Production of monoclonal antibodies against fullerene C60 and development of a fullerene enzyme immunoassay

Abstract

The aim of the present study was to produce monoclonal anti-fullerene C₆₀ antibodies and to develop the enzyme immunoassay for the detection in the first use of free fullerene C₆₀ both in solutions and in multicomponent biological probes. The immunization of mice with the conjugate of fullerene C₆₀ carboxylic derivative with thyroglobulin synthesized by carbodiimide activation led to the production of eight clones of anti-fullerene antibodies. The specificity of the antibody-fullerene binding was confirmed. Indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed for the determination of water-soluble protein–conjugated fullerene, the fullerene aminocaproic acid, fullerenol and for pristine fullerene in solution. To solubilize extremely hydrophobic free fullerene C₆₀ a specially selected water–organic mixture compatible with immunoassay was proposed. The detection limit of free fullerene C₆₀ in solution was 2 μg L⁻¹. Fullerene C₆₀ was also detected by ELISA in organ homogenates of rats intraperitoneally or intragastrically administered with fullerene. To reduce the influence of biomatrices on the assay results a technique was developed for the biological sample pretreatment by the extraction of C₆₀ from bioprobe by toluene followed by the evaporation of toluene and dissolution of the fullerene-containing extract in the selected water-organic media. The ELISA procedure in the first use allowed the detection of fullerene C₆₀ in different tissues.