Mass cytometry (MC) is a bioanalytical technique that uses metal-tagged antibodies (Abs) for high-dimensional single-cell immunoassays. Currently, this technology can measure over 40 parameters simultaneously on individual cells using metal-chelating polymer (MCP) based reagents. However, MC can in principle detect up to 135 parameters with the development of new elemental mass tags. Here we report the development of a tantalum oxide nanoparticle (NP)-based mass tag for MC immunoassays. Uniform-sized amine-functionalized tantalum oxide NPs (d ∼ 5.7 nm) were synthesized via a one-pot two-step reverse microemulsion method. These amine-functionalized NPs were further modified with azide groups by reacting with azide-PEG2k succinimidyl carboxymethyl ester (NHS-PEG2k -N3) cross-linkers. The Ab-NP conjugates were prepared by reacting azide-functionalized NPs with dibenzocyclooctyne (DBCO)-functionalized primary or secondary Abs (DBCO-Ab) followed by fast protein size exclusion liquid chromatography (FPLC) purification. Three Ab-NP conjugates (TaO2-PEG2k -goat antimouse, TaO2-PEG2k -CD25, TaO2-PEG2k -CD196) were fabricated and tested in MC immunoassays. For the TaO2-PEG2k -goat antimouse conjugate, we showed that it can effectively detect abundant CD20 biomarkers on Ramos cells. For TaO2-PEG2k -CD25 and TaO2-PEG2k -CD196 conjugates, we demonstrated that these Ab-NP conjugates could be integrated into the commercial Ab staining panels for high-dimensional single-cell immune profiling of human peripheral blood mononuclear cells.

The synthesis of a polylysine polymer functionalized with the previously reported astonishingly inert [In(cb-te2pa)] + chelate was performed. A biotin end group allowed the conjugation to biotinylated beads by the intermediary of a Fluorescein isothiocyanate / neutravidin receptor. High quality Imaging Mass Cytometry TM trials, based on 115 In detection were performed to highlight the behavior of the material. Anti-CD20 antibody was labeled by the so obtained In(III) modified polylysine using the biotin/neutravidin interaction. Ramos (CD20[+]) and HL-60 (CD20[-]) cell lines were co-stained with the In(III) modified bioconjugate by finding the best staining conditions. Both immunofluorescence IF-M and Mass Cytometry analyses confirmed the specific binding of anti-CD20 onto Ramos cells. CyTOF R histograms constructed on the 115 In detection allowed to define and to separate, with a good signal-to-noise ratio, two populations (Ramos and HL-60). The inertness of In(III)-MCP-Nav over a three-month storage period was proved by performing new functionality tests involving Jurkat cells (CD20[-]) as well as the possibility of multiplexing trials involving the 115 In channel. The results assure a promising future use of the previously announced [In(cb-te2pa)] + complex based polymers for mass cytometry. Electronic Supplementary Information (ESI) available: [1H NMR of TFA-Lys-NCA, Poly(TFA-L-Lys)-Btn, Poly(L-Lys)-Btn and (PEGpoly(L-Lys)-Btn); GPC chromatogram of Poly(L-Lys)-Btn; NMR integration for PEG number evaluation;. Selected areas of blanks' and beads' 115 In integral intensities on IMC images of indium beads; Average 154 Sm counts per cell for Jurkat (CD45[+]) and Ramos (CD45[+]) cells; Overlayed histograms of Jurkat and Ramos cells].

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A metal containing polymer (MCP) based on a polylysine functionalized by In(III) chelates was synthesized. The chelator is based on a constrained dipicolinate cyclam that forms a highly inert In(III) complex. The MCP was conjugated to anti CD20 antibody using the very strong neutravidin/biotin interaction. Two cell lines, one expressing CD20 the other not, were stained with the modified antibody and analysed by mass cytometry using the In-115 channel. The results showed a specific antigen-antibody recognition and images by mass cytometry imaging could be obtained thanks to In-115 detection. Finally, overtime stability tests of the bioconjugate as well as multiplex experiments using the In-115 channel underline the high potentiel of this new In based MCP. <br><p></p>

Monitoring the immune response in the setting of infectious disease and cancer is critical to assess disease status and targets of immune therapy. CyTOF® mass cytometry enables multiplex cellular phenotyping with more than 50 markers, making it ideal for comprehensive immune profiling. CyTOF technology utilizes antibodies tagged with unique monoisotopic metals, resulting in distinct signals that provide a high-resolution multiparametric landscape of a single cell. The Maxpar® Direct™ Immune Profiling Assay™ is a pre-titrated, dried-down, 30-marker antibody cocktail for immune profiling of human whole blood and PBMC by CyTOF. Paired with Maxpar Pathsetter™ software, stained samples are automatically resolved into 37 immune populations including major lineages and their subsets. In this study, we expanded the 30-marker assay to a 44-marker panel including exhaustion markers such as PD-1 and CTLA-4, co-stimulation markers 4-1BB and ICOS, and intracellular cytoplasmic markers IFN-γ, TNF-α, IL-2, perforin and granzyme B to assess cellular function in PMA/ionomycin-stimulated whole blood cultures. We modified the existing Maxpar Pathsetter model to automate the analysis of the expanded panel and report on additional functional parameters such as T cell exhaustion and cytokine production. Next, we applied this panel to whole blood stimulated with CMV peptides to investigate antigen-specific immune responses in a viral infection model in concert with in-depth phenotypic assessment. Collectively, we demonstrate the flexibility of the Maxpar Direct Immune Profiling Assay to incorporate additional surface and intracellular markers to study antigen-specific immunity in the context of whole blood immune profiling. For RUO.