NPC2 ELISA Kit (Human)

Intra-assay Precision (Precision within an assay) : 3 samples with low, middle and high level Niemann Pick Disease Type C2 (NPC2) were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays) : 3 samples with low, middle and high level Niemann Pick Disease Type C2 (NPC2) were tested on 3 different plates, 8 replicates in each plate. CV (%) = SD/meanX100 Intra-Assay: CV<10% Inter-Assay: CV<12%

EDDM1; epididymal protein 1; Epididymal secretory protein E1; epididymis secretory sperm binding protein; HE1; human epididymis-specific protein 1; Niemann-Pick disease type C2 protein; NPC intracellular cholesterol transporter 2; tissue-specific secretory protein.

Intracellular cholesterol transporter which acts in concert with NPC1 and plays an important role in the egress of cholesterol from the endosomal/lysosomal compartment. Both NPC1 and NPC2 function as the cellular 'tag team duo' (TTD) to catalyze the mobilization of cholesterol within the multivesicular environment of the late endosome (LE) to effect egress through the limiting bilayer of the LE. NPC2 binds unesterified cholesterol that has been released from LDLs in the lumen of the late endosomes/lysosomes and transfers it to the cholesterol-binding pocket of the N-terminal domain of NPC1. Cholesterol binds to NPC1 with the hydroxyl group buried in the binding pocket and is exported from the limiting membrane of late endosomes/ lysosomes to the ER and plasma membrane by an unknown mechanism. The secreted form of NCP2 regulates biliary cholesterol secretion via stimulation of ABCG5/ABCG8-mediated cholesterol transport.

The test principle applied in this kit is Sandwich enzyme immunoassay. The microtiter plate provided in this kit has been pre-coated with an antibody specific to Niemann Pick Disease Type C2 (NPC2) . Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to Niemann Pick Disease Type C2 (NPC2) . Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain Niemann Pick Disease Type C2 (NPC2), biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm +/- 10nm. The concentration of Niemann Pick Disease Type C2 (NPC2) in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Reconstitution & Storage Instructions Western Blotting/Immunoblotting (WB/IB) Protocol Immunohistochemistry (IHC) Protocol Immunocytochemistry (ICC) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Blocking Peptide Competition Protocol (BPCP) Immunoprecipitation (IP) Protocol Antibody Array (AA) Protocol Reconstitution & Storage Instructions Reconstitution & Storage Instructions Western Blotting/Immunoblotting (WB/IB) Protocol Western Blotting/Immunoblotting (WB/IB) Protocol Immunohistochemistry (IHC) Protocol Immunohistochemistry (IHC) Protocol Immunocytochemistry (ICC) Protocol Immunocytochemistry (ICC) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Blocking Peptide Competition Protocol (BPCP) Blocking Peptide Competition Protocol (BPCP) Immunoprecipitation (IP) Protocol Immunoprecipitation (IP) Protocol Antibody Array (AA) Protocol Antibody Array (AA) Protocol

Matrix Recovery Range (%) Average (%) serum (n=5) 94-101 97 EDTA plasma (n=5) 86-95 89 heparin plasma (n=5) 88-102 94 Matrix Recovery Range (%) Average (%) Matrix Recovery Range (%) Average (%) serum (n=5) 94-101 97 serum (n=5) 94-101 97 EDTA plasma (n=5) 86-95 89 EDTA plasma (n=5) 86-95 89 heparin plasma (n=5) 88-102 94 heparin plasma (n=5) 88-102 94

Component Amount Anti-NPC2 Microplate 96 Wells (12 x 8 Well Strips) NPC2 Lyophilized Standard 2 x 50 ng 100X Biotinylated NPC2 Detector Antibody 120 uL Avidin/HRP Conjugate 120 uL Standard Diluent 1 x 20 mL Detector Antibody Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL 30X Wash Buffer 1 x 20 mL TMB Substrate 1 x 9 mL Stop Solution 1 x 6 mL Component Amount Component Amount Anti-NPC2 Microplate 96 Wells (12 x 8 Well Strips) Anti-NPC2 Microplate 96 Wells (12 x 8 Well Strips) NPC2 Lyophilized Standard 2 x 50 ng NPC2 Lyophilized Standard 2 x 50 ng 100X Biotinylated NPC2 Detector Antibody 120 uL 100X Biotinylated NPC2 Detector Antibody 120 uL Avidin/HRP Conjugate 120 uL Avidin/HRP Conjugate 120 uL Standard Diluent 1 x 20 mL Standard Diluent 1 x 20 mL Detector Antibody Diluent 1 x 12 mL Detector Antibody Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL 30X Wash Buffer 1 x 20 mL 30X Wash Buffer 1 x 20 mL TMB Substrate 1 x 9 mL TMB Substrate 1 x 9 mL Stop Solution 1 x 6 mL Stop Solution 1 x 6 mL

This assay has high sensitivity and excellent specificity for detection of Niemann Pick Disease Type C2 (NPC2) . No significant cross-reactivity or interference between Niemann Pick Disease Type C2 (NPC2) and analogues was observed.