Fluorescent Neoglycoproteins:  Antibody-Aminodextran-Phycobiliprotein Conjugates

Abstract

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.

Polysaccharide−Oligoamine Based Conjugates for Gene Delivery

“…The drastic decrease in molecular weights of conjugates in comparison with the starting polymers is explained by the extensive aminolysis of the glycoside linkages during conjugation. 31 Also, transfection efficiencies of these similar conjugates were evaluated in EPC cells applying pLNC-luc as the reporter gene. Average transfection (relative to DOTAP/Chol 1:1, control) was found to be 100 ( 20%, indicating excellent reproducibility in transfectivities.…”

Covalently Bound Antibody on Polystyrene Latex Beads: Formation, Stability, and Use in Analyses of White Blood Cell Populations

“…The surface coverage results indicate that polyamine-coated polystyrene latex particles are capable of increasing the available surface for conjugation of antibody up to about twofold. Also, as shown by Doppler electrophoretic light scattering measurements (33), the aminodextran coating alters the surface charge of the particles from slightly negative due to terminal sulfate groups on polymer chains to positive due to protonated amino groups at pH 7.2-7.3 of the 1 × PBS medium. This positively charged particle surface is then more attractive to CD4 antibody, which has a slight negative charge at near-neutral pH values of the medium.…”

“…Covalent attachment of activated beads to activated antibody occurs through one or more long, bridging linkers consisting of diaminopropane and maleimide. The same conjugation procedure was previously used successfully to yield active antibody in antibodydextran conjugates for use in activation and proliferation of T cells (47) and in amplification of phycobiliprotein fluorescence (33).…”

Defined Protein Conjugates as Signaling Agents in Immunoassays

“…Large aggregates of phycoerythrins can be prepared by covalent linkage (10 ) or by chemically stabilizing naturally occurring phycobilisomes, a naturally aggregated form of phycoerythrin isolated from algae, and the aggregates conjugated to binding proteins to produce reagents with very high fluorescence response (11,12 ). Detection reagents can also be prepared by linking antibodies and reporter proteins directly to the same reactive polymer or polystyrene microspheres (13)(14)(15)(16). Signaling capacity for smallmolecule reporters has been increased by use of similarly labeled secondary antibodies that bind to labels on the first antibody (17 ), and by linkage of dendrimers to the binding protein to increase labeling capacity (18 ).…”