Multi-parameter immunofluorescent labeling is the method of choice for the flow-based sorting of targeted cell populations from heterogeneous samples. However, existing antibody-fluorochrome conjugates used for flow sorting limit the types of downstream analyses that can be applied to the isolated cells, as their continued presence on the cells blocks specific cellular epitopes and restricts the choice of downstream fluorescence detection channels. Herein, we present a new type of antibody-fluorochrome conjugates that facilitates a highly specific multi-parameter cell staining for flow sorting as well as subsequent complete release of the conjugate probes from the sample. These new conjugates rely on recombinantly engineered antibody fragments that individually possess low epitope binding affinities. A tailor-made covalent conjugation chemistry allows for their multimerization as well as fluorochrome labeling, generating fluorescent probes comparable to conventional antibody conjugates. Importantly, upon addition of a release reagent, the antibody-fluorophore conjugates can be rapidly released from the cell surface. Accordingly, previously blocked epitopes are free for re-labeling, which provides maximal flexibility for renewed epitope targeting in downstream applications. As many targeted cell populations require multi-parameter detection of several antigens for their isolation, the recombinant engineered antibodies were conjugated with different fluorochromes. The versatility of this technology is presented in multi-parameter panels enabling clear distinction and discrimination of different cell populations of different starting material, e.g. peripheral blood and tumor cells. The applicability of the new REAlease® antibody fluorochrome conjugates is also demonstrated by flow sorting of human naïve regulatory T cells. In addition, we show that the fast and efficient releasability of the new conjugates can serve as an optimal basis for cyclic immunofluorescence microscopy using a new MACSima™ imaging platform, which permits cell analysis with potentially unlimited number of parameters. Citation Format: Jennifer Pankratz, Sabine Schmachtenberg, Nicole Jansen, Marie Hansen, Susanne Krauthäuser, Ali Kinkhabwala, Karolina Schlegel, Svenja Meiler, Christiane Siewert, Alina Bartholomäus, Dmytro A. Yushchenko, Christian Dose. REAlease® technology: Controlled release of antibody-fluorochrome conjugates for maximal flexibility in flow sorting and fluorescence microscopy applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4048.

The potential of flow cytometry and fluorescence light microscopy in the analysis of complex biological systems is still limited by the range of bright fluorochromes. The sensitivity of fluorescent probes in cell staining is usually limited by several factors including i) the ability of the fluorochrome to absorb energy and convert it into fluorescence light, ii) the extent to which the intrinsic autofluorescence of the system interferes with the fluorochrome emission, and iii) for certain applications, the resistance of the fluorochrome to fixation agents. Here we present a new set of fluorescent dyes, VB515 and VB667, which are detected in the channels of flow cytometers and fluorescence microscopes. We demonstrate that these fluorescent molecules are superior to commercially available alternatives, e.g., FITC, Alexa Fluor® 488, VioBright™ FITC, BD™ Horizon Brilliant™ Blue 515, or Alexa Fluor 647, and can even exceed the fluorescence signal of Allophycocyanine (APC)- and Phycoerythrin (PE)-conjugated antibodies. While offering higher brightness than APC- and PE, VB515 and VB667-conjugated antibodies also provide higher stability towards fixation agents in comparison to their protein counterparts. They are thus ideally suited for applications requiring high sensitivity for the detection of especially low expressed cell markers. With these new fluorochromes a multicolor panel and a gating strategy has been developed for the flow analysis of regulatory T (Treg) cells derived from PBMC and lysed blood. Treg cells play a crucial role in the suppression of non-specific or excessive immune responses. Therefore, the detailed characterization of these cells and their function is of great interest for basic and translational research.

