Direct detection of the binding of avidin and lactoferrin fluorescent probes to heparinized surfaces.
We describe the use of two heparin-binding proteins, avidin and lactoferrin, as probes for monitoring the amount of heparin immobilized to plastic surfaces. The proteins were derivatized with either fluorescent labels or europium chelates, enabling sensitive, fast, reproducible, and robust assays, and were used to measure the amount of protein bound to heparinized microplates, with particular attention to plates that have been coated with bovine serum albumin (BSA)-heparin conjugate. This direct method unequivocally shows that BSA-heparin affords an economical, convenient, and reliable method for coating both polystyrene microtiter plates and magnetic beads with heparin. We demonstrate that assays using directly labeled proteins overcome the problems of dissociation of the heparin-protein complex, which can occur during incubation and washing steps associated with antibody-based detection methods, and the loss in binding capacity caused by certain blocking regimes. We suggest that labeled avidin and lactoferrin are convenient probes for heparinized surfaces with the potential for much wider applicability than that presented here.
Kett
Warren C
WC
Molecular Immunology, School of Biomedical Sciences, Curtin University of Technology, Perth, WA 6000, Australia.
Osmond
Ronald I W
RI
Stevenson
Sandra M
SM
Moe
Lily
L
Coombe
Deirdre R
DR
D001360:Avidin / Q000378:metabolism*; D003911:Dextrans / Q000378:metabolism; D004355:Drug Stability; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism*; D007781:Lactoferrin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism*; D015336:Molecular Probe Techniques; D004369:Pentetic Acid / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000494:pharmacology
D003911:Dextrans; D005456:Fluorescent Dyes; C036963:fluorescein isothiocyante avidin; C503485:lactoferrin-DTPA-Eu conjugate; D001360:Avidin; D012710:Serum Albumin, Bovine; D004369:Pentetic Acid; D006493:Heparin; D007781:Lactoferrin; D016650:Fluorescein-5-isothiocyanate