroughly characterized by (SPOT) epitope mapping and immunoprecipitation/mass spectrometry. We determined whether aggregation affected the binding capacities of the antibodies to synthetic peptides and whether components of the CSF affected the ability of the antibodies to bind synthetic Abeta1-40 and Abeta1-42 peptides. The stability of Abeta40 and Abeta42 in CSF during different temperature conditions was also studied to optimize sample handling from lumbar puncture to Abeta assay.


RESULTS
The detection range for the ELISAs were 20-250 pM. The intra-assay variations were 2% and 3%, and the inter-assay variations were 2% and 10% for Abeta40 and Abeta42, respectively. The antibodies specifically detected the expected peptides with equal affinity for soluble and fibrillar forms of the peptide. The presence of CSF obstructed the recognition of synthetic peptides by the antibodies and the immunoreactivity of endogenous CSF Abeta decreased with increasing storage time and temperature.


CONCLUSIONS
This study describes highly sensitive ELISAs with thoroughly characterized antibodies for quantification of Abeta40 and Abeta42, an important tool for the understanding of the pathogenesis of Alzheimer's disease. Our results pinpoint some of the difficulties associated with Abeta quantification and emphasize the importance of using a well-documented assay.
Jensen
M
M
Karolinska Institutet, NEUROTEC Department, Huddinge, Sweden. malene@kfcmail.hs.sll.se
Hartmann
T
T
Engvall
B
B
Wang
R
R
Uljon
S N
SN
Sennvik
K
K
Näslund
J
J
Muehlhauser
F
F
Nordstedt
C
C
Beyreuther
K
K
Lannfelt
L
L
D000418:Albumins / Q000378:metabolism; D000544:Alzheimer Disease / Q000378:metabolism*; D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology / Q000648:ultrastructure; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D002555:Cerebrospinal Fluid / Q000737:chemistry / Q000378:metabolism; D017077:Culture Media, Conditioned / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D018604:Epitope Mapping; D006801:Humans; D013058:Mass Spectrometry; D008854:Microscopy, Electron; D010446:Peptide Fragments / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology / Q000648:ultrastructure; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012995:Solubility; D013048:Specimen Handling; D013696:Temperature
D000418:Albumins; D016229:Amyloid beta-Peptides; D000911:Antibodies, Monoclonal; D017077:Culture Media, Conditioned; D010446:Peptide Fragments; C078592:amyloid beta-protein (1-40); C075222:amyloid beta-protein (1-42)