State-of-the-art sensitivity and target specificity of fluorochrome-conjugated antibodies limit applications in flow cytometry. Identification of cell subsets can be challenging due to low expression levels of surface and intracellular markers. The combination of Vio®Dyes and REAfinity™ antibodies provides researchers with the most powerful and reliable tools for flow cytometric cells analysis. REAfinity™ are recombinant antibodies of human IgG1 isotype with a mutated region within the Fc part, which prevents Fcγ receptor interactions. They are characterized by high antigen specificity and consistent quality compared to conventional hybridoma-derived monoclonal antibodies. Vio®Dyes, e.g. Vio®515, VioBright®515, Vio®667, VioBright®667, and PE-Vio®615, allow due to their excellent fluorescent properties the detection of low expressed markers and provide new choices for multi-parameter analysis. To demonstrate the potential of the combination of REAfinity™ antibodies and Vio®Dyes, we developed detection panels and protocols for the characterization of FOXP3+, HELIOS+, and CD45RA+ in CD4+ CD25+ CD127low/− human regulatory T cells (Tregs). Tregs were detected and enumerated in whole blood, PBMC, and in samples of magnetically enriched Tregs utilizing the newly developed reagents. In addition, we set-up new and fast staining protocols using 96-well plates or magnetic columns, which allow improved identification of Treg subsets. In summary, the portfolio of Vio®Dyes conjugated REAfinity and special protocols open up new ways for designing and implementing multicolor panels towards highly sensitive, reproducible, and rapid cell profiling approaches.

Multi-parameter immunofluorescent labeling is the method of choice for flow sorting of cell populations out of heterogeneous mixtures. However, downstream applications of isolated cells are limited since fluorescence channels and epitopes are blocked by antibody fluorochrome conjugates utilized for the flow sorting experiment. Here, we describe for the first time new types of antibody fluorochrome conjugates that enable a highly specific multi-parameter cell labeling. Unlike conventional fluorochrome-conjugated antibodies, the introduced conjugates rely on recombinantly engineered antibodies and a conjugation chemistry that facilitates the release of the whole conjugates from the cell surface after the flow sorting step. Thereby, previously blocked epitopes are free for relabeling, which provides maximal flexibility for renewed epitope targeting in downstream applications. We demonstrate this flexibility in the context of a workflow for the isolation of highly pure human regulatory T cells (Treg). Tregs labeled with conventional CD4-VioBlue, CD25-PE and the new releasable CD127-APC were enriched by flow sorting. Final identification of the target cells was achieved by intranuclear labeling of the transcription factor FoxP3, a low expressed marker which requires a bright fluorochrome and fluorescence channel with high sensitivity for reliable detection. The selective removal of the CD127-APC conjugate facilitated the reuse of the APC-channel and enables maximum sensitivity for the cell sorting and analysis step. In summary, this new generation of releasable antibody fluorochrome conjugates for multi-parameter cell labeling paves the way for so far impossible workflows in research and clinical applications.

Multi-parameter immunofluorescent labeling is the method of choice for the flow-based sorting of targeted cell populations from heterogeneous samples. However, existing antibody-fluorochrome conjugates used for flow sorting limit the types of downstream analyses that can be applied to the isolated cells, as their continued presence on the cells blocks specific cellular epitopes and restricts the choice of downstream fluorescence detection channels. Herein, we present a new type of antibody-fluorochrome conjugates that facilitates a highly specific multi-parameter cell staining for flow sorting as well as subsequent complete release of the conjugate probes from the sample. These new conjugates rely on recombinantly engineered antibody fragments that individually possess low epitope binding affinities. A tailor-made covalent conjugation chemistry allows for their multimerization as well as fluorochrome labeling, generating fluorescent probes comparable to conventional antibody conjugates. Importantly, upon addition of a release reagent, the antibody-fluorophore conjugates can be rapidly released from the cell surface. Accordingly, previously blocked epitopes are free for re-labeling, which provides maximal flexibility for renewed epitope targeting in downstream applications. The versatility of this technology is presented in multi-parameter panels enabling clear distinction and discrimination of different cell populations. The applicability of the new REAlease® antibody fluorochrome conjugates is also demonstrated by flow sorting of human naïve regulatory T cells.