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

The on-line coupling of liquid chromatography (LC) to a biochemical de tection (BCD) technique based fluorescein-labeled ligands as reporter molecules is described. In a first step, affinity proteins such as ant ibodies 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 tit rate the remaining free binding sites. Prior to detection of the label ed ligand/protein complex, free and bound label are separated on the b asis 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 t o trap the free labeled ligand at the hydrophobic inner surface of the pores. The high molecular-weight labeled ligand/protein complex passe s this column unretained and is detected by means of fluorescence dete ction. The interaction between biotin and avidin was chosen as a model system. A detection limit of 160 fmol was obtained for biotin using r eversed-phase LC-BCD. An equilibrium and kinetic model is described wh ich relates the detector response to the concentration of affinity pro tein, fluorescent label, and reaction time.