Natalizumab RO with QSC beads: triplicate experiment

Demonstrate a method for standardization across channels in mass cytometry receptor occupancy (RO) assays, where drug and receptor are detected in mass cytometry channels with different sensitivity. We exemplify this in a RO assay of the MS-therapeutic antibody natalizumab. Conclusion: RO is the ratio between bound drug and total target receptor. Estimation of RO is inaccurate when drug and receptor are detected in mass cytometry channels with different sensitivity. Accurate values are obtained by standardizing with QSC beads coated with known numbers of the antibodies used to detect drug and receptor in the sample. Negative controls: - Unstained PBL sample (preexisting metal isotopes in the detection range of the mass cytometer) - Mass-minus-one controls stained with the whole panel minus antibody for detection of drug or receptor (spillover) - Untreated healthy donor PBL (specificity of anti-IgG4). Positive control: in vitro drug saturated patient sample with expected RO of 100%. Competitivity: equal detection of receptor in the abcence and precence of drug.