ONLINE COUPLING OF LIQUID-CHROMATOGRAPHY TO BIOCHEMICAL ASSAYS BASED ON FLUORESCENT-LABELED LIGANDS

The on-line coupling of liquid chromatography (LC) to a biochemical detection (BCD) technique based fluorescein-labeled ligands as reporter molecules is described. In a first step, affinity proteins such as antibodies or avidin are added to the LC effluent to react with ligands (analytes) eluting from the LC column. Unbound affinity proteins react, in a second step, with an excess of fluorescein-labeled ligand to titrate the remaining free binding sites. Prior to detection of the labeled ligand/protein complex, free and bound label are separated on the basis of the considerable difference in molecular weight, A short (10 x 4.0 mm i.d.) column packed with a restricted-access support is used to trap the free labeled ligand at the hydrophobic inner surface of the pores. The high molecular-weight labeled ligand/protein complex passes this column unretained and is detected by means of fluorescence detection. The interaction between biotin and avidin was chosen as a model system. A detection limit of 160 fmol was obtained for biotin using reversed-phase LC-BCD. An equilibrium and kinetic model is described which relates the detector response to the concentration of affinity protein, fluorescent label, and reaction time.