It is a solid phase sandwich Enzyme Linked-Immuno-Sorbent Assay (ELISA) for the quantitative determination of APP protein in cell lysates. A monoclonal antibody specific for APP (regardless of phosphorylation state) has been coated onto the multiwell plate. APP standards, controls and unknown samples are incubated 2 hours at RT and APP antigen binds to the immobilized (capture) antibody. Anti-APP biotinylated detection antibody is added and incubated 1 hr at RT binding to the immobilized APP. After removal of excess detection antibody, Streptavidin-(HRP) is added. This binds to the detection antibody to complete the four-member sandwich. A substrate solution is added, which is acted upon by the bound enzyme to produce color. The intensity of this colored product is directly proportional to the concentration of APP present in the original specimen and the O.D. measured at 450 nm are used to calculate the concentration of APP.
Features and Benefits
APP ELISA is a sensitive (<0.4 ng/mL and 8x more sensitive than immunoblotting) and specific (100 ng/mL and no cross-reactivity with Tau, α-synuclein, β-synuclein, Aβ1-40, and Aβ1-42) alternative to immunoblotting and RIA.
APP ELISA is designed to detect and quantify both natural and recombinant human APP protein in CSF and cell lysates. The capture antibody binds to the N-terminus of human APP, and the detection antibody recognizes the N-terminus of amyloid β peptide. The assay detects the isoforms APP770, APP751 (soluble APP), APP733, and APP695. It also recognizes soluble APPα, but not soluble APPβ.