Amyloid Precursor Protein (APP) ELISA, Human Product Number CS0500 Storage Temperature 2-8 °C Technical Bulletin Product Description Amyloid Precursor Protein (APP) ELISA 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 wells of the multiwell plate strips provided. APP standard dilutions, control specimens, and unknown samples are pipetted into these wells. During the first incubation, the APP antigen binds to the immobilized (capture) antibody. After washing, a biotinylated monoclonal antibody specific for Hu APP is added. During the second incubation, this antibody binds to the immobilized Hu APP captured during the first incubation. After removal of excess second antibody, Streptavidin-Peroxidase is added. This binds to the biotinylated antibody to complete the four-member sandwich. After a third incubation and washing to remove all the unbound enzyme, 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. The optical density measured at 450 nm in the multiwell plate reader is used to calculate the concentration of APP. APP ELISA is designed to detect and quantify the level of APP protein in human cerebral spinal fluid samples (CSF) and human cell lysates. The assay will recognize both natural and recombinant human APP. The capture antibody for this assay binds to the N-terminal part of human APP and the detection antibody recognizes the N-terminal part of amyloid β peptide (Figure 1 ). Therefore, this ELISA kit will detect the isoforms of APP770, APP751 (soluble APP), APP733 and APP695. This kit detects soluble APPα, which is cleaved by α-secretase, but not soluble APPβ that is cleaved by β-secretase. Figure 1 Amyloid Precursor Protein (APP) is a large transmembrane protein encoded by a single gene located on human chromosome 21 . APP is found as an immature form, a protein with Mr = 95 kDa and also as the APP holoprotein, a post-translationally modified protein with Mr = 1 00-1 20 kDa. APP localizes to the Golgi apparatus, endosome, and cell membrane. Within the Golgi apparatus, APP is oriented such that its N-terminus is within the lumen while the C-terminus extends into the cytoplasm. Within the cell membrane, APP contains a large ectodomain that corresponds to the N-terminus, a transmembrane domain, and a short cytoplasmic tail, which corresponds to the C-terminus. APP is proteolytically cleaved through the action of several proteases. β-secretase, an enzyme activity recently attributed to BACE, cleaves APP to generate the APP N-terminal fragment (sAPPβ) with Mr = 1 00 kDa and a C-terminal fragment (C99) with Mr = 1 2 kDa. The smaller 1 2 kDa fragment can be further cleaved by presenilin-dependent γ-secretase. This second cleavage produces the insoluble β-amyloid (Aβ), a peptide of ~4 kDa comprised of 40-43 amino acid residues. Alternatively, APP can be cleaved by α-secretase to create a large soluble fragment known as sAPPα and a smaller fragment comprised of 83 amino acid residues, known as C83 (Mr = 1 0 kDa), which is retained in the membrane. Cleavage of C83 by γ-secretase generates a small fragment known as p3. Thus cleavage of APP by α-secretase precludes the formation of β-amyloid. The function of APP and the roles of the various fragments resulting from the proteolytic cleavage of APP in behavior, cognition, and
