title: Comparative study of random and oriented antibody immobilization techniques on the binding capacity of immunosensor.
abstract: 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.
authors.affiliation: Nanotechnas-Centre of Nanotechnology and Material science, Vilnius University, Naugarduko 24, 03225 Vilnius, Lithuania.
authors.forename: A
authors.initials: A
authors.lastname: Kausaite-Minkstimiene
authors.forename: A
authors.initials: A
authors.lastname: Ramanaviciene
authors.forename: J
authors.initials: J
authors.lastname: Kirlyte
authors.forename: A
authors.initials: A
authors.lastname: Ramanavicius
mesh_terms: D055910:Antibodies, Immobilized / Q000737:chemistry* / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods; D003911:Dextrans / Q000737:chemistry; D005227:Fatty Acids / Q000737:chemistry; D006046:Gold / Q000737:chemistry; D019382:Human Growth Hormone / Q000276:immunology; D006801:Humans; D020100:Hydrogels / Q000737:chemistry; D007118:Immunoassay / Q000379:methods*; D009419:Nerve Tissue Proteins / Q000737:chemistry; D011485:Protein Binding; D013438:Sulfhydryl Compounds / Q000737:chemistry; D020349:Surface Plasmon Resonance
chemical_list: C505222:11-mercaptoundecanoic acid; D055910:Antibodies, Immobilized; D003911:Dextrans; D005227:Fatty Acids; C029152:G-substrate; D020100:Hydrogels; D009419:Nerve Tissue Proteins; D013438:Sulfhydryl Compounds; D019382:Human Growth Hormone; D006046:Gold; C014392:carboxymethyl dextran