Preparation and purification of antisera against different regions or isoforms of beta-amyloid precursor protein.
We describe a procedure for the production and peptide affinity purification of polyclonal antisera against synthetic peptides representing different domains of beta-amyloid precursor protein (APP). Rabbits were immunised with keyhole limpet haemocyanin coupled to synthetic peptides representing the amino-terminal APP18-32, Kunitz-type protease inhibitor (KPI) region APP301-316, the A beta region APP670-686, and the carboxy-terminal APP756-770 of APP770 for the production of antisera anti-AP-1, anti-AP-2, anti-AP-4 and anti-AP-5, respectively. Each antiserum was purified to specific antibody using the respective cognate peptides immobilised on affinity columns as ligand, using the 1-ethyl-3-(dimethylaminopropyl)carbodiimide-diaminodipropylamine method. Purified antibodies of these four antisera were highly specific and in enzyme-linked immunosorbent assays (ELISA) reacted only to the corresponding peptide. These purified antisera have been used in Western blot, immunohistochemical and immunoprecipitation techniques to facilitate the understanding of the regulation of APP and amyloid beta-protein (A beta). The A beta is formed by proteolysis of APP, and its deposition leading to the formation of senile plaques in the brain is considered to be a key step in the pathogenesis of Alzheimer's disease.
Jen
A
A
MRC Laboratory, Imperial College School of Medicine, Charing Cross Hospital, London, UK.
Wickenden
C
C
Rohan de Silva
H A
HA
Patel
A J
AJ
D000544:Alzheimer Disease / Q000473:pathology / Q000503:physiopathology; D016564:Amyloid beta-Protein Precursor / Q000032:analysis* / Q000276:immunology; D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D000918:Antibody Specificity; D015153:Blotting, Western / Q000379:methods; D001921:Brain / Q000473:pathology; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D007202:Indicators and Reagents; D010446:Peptide Fragments / Q000032:analysis* / Q000138:chemical synthesis / Q000276:immunology; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D011817:Rabbits
D016564:Amyloid beta-Protein Precursor; D000906:Antibodies; D007202:Indicators and Reagents; D010446:Peptide Fragments; D010455:Peptides