Intended use

This immunoassay kit allows for the specific measurement of Human Annexin V concentrations in cell culture supernates, serum and plasma.

 

Introduction

Annexin V is a calcium binding protein which is widely present in various cells and tissues. Annexin V, belonging to a recently discovered family of proteins, the annexins, with anticoagulant properties has proven to be a useful tool in detecting apoptotic cells since it preferentially binds to negatively charged phospholipids like PS in the presence of Ca2+ and shows minimal binding to phosphatidylcholine and sphingomyeline. Changes in phosphatidylserine（PS） asymmetry, which is analyzed by measuring Annexin V binding to the cell membrane, were detected before morphological changes associated with apoptosis have occurred and before membrane integrity has been lost. By conjugating FITC to Annexin V it is possible to identify and quantitate apoptotic cells on a single-cell basis by flow cytometry. Staining cells simultaneously with FITC-Annexin V (green fluorescence) and the non-vital dye propidium iodide (red fluorescence) allows (bivariate analysis) the discrimination of intact cells (FITC-PI-), early apoptotic (FITC+PI-) and late apoptotic or necrotic cells (FITC+PI+).

 

Test principle

This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for Annexin V has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any Annexin V present is bound by the immobilized antibody. An enzyme-linked monoclonal antibody specific for Annexin V is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of Annexin V bound in the initial step. The color development is stopped and the intensity of the color is measured.