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

Abstract

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.