title: Highly sensitive detection of human cardiac myoglobin using a reverse sandwich immunoassay with a gold nanoparticle-enhanced surface plasmon resonance biosensor.
abstract: A highly sensitive reverse sandwich immunoassay for the detection of human cardiac myoglobin (cMb) in serum was designed utilizing a gold nanoparticle (AuNP)-enhanced surface plasmon resonance (SPR) biosensor. First, a monoclonal anti-cMb antibody (Mab1) was covalently immobilized on the sensor surface. AuNPs were covalently conjugated to the second monoclonal anti-cMb antibody (Mab2) to form an immuno-gold reagent (Mab2-AuNP). The reverse sandwich immunoassay consists of two steps: (1) mixing the serum sample with Mab2-AuNP and incubation for the formation of cMb/Mab2-AuNP complexes and (2) sample injection over the sensor surface and evaluation of the Mab1/cMb/Mab2-AuNP complex formation, with the subsequent calculation of the cMb concentration in the serum. The biosensor signal was amplified approximately 30-fold compared with the direct reaction of cMb with Mab1 on the sensor surface. The limit of detection of cMb in a human blood serum sample was found to be as low as 10 pM (approx. 0.18 ng mL(-1)), and the inter-assay coefficient of variation was less than 3%. Thus, the developed SPR-based reverse sandwich immunoassay has a sensitivity that is sufficient to measure cMb across a wide range of normal and pathological concentrations, allowing an adequate estimation of the disease severity and the monitoring of treatment.
authors.affiliation: IBMC RAMS, Pogodinskaya Street, 10, 119121 Moscow, Russian Federation. oksana_gnedenko@pochta.ru
authors.forename: Oksana V
authors.initials: OV
authors.lastname: Gnedenko
authors.forename: Yury V
authors.initials: YV
authors.lastname: Mezentsev
authors.forename: Andrey A
authors.initials: AA
authors.lastname: Molnar
authors.forename: Andrey V
authors.initials: AV
authors.lastname: Lisitsa
authors.forename: Alexis S
authors.initials: AS
authors.lastname: Ivanov
authors.forename: Alexander I
authors.initials: AI
authors.lastname: Archakov
mesh_terms: D055910:Antibodies, Immobilized / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D006046:Gold / Q000737:chemistry*; D006801:Humans; D007118:Immunoassay / Q000379:methods; D057230:Limit of Detection; D009206:Myocardium / Q000737:chemistry; D009211:Myoglobin / Q000032:analysis / Q000097:blood* / Q000276:immunology; D053758:Nanoparticles / Q000737:chemistry*; D020349:Surface Plasmon Resonance / Q000379:methods*
chemical_list: D055910:Antibodies, Immobilized; D000911:Antibodies, Monoclonal; D009211:Myoglobin; D006046:Gold