Electrochemical immunosensor for detection of antibodies against influenza A virus H5N1 in hen serum

This paper describes the development of an immunosensor for detection of anti-hemagglutinin antibodies. Its preparation consists of successive modification steps of glassy carbon electrodes: (i) creation of COOH groups, (ii) covalent immobilization of protein A with ED/NHS coupling reaction, (iii) covering with anti-His IgG monoclonal antibody, (iv) immobilization of the recombinant His-tagged hemagglutinin (His(6)-H-5 HA), (v) filling free space with BSA. The interactions between two variants of recombinant HA (short and long) from highly pathogenic avian influenza virus H5N1 and the anti-H-5 HA monoclonal antibody (Mab 6-9-1) have been explored with electrochemical impedance spectroscopy (EIS). The impedimetric immunosensor displayed a very good detection limit (LOD) of 2.1 pg/mL, the quantification limit (LOQ) of 63 pg/mL and a dynamic range from 4 pg/mL to 20 pg/mL. In addition, this analytical device was applied for detection of antibodies against His(6)-H-5 HA in serum of vaccinated hen using serial 10-fold dilutions of serum. The immunosensor proposed was able to detect antibody in hen serum diluted up to 7 x 10(7)-fold. The sensitivity of immunosensor was about four orders of magnitude much better than ELISA. (C) 2013 Elsevier B.V. All rights reserved.