A comparative study of four different antibody immobilization techniques that are suitable for modification of surface plasmon resonance (SPR) chip (SPR-chip) is reported. Antibodies against human growth hormone (anti-HGH) were used as the model system. The evaluated SPR-chip modification techniques were (i) random immobilization of intact anti-HGH (intact-anti-HGH) via self-assembled monolayer (SAM) based on 11-mercaptoundecanoic acid (MUA); (ii) random immobilization of intact-anti-HGH within carboxymethyl dextran (CMD) hydrogel by direct covalent amine coupling technique; (iii) oriented coupling of intact-anti-HGH via Fc-fragment to protein-G layer assembled on SAM consisting of MUA (MUA/pG); (iv) oriented immobilization of fragmented anti-HGH antibodies (frag-anti-HGH) via their native thiol-groups directly coupled to the gold. To liberate these thiol groups, the intact-anti-HGH was chemically "divided" into two frag-anti-HGH fragments by chemical reduction with 2-mercaptoethylamine (2-MEA). Optimal concentration of 2-MEA for preparation of anti-HGH was 15 mM. The surface concentration of immobilized antibodies and the antigen binding capacity for all four differently modified SPR-chips was evaluated and compared. The maximum surface concentration of immobilized intact-anti-HGH was obtained by immobilizing the antibody within CMD-hydrogel. The maximal antigen binding capacity was obtained by SPR-chip based on intact-anti-HGH immobilized via MUA/pG. The immobilization based on application of frag-anti-HGH was found to be the most suitable for design of SPR-immunosensor for HGH detection, due to its sufficient antigen binding capacity, simplicity, and low cost in respect to the currently evaluated techniques.

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In this study, the combination of autofluorescent proteins and fluorescence quenching polymers was shown to be a design which can increase the selectivity and sensitivity of immunosensors. With this objective, the conducting polymer polypyrrole (Ppy) was used as a matrix for immobilization of proteins, which enables biological recognition of the analyte, and as a fluorescence quencher, which increases the selectivity of fluorescence-based detection. In this study, bovine leukemia virus proteins gp51 were immobilized within the Ppy matrix and formed a polymeric layer with affinity for antibodies against protein gp51 (anti-gp51). The anti-gp51 antibodies are present at high levels in the blood serum of cattle infected by bovine leukemia virus. Secondary antibodies labeled with horseradish peroxidase (HRP) were used as specific fluorescent probes for detection of a particular target, because the fluorescence of HRP was readily detectable at the required sensitivity. The Ppy was used as fluorescent background, because its fluorescence was almost undetectable when excited by near UV light at 325 nm. Moreover the Ppy quenched the fluorescence of some fluorescent agents including fluorescein-5(6)-isothiocyanate (fluorescein), rhodamine B, and HRP by almost 100% when these fluorescent agents were adsorbed on the surface of Ppy. It is predicted that Ppy-induced fluorescence quenching could be used in the design of immunosensors to increase selectivity and sensitivity.

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