Surface plasmon resonance biosensor for direct detection of antibodies against human growth hormone.
A direct, label-free detection method of antibodies against the human growth hormone (anti-HGH) using a surface plasmon resonance (SPR) biosensor is reported. The sensing surface of the surface plasmon resonance biosensor chip (SPR-chip) was modified by covalent coupling of the human growth hormone (HGH) to the self-assembled monolayer of 11-mercaptoundecanoic acid (MUA). HGH was immobilized via primary amine groups after activation of the MUA carboxyl groups with a mixture of N-hydroxysuccinimide (NHS), and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC). The specific binding of monoclonal anti-HGH antibody on the HGH-modified surface was examined in the concentration range from 0.25 nM to 10 microM. The experimentally observed detection minimum for anti-HGH was 2.47 nM. A single immunoassay cycle could be done within 30 min including the HGH and anti-HGH association, HGH/anti-HGH complex dissociation and surface regeneration steps. The SPR biosensor response for repeatable detections of anti-HGH was highly reproducible and very stable. On the SPR-chip the formed HGH and anti-HGH complex (HGH/anti-HGH) could be gently dissociated and the sensing surface might be regenerated by 50 mM NaOH and 0.5% sodium dodecylsulfate (SDS) solution. Any changes in the original baseline level were detected during the 40 detection-regeneration cycles. This means that damage of the immobilized HGH-based sensitive layer during regeneration was minimal. It was demonstrated that the developed SPR-chip could be stored for at least 21 days before use without considerable loss of sensitivity towards anti-HGH.
Kausaite-Minkstimiene
Asta
A
Centre of Nanotechnology and Material science-Nanotechnas, Faculty of Chemistry, Vilnius University, Naugarduko 24, LT-03225, Vilnius 6, Lithuania. astakausaite@imi.lt
Ramanaviciene
Almira
A
Ramanavicius
Arunas
A
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D003429:Cross Reactions; D005227:Fatty Acids / Q000737:chemistry; D019382:Human Growth Hormone / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D055767:Immobilized Proteins / Q000737:chemistry / Q000276:immunology; D051379:Mice; D015203:Reproducibility of Results; D013438:Sulfhydryl Compounds / Q000737:chemistry; D020349:Surface Plasmon Resonance / Q000379:methods*; D013997:Time Factors
C505222:11-mercaptoundecanoic acid; D000906:Antibodies; D005227:Fatty Acids; D055767:Immobilized Proteins; D013438:Sulfhydryl Compounds; D019382:Human Growth Hormone