Fluorescent neoglycoproteins: antibody-aminodextran-phycobiliprotein conjugates.
New, highly amino-substituted dextran or aminodextran (hereafter denoted Amdex) of various sizes between about 20 and 1000 kDa molecular mass and degrees of amino-substitution between 7 and 40% were prepared and characterized by elemental analyses and polyacrylamide gel electrophoresis. These aminodextrans together with others commercially available were shown by static light scattering, viscosity, and refractive index measurements to adopt a globular structure in aqueous salt solutions. Antibody and fluorescent protein dye, phycoerythrin, or its tandems with cyanin 5. 1 and TEXAS RED, were covalently conjugated to the aminodextrans. The conjugates contained multiple dye molecules and were shown by dynamic light scattering and scanning electron microscopy to assume either globular structure or aggregates thereof. Streptavidin could be substituted for antibody to prepare streptavidin-aminodextran-PE conjugates, which were then used with biotinylated antibody to label subpopulations of white blood cells. The conjugates yielded up to 20-fold amplification of fluorescence intensity over direct antibody-dye conjugates in labeling white blood cells for flow cytometry.
Siiman
O
O
Advanced Technology, and Reagents Applications & Development, Beckman Coulter, Inc., Miami, Florida 33196-2500, USA. olavi.siiman@coulter.com
Wilkinson
J
J
Burshteyn
A
A
Roth
P
P
Ledis
S
S
D000911:Antibodies, Monoclonal / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D019921:Biotinylation; D016827:CD8 Antigens / Q000276:immunology; D002219:Carbamates / Q000737:chemistry; D004396:Coloring Agents; D003911:Dextrans / Q000737:chemistry*; D005434:Flow Cytometry; D005453:Fluorescence; D005456:Fluorescent Dyes; D007962:Leukocytes; D008027:Light; D045342:Light-Harvesting Protein Complexes; D008855:Microscopy, Electron, Scanning; D009842:Oligopeptides / Q000737:chemistry; D010799:Phycoerythrin / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry*; D012542:Scattering, Radiation; D015842:Serine Proteinase Inhibitors / Q000737:chemistry; D014783:Viscosity; D014966:Xanthenes / Q000737:chemistry
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D016827:CD8 Antigens; D002219:Carbamates; D004396:Coloring Agents; D003911:Dextrans; D005456:Fluorescent Dyes; D045342:Light-Harvesting Protein Complexes; D009842:Oligopeptides; D010940:Plant Proteins; D015842:Serine Proteinase Inhibitors; D014966:Xanthenes; C075359:tetrapeptide carbamate; D010799:Phycoerythrin; C034657:Texas red