Phycobiliprotein-Fab conjugates as probes for single particle fluorescence imaging.
METHODS
Single particle fluorescence imaging (SPFI) is a recently developed method that has provided a powerful approach to observing receptor movement and associations at high spatial resolution. It provides a noninvasive alternative to the existing biochemical techniques. It can also quantify and resolve molecular interactions at the cell surface at a nanometer scale. Probes that have been used in the past to study mobility and associations of cell surface receptors have many limitations. These include concerns about the specificity of the probes, the possibility that their size interferes with the receptor once bound to it, the nonuniform fluorescence, and the questionable stoichiometry.


RESULTS
In this study, we have generated phycobiliprotein-Fab conjugates, and have shown that they are a significant advance on existing probes for SPFI studies. They are small in size, highly specific, highly fluorescent, of known stoichiometry, photostable, emit uniform fluorescence, and are generally well defined.


CONCLUSIONS
It is highly important that when studying receptor mobility or associations, fully characterized probes are used. Phycoerythrin(PE)-Fab probes provide us with the perfect tool for SPFI, and a system with a wide range of applicability to study any cell surface receptor against which a monoclonal antibody exists.
Triantafilou
K
K
Department of Biological Sciences, University of Essex, Central Campus, Colchester, United Kingdom. ktrian@essex.ac.uk
Triantafilou
M
M
Wilson
K M
KM
D000911:Antibodies, Monoclonal / Q000276:immunology; D002460:Cell Line; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000302:isolation & purification / Q000494:pharmacology*; D006684:HLA-DR Antigens / Q000032:analysis / Q000276:immunology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology* / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000276:immunology / Q000302:isolation & purification; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence / Q000379:methods*; D010206:Papain / Q000737:chemistry; D010799:Phycoerythrin / Q000302:isolation & purification / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D006684:HLA-DR Antigens; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D010799:Phycoerythrin; D010206:Papain