RESUMO
An immunoliposome (80 nm in diameter) encapsulating a Ru complex with two aminobutyl moieties was prepared to detect the presence of hemagglutinin molecules, which play an important role in influenza virus infection. The highly sensitive detection was accomplished by electrochemiluminescence (ECL) from the Ru complex adsorbed onto Au electrodes after competitive immunoreactions. This method clarified that the adsorption of the Ru complex onto the electrode was an important factor in obtaining high sensitivity. Optimization of the analytical conditions enabled determination of the hemagglutinin molecules of the influenza virus in the concentration range of 3 x 10(-14) (6 x 10(-19) mol/50 microL sample) to 2 x 10(-12) g/mL. The sensitivity was far superior to that obtained by conventional ELISA as well as to that obtained by biosensors and reported thus far.
Assuntos
Técnicas Biossensoriais/instrumentação , Hemaglutininas/análise , Imunoensaio/métodos , Lipossomos/imunologia , Medições Luminescentes/métodos , Compostos Organometálicos/imunologia , Orthomyxoviridae/química , Compostos de Rutênio/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Calibragem , Eletroquímica , Eletrodos , Ouro/química , Hemaglutininas/imunologia , Dados de Sequência Molecular , Compostos Organometálicos/química , Orthomyxoviridae/imunologia , Orthomyxoviridae/isolamento & purificação , Peptídeos/química , Compostos de Rutênio/química , Sensibilidade e EspecificidadeRESUMO
The characterization of a polyclonal antibody produced via immunization with an [Os(bpy)(2)dcbpy] hapten is described. Bpy is 2,2'-bipyridine and dcbpy is 2,2'-bipyridine-4,4'-dicarboxylic acid. The cross-reactivity of the antibody for the Ru(II) analogue of the hapten was also investigated. Large increases in the emission and luminescent lifetime of a series of Os and Ru complexes were observed on binding of the antibody. Association equilibrium constants were derived from luminescence titration data and were found to be 5.6 x 10(8) and 5.0 x 10(8)M(-1) for [Os(bpy)(2)dcbpy] and [Ru(bpy)(2)dcbpy], respectively. Spectroscopic changes were likely due to the exclusion of H(2)O from the complex/antibody binding cleft and blocking of vibrational relaxation pathways of the Os/Ru excited state. D(2)O/H(2)O experiments confirmed that the antibody protected approx. 82% of [Os(bpy)(2)dcbpy] and 80% of [Ru(bpy)(2)dcbpy] from excited state deactivation by the aqueous solvent.
