High-Performance Liquid Affinity Chromatography: Rapid Immunoanalysis of Transferrin in Serum

• We describe a new method for quantitatively measuring substances ofclinical interest by high-performance liquid affinity chromatography(HPLAC). As a model system we selected analysis for transferrin in humanserum with immobilized antibodies in a high-performance liquidchromatographic system. SelectiSpher-10 Activated Tresyl columns (5 or 10 x0.5 cm) were used for in situ coupling of polyclonal antibodies totransferrin. The amount of transferrin eluted was determined by integratingthe eluted peak at 280 nm. The whole analytical procedure-- includinginjection of sample, washing, elution, and analysis of data-- takes only 7min. We characterized the HPLAC system for analysis of transferrin inseveral ways: intra-assay CV approximately 3%; inter- assay CV 2-9%; linearresponse up to 1 mg/mL column volume; detection limit approximately 3micrograms; analytical recovery 98% +/- 2%; purity of eluted sample greaterthan 95% (SDS-PAGE). The HPLAC method was compared with "rocket"immunoelectrophoresis, a commonly used method of analysis for transferrin,and there was excellent correlation between the two methods (r = 0.96, n =60). Benefits of this HPLAC technique include high precision, rapidanalysis, and simplified sample handling. 

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Biotechnology

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