Time-resolved fluorometric assay for the detection of endostatin in chromatographically separated extracts of natural peptides.
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.
John
Harald
H
IPF PharmaCeuticals GmbH, Feodor-Lynen-Str. 31, D-30625 Hanover, Germany. H.John@ipf-pharmaceuticals.de
Schulz
Stefanie
S
Forssmann
Wolf-Georg
WG
Ständker
Ludger
L
D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D003094:Collagen / Q000032:analysis* / Q000737:chemistry; D043170:Collagen Type XVIII; D043169:Endostatins; D015200:Fluoroimmunoassay / Q000379:methods*; D005470:Fluorometry; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry
D000970:Antineoplastic Agents; D043170:Collagen Type XVIII; D043169:Endostatins; D010446:Peptide Fragments; D003094:Collagen