RESUMO
Immunoassay for recombinant methioninase (rMETase), an anti-cancer agent, in biological sample was developed. Antisera were produced by immunizing rabbits with rMETase. The antisera were evaluated using radioiodine-labeled rMETase and good antisera for sensitive immunoassay were obtained. Horseradish peroxidase (HRP) was coupled to reduced IgG of K232 antiserum through bridging agent, N-(epsilon -maleimidocaproyloxy) sulfosuccinimide ester (sulfo-EMCS), to prepare enzyme-labeled antibody. IgG fraction of K231 antiserum was immobilized on microplate well. Two-site sandwich immunoenzymometric assay (IEMA) was developed using these antibodies and had good standard curve between 0.4 and 12 ng per well. For determination of rMETase in mouse plasma, sample was diluted 100-fold with dilution buffer containing protease inhibitors, because about 10% of rMETase immunoreactivity was lost for 2 h at room temperature. rMETase in mouse plasma could be determined by the proposed method in the range of 0.5-8 microg/ml and the method was validated. This novel IEMA, in substitution for the measurement of its enzyme activity, should be very useful not only for preclinical studies of rMETase but also for the clinical studies.
