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Abstract
We present a heterogeneous non-competitive immunological detection assay for peptide and protein antigens from crude extracts of biological sources. This time-resolved fluoroimmunoassay (TR-FIA) has been designed in a solid-phase mode using 96-well microtiter plates. Using the rare-earth metal europium as a fluorescent marker, a highly sensitive, selective and efficient procedure was developed. This technique prevents from interferences of intrinsic protein fluorescence which is highly important for antigen measurement in complex matrices. The TR-FIA has been applied for the detection of circulating forms of the potential anti-tumor agent endostatin, a C-terminal fragment of collagen XVIII, and its close homolog collagen XV (restin) from hemofiltrate. Endostatin was detected with a limit of detection of 3 ng (150 fmol/well) and a broad dynamic range from 10–1000 ng/well.
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