Preparation and purification of antisera against different regions or isoforms of β-amyloid precursor protein

• Published in: Brain research. Brain research protocols 1997-12, Vol.2 (1), p.23-30
• Main Authors: Jen, Angela, Wickenden, Colin, de Silva, H.A.Rohan, Patel, Ambrish J
• Format: Article
• Language: English
• Subjects: Affinity chromatography; Alzheimer Disease - pathology; Alzheimer Disease - physiopathology; Alzheimer's disease; Amyloid beta-Protein Precursor - analysis; Amyloid beta-Protein Precursor - immunology; Amyloid β-protein; Animals; Antibodies - isolation & purification; Antibody purification; Antibody Specificity; APP isoform; Blotting, Western - methods; Brain - pathology; Chromatography, Affinity; Enzyme-Linked Immunosorbent Assay - methods; Humans; Immunohistochemistry - methods; Indicators and Reagents; Peptide Fragments - analysis; Peptide Fragments - chemical synthesis; Peptide Fragments - immunology; Peptides - chemical synthesis; Peptides - immunology; Rabbits; β-Amyloid precursor protein
• ISSN: 1385-299X
• DOI: 10.1016/S1385-299X(97)00023-8

We describe a procedure for the production and peptide affinity purification of polyclonal antisera against synthetic peptides representing different domains of β-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 β 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 β-protein (A β). The A β 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.