Immunophenotyping using gold or silver nanoparticle-polystyrene bead conjugates with multiple light scatter.

Abstract

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. A modified flow instrument, including forward light scatter (FS), side light scatter (SS), light scatter at other intermediate angle ranges, LMALS (10-20 degrees ) and UMALS (20-65 degrees ) was used for simultaneous bead probe measurements. A conventional flow cytometer was used in simultaneous bead-fluorescent marker experiments. 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. 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.