Spherical polystyrene latex beads of about 2.0 µm diameter were coated with islands of silver or gold metal, about 5-200 nm in diameter, by reduction of aqueous silver or gold ions in the presence of sugar-coated polystyrene latex beads. The metal islands are held on the bead surface by a polymeric sugar derivative, aminodextran, covalently bound to the polystyrene aldehyde/sulfate bead. Images of the gold or silver nanoparticle-coated polystyrene beads, obtained with an optical microscope, show that gold, silver, and uncoated polystyrene beads can be distinguished by their different colors, red-purple, black, and colorless, respectively. Also, scanning electron micrographs of coated versus uncoated polystyrene beads show a distinct granular bead surface when metal nanostructures are present versus a smooth bead surface when they are absent. Nanoparticles of gold greater than about 50 nm in diameter on polystyrene beads showed enhanced 90°or side light scatter (resonant Rayleigh scattering) with excitation at 633 nm but no enhancement (same light scatter intensity as uncoated polystyrene beads) at excitation wavelengths of 544, 488, and 458 nm. On the other hand, nanoparticles of silver greater than about 50 nm in diameter on polystyrene beads showed enhanced 90°or side light scatter with excitation wavelengths of 458, 488, 544, and 633 nm but no enhancement with 351-365 nm excitation. Amplification of elastic scatter from both silver and gold colloids was maximally achieved with structures of 50-200 nm diameter, as shown in forward versus side scatter histograms obtained by flow cytometry. Side scatter enhancements of 2-to 10-fold were observed for gold-or silver-coated polystyrene beads over uncoated polystyrene beads of the same diameter. The origin of this wavelengthdependent, light scattering amplification that was observed by flow cytometry has been identified with excitation into surface plasmon resonance bands of gold and silver nanoparticles on polystyrene latex beads.

Background The type of antibody‐conjugated polystyrene (PS) latex beads for use as light scatter shift agents for targeted lymphocyte populations in whole blood has been expanded to include gold and silver nanoparticle‐aminodextran‐PS latex bead conjugates with antibodies. The linkers between antibody and colloidal metal were an aminotrithiol ligand or aminodextran polymer molecules. Methods A modified flow instrument, including forward light scatter (FS), side light scatter (SS), light scatter at other intermediate angle ranges, LMALS (10–20°) and UMALS (20–65°) was used for simultaneous bead probe measurements. A conventional flow cytometer was used in simultaneous bead‐fluorescent marker experiments. Results Two mutually exclusive cell populations, CD4+ and CD8+ lymphocytes, have been simultaneously enumerated in blood by using a mixture of CD4‐PS, CD8‐Au‐PS or CD4‐Au‐PS, CD8‐PS beads, and one laser line, 633 nm, excitation. Similar measurements were made with mixtures of CD4‐PS, CD8‐Ag‐PS or CD4‐Ag‐PS, CD8‐PS beads. Also, simultaneous use of bead and fluorescent markers mixed with whole blood was demonstrated with CD4‐PS beads and with the CD4‐RD1/CD8‐FITC dual marker. Conclusions Enumeration of CD4 and CD8 lymphocytes in whole blood by light scatter parameters only compared well with standard analyses with fluorescent markers. In simultaneous bead‐fluorescent marker labeling of lymphocytes, the labeled bead had to be mixed first with cells in whole blood. Cytometry 41:298–307, 2000. © 2000 Wiley‐Liss, Inc.

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.

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Conjugates of nickel beads with CD8 and anti-red blood cell KC16 antibody were prepared by using the aminotrithiolate "spider" ligand, tris(3-mercaptopropyl)-N-glycylaminomethane, in its new function as a linker between the surface of nickel beads and antibody via activation of spider ligand attached to nickel beads with the common, heterobifunctional cross-linker, sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC). Raw nickel beads were cleaned by either mild sonication in a bath or by stronger probe sonication to remove surface nickel oxide layers, before attachment of the spider ligand. Scanning electron micrographs of the nickel beads before and after probe sonication showed a marked change from a corrugated to a smooth bead surface. Analyses of the supernatants of conjugation mixtures for antibody gave surface densities of 2.5-5.2 mg/m(2) for CD8 and 0.6-12 mg/m(2) for KC16 antibody runs. The antibody-spider-nickel bead conjugates were used in magnetic bead depletions of targeted CD8+ lymphocytes or red blood cells (rbcs) in whole blood of normal donors. For CD8 cell depletions, the undepleted controls and supernatants of depleted samples were analyzed for CD8/CD4 cell populations by flow cytometry with appropriate fluorescent antibody markers. Enumeration of red blood cells, white blood cells (wbcs), and platelets (plts) in undepleted controls and supernatants of depleted samples were carried out on appropriate hematology counters. Whole blood titer results with various lots of either CD8-spider-nickel or KC16-spider-nickel bead conjugates showed varying degrees of depletion ability as indicated by bead-to-cell ratios of 2-32 for CD8 beads and by rbc-to-bead ratios of 1.2-10 for KC16 beads. Moreover, varying degrees of specificity of CD8 beads for CD8+ cells over CD4+ cells and of KC16 beads for rbcs over white blood cells and platelets were observed from the normalized nontargeted cell population figures in undepleted controls versus supernatants of depleted samples.

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Values of receptors per cell and binding constants obtained by the new method from flow cytometric analyses of mixtures of whole blood with FITC- or RD1-labeled CD4, CD8, and CD3 antibodies compare well with literature values determined by other methods.

Background Shed receptors from the surface of white blood cells in whole blood have been quantitated using the long and tedious enzyme‐linked immunosorbent assay (ELISA) method. A simple rapid flow cytometric method of analysis for shed antigen in the presence of cell‐bound antigen can be advantageous. Methods Magnetic bead depletion of neutrophils in whole blood with CD16b antibody‐conjugated beads as measured by flow cytometric analysis of the remaining cell suspension was inhibited by the presence of soluble CD16b antigen in the blood plasma of normal donors. We describe a competitive binding assay between labeled and unlabeled CD16b antibody for receptors shed from the surface of formed bodies (cells) into solution. Also presented is a new method of obtaining the amount of soluble antigen in a sample. We determine the total unlabeled and labeled ligand concentration at which the fluorescence intensity of the labeled ligand matches the fluorescence intensity in a control run with only the labeled ligand. Results Normal blood donors showed serum concentration levels of shed CD16b antigen in the range of 1–50 nM as determined by a flow cytometric competitive binding assay. These figures compared favorably with parallel determinations using magnetic bead depletion of targeted neutrophils for washed and unwashed whole blood samples to evaluate the concentration of shed CD16b antigen. Conclusions The competitive antibody binding assay for shed and cell‐bound CD16b antigen can be applied to similar GPI‐linked antigens, for which purified antibody and fluorescent antibody against the same antigenic receptor are available. Cytometry 44:30–37, 2001. © 2001 Wiley‐Liss, Inc.