Reagents

Synthesis of element-labeled tags. An acrylic acid polymer with low chain-length dispersity is functionalized with NHS reactive groups at regular intervals. Functionalized DTPA appends chelators to the reactive groups. An end-terminus thiol allows linkage to bismaleimide which in turn complexes the polymer to reduced thiols in the Fc fragment of the antibody. The unlabeled Ab-tag can be stored and labeled with enriched lanthanide elements prior to application to the sample.

Preparation of tagged antibody. The antibody is subjected to mild reduction to convert the disulfides in the Fc fragment to thiols. The unlabeled polymeric tag is conjugated to the antibody through a bismaleimide linker. A solution of an enriched isotopic lanthanum chloride is added in the last step.

Cell labeling protocol. Cell samples are fixed and permeabilized to allow staining of intracellular markers, and then incubated with element-tagged antibodies. After stringent washing to remove unbound tagged antibodies, the sample can be homogenized by acid degradation for bulk assay, or the cells can be resuspended for cytometric analysis.

Cell enumeration using DNA metallointercalator. An intercalator containing a metal atom quantitatively binds between the base pairs in the DNA groove. Through mass-balance interaction, the metallointercalator quantifies the DNA, thus providing for cell enumeration in bulk analysis mode or distinction of single cells in cytometry mode. A Rh-containing metallointercalator is shown at the left, and an Ir-containing metallointercalator is shown on the right. The intercalating moiety is shown approaching the DNA binding groove.