Detection of human Annexin V using anti- Annexin V polyclonal antibodies produced in rabbit

Abstract

Annexin -V (ANXV), a 35- kDa protein, binds with high affinity to phosphatidylserine (PS) in the presence of calcium ions. It has been considered as sensitive and specific probe to mark apoptosis. ANXV correlates to several cellular and physiological processes and is found in serums of many patients.  Antibodies are essential tools of biomedical and biochemical researches. Polyclonal antibodies are produced against different epitopes of antigens. In this work, soluble recombinant human ANXV was produced in Escherichia coli. ANXV was then purified by FPLC. The specific polyclonal antibody was produced in a rabbit after immunization with highly pure recombinant ANXV. The immune response was then evaluated and rabbit IgG were tittered. Antibody purification, using protein A sepharose affinity chromatography column, was performed to obtain reactive and pure anti−ANXV IgG. The potency and specificity of the purified IgG were examined by ELISA and immunoblotting. Anti− ANXV antibodies have interesting potentials, in the detection and measurement of ANXV and could be used as alternative to the commercial one. Further conjugation of Anti− ANXV antibodies would be useful for in vivo studies.