Notes: This paper describes a new method for assessing receptor-mediated antibody internalization, a key mechanism underlying several anti-cancer antibody therapeutics. The method allows uses a hydrophilic, bright pH sensor dye (pHAb dye), which becomes fluorescent at acidic pH. Upon antibody binding to a receptor, the dyes conjugated to the antibody are not fluorescent due to the neutral pH of the media environment. Upon antibody internalization and trafficking into endosomal and lysosomal vesicles, the pH drops and dyes become highly fluorescent. The authors show the effectiveness of the method using two different therapeutic antibodies – Trastuzumab (anti-HER2) and Cetuximab (anti-EGFR). (4633)

Notes: The pHAb Thiol Reactive Dye was conjugated to two antibodies to test whether antibody-induced internalization of CD56 reached lysosomes. (4937)

Notes: Various anti-tumor antibodies were chemically conjugated with pHAb dye on Magne™ Protein A beads to determine antibody internalization. (4938)

Notes: This paper describes an improved method for on-bead conjugation of antibodies that overcomes the limitations of solution-based protocols (requirement for purified, high-concentration antibodies and the need for multiple buffer changes). The method uses HaloTag technology to immobilize protein A and G onto high-capacity magnetic beads. Antibodies are then captured and labeled on-bead. The authors demonstrate that the method is compatible with amine- and thiol-based chemistries as well as with  mouse and human antibody isotypes, both purified and in cell media. Protein G and Protein A-HaloTag fusion proteins were constructed by cloning the coding sequences for the Fc-binding domains between N-terminal HQ and C-terminal HaloTag sequences. To create Protein A and G beads, the purified Protein G-HaloTag and Protein A-HaloTag constructs were covalently attached to Magne HaloTag Beads--magnetic cellulose beads activated with HaloTag ligands. (4586)