Tris(3-mercaptopropyl)-N-glycylaminomethane as a new linker to bridge antibody with metal particles for biological cell separations

Conjugates of nickel beads with CD8 and anti-red blood cell KC16 antibody w ere prepared by using the aminotrithiolate "spider" ligand, tris(3-mercapto propyl)-N-glycylaminomethane, in its new function as a linker between the s urface of nickel beads and antibody via activation of spider ligand attache d to nickel beads with the common, heterobifunctional cross-linker, sulfosu ccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC). Raw nickel beads were cleaned by either mild sonication in a bath or by strong er probe sonication to remove surface nickel oxide layers, before attachmen t of the spider ligand. Scanning electron micrographs of the nickel beads b efore and after probe sonication showed a marked change from a corrugated t o a smooth bead surface. Analyses of the supernatants of conjugation mixtur es for antibody gave surface densities of 2.5-5.2 mg/m(2) for CD8 and 0.6-1 2 mg/m(2) for KC16 antibody runs. The antibody-spider-nickel bead conjugate s were used in magnetic bead depletions of targeted CD8+ lymphocytes or red blood cells (rbcs) in whole blood of normal donors. For CD8 cell depletion s, the undepleted controls and supernatants of depleted samples were analyz ed for CD8/CD4 cell populations by now cytometry with appropriate fluoresce nt antibody markers. Enumeration of red blood cells, white blood cells (wbc s), and platelets (plts) in undepleted controls and supernatants of deplete d samples were carried out on appropriate hematology counters. Whale blood titer results with various lots of either CD8-spider-nickel or KC16-spider- nickel bead conjugates showed varying degrees of depletion ability as indic ated 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 cel l population figures in undepleted controls versus supernatants of depleted samples.