Determination of antibodies against human growth hormone using a direct immunoassay format and different electrochemical methods.

A direct immunoassay format with human growth hormone (hGH) immobilized on a self assembled monolayer modified surface plasmon resonance (SPR) chip was chosen to detect specific antibodies (anti-hGH) using different electrochemical techniques. Atomic force microscopy imaging and SPR were used as control methods for the evaluation and confirmation of the antigen-antibody complex formation. The applicability and sensitivity of candidate electrochemical techniques to develop an accurate and sensitive electrochemical immunosensor were investigated. Four electrochemical methods for anti-hGH determination - pulse amperometry (PA), cyclic voltammetry (CV), square wave voltammetry (SWV) and differential pulse voltammetry (DPV), were compared. Higher sensitivity of the developed immunosensor was observed using PA and CV: analytical signal registered using the PA method was 2.50 times higher in comparison with CV, 16.3 times higher in comparison with SWV and 24.5 times higher in comparison with the DPV method. In the case of PA detection method, the limit of detection was lower (75 nM) than that of the CV method (108 nM).

Comparative study of surface plasmon resonance, electrochemical and electroassisted chemiluminescence methods based immunosensor for the determination of antibodies against human growth hormone.

An indirect immunoassay format with human growth hormone (hGH) immobilized on the self-assembled monolayer (SAM) modified surface plasmon resonance (SPR) chip has been shown to detect specific anti-hGH antibodies using the combination of three different physical phenomena in the same channel of the SPR analyzer. For the enhancement of analytical signal and sensitivity of the immunosensor horseradish peroxidase (HRP) labeled secondary antibodies, specifically interacting with the formed immune complexes, were used. The electroassisted chemiluminescence (ECL) protocol offered the limit of detection (LOD) as low as 0.061 nM and this result was very similar to that obtained by SPR, which was 0.051 nM. In the case of anti-hGH detection using pulsed amperometry (PA) with 3,3',5,5'-tetramethylbenzidine (TMB) and H(2)O(2) in the electrochemical system the LOD was the lowest - 0.027 nm. Lower reproducibility of the analytical signal and higher limit of detection was observed using cyclic voltammetry (CV) where LOD was 0.056 nM. PA detection shows 1.89, 2.07 and 2.26 times higher sensitivity if compared with SPR, CV and ECL, respectively. This work demonstrates successful simultaneous exploitation of several techniques to detect the specific anti-hGH antibodies using indirect immunoassay format on the same area of the SPR-chip.