TY - JOUR A1 - Kuhne, Maren A1 - Dippong, Martin A1 - Flemig, Sabine A1 - Hoffmann, Katrin A1 - Petsch, K. A1 - Schenk, J.A. A1 - Kunte, Hans-Jörg A1 - Schneider, Rudolf T1 - Comparative characterization of mAb producing hapten-specific hybridoma cells by flow cytometric analysis and ELISA N2 - A novel method that optimizes the screening for antibody-secreting hapten-specific hybridoma cells by using flow cytometry is described. Cell clones specific for five different haptens were analyzed. We selectively double stained and analyzed fixed hybridoma cells with fluorophore-labeled haptens to demonstrate the target-selectivity, and with a fluorophore-labeled anti-mouse IgG antibody to characterize the level of surface expression of membrane-bound IgGs. ELISA measurements with the supernatants of the individual hybridoma clones revealed that antibodies from those cells, which showed the highest fluorescence intensities in the flow cytometric analysis, also displayed the highest affinities for the target antigens. The fluorescence intensity of antibody-producing cells corresponded well with the produced antibodies' affinities toward their respective antigens. Immunohistochemical staining verified the successful double labeling of the cells. Our method makes it possible to perform a high-throughput screening for hybridoma cells, which have both an adequate IgG production rate and a high target affinity. KW - Immunization KW - Hapten KW - Monoclonal antibodies KW - Hybridoma KW - Flow cytometry KW - ELISA KW - Estradiol KW - Estrone KW - Digoxigenin KW - Zearalenone KW - Aflatoxin KW - CLSM PY - 2014 DO - https://doi.org/10.1016/j.jim.2014.07.004 SN - 0022-1759 SN - 1872-7905 VL - 413 SP - 45 EP - 56 PB - Elsevier B.V. CY - Amsterdam AN - OPUS4-32322 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CHAP A1 - Resch-Genger, Ute A1 - Hoffmann, Katrin A1 - Pauli, Jutta ED - Brahme, A. T1 - Signal-relevant properties of fluorescent labels and optical probes and their determination N2 - Luminescence techniques are among the most populär analytical and detection tools in the life Sciences (Das et al., 2012; Kobayashi et al., 2010; Lakowicz, 2006; Weissleder and Pittet, 2008). The ever-growing interest in these methods that yield fluorophore-specific quantities such as emission and excitation spectra, fluorescence quantum yields, fluorescence lifetimes, and emission anisotropies is due to their comparable ease of use; noninvasive character; potential for combining spectrally, temporally, and spatially resolved ineasurements; and suitability for multiplexing and remote sensing (Resch-Genger et al., 2005a). Moreover, fluorescence detection techniques can provide sensitivity down to the single-molecule level. As the vast majority of analytes or targets of interest are nonfluorescent and most endogenous chromophores typically display rather low fluorescence quantum yields, the tremendous analytical potential of photoluminescence techniques initiated also the development of an ever-growing number of fluorescent labels and probes as readily available tools for a broad variety of analytes and applications and the labeling of biomolecules such as oligonucleotides, antibodies, or peptides as detailed in the chapter "Fluorescent Reporters and Optical Probes: Dye Conjugation Chemistry, Spectroscopic Properties, and Design Concepts" (Berezin and Achilefu, 2010; Escobedo et al., 2010; Luo et al., 2011; Resch-Genger and Licha, 2011; Resch-Genger et al., 2008). Prominent examples of such dye-biomolecule conjugates are fluorophore-labeled secondary antibodies, for example, for immunohistochemistry or flow cytometry, and conventional contrast agents, consisting of a fluorescent reporter and a biomarker-specific biomolecule like a peptide, antibody, or antibody fragment for in vivo near-infrared (NIR) fluorescence imaging of pathological changes in the spectral window of 650-950 nm. These contrast agents are often referred to as targeted optical probes. In addition, more sophisticated Systems are increasingly emerging, the optical properties of which can be modulated selectively by Chemical or biological inputs (Elias et al., 2008; Kobayashi et al., 2011; Resch-Genger and Licha, 2011). PY - 2014 SN - 978-0-444-53632-7 DO - https://doi.org/10.1016//B978-0-444-53632-7.00408-1 VL - 4 SP - Chapter 4.02, 15 EP - 26 PB - Elsevier CY - Amsterdam AN - OPUS4-31433 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Hoffmann, Katrin A1 - Behnke, Thomas A1 - Grabolle, Markus A1 - Resch-Genger, Ute T1 - Nanoparticle-encapsulated vis- and NIR-emissive fluorophores with different fluorescence decay kinetics for lifetime multiplexing N2 - Bioanalytical, clinical, and security applications increasingly require simple, efficient, and versatile strategies to measure an ever increasing number of analytes or events in parallel in a broad variety of detection formats as well as in conjunction with chromatographic separation techniques or flow cytometry. An attractive alternative to common optical multiplexing and encoding methods utilizing spectral multiplexing/color encoding and intensity encoding is lifetime multiplexing, which relies on the discrimination between different fluorescent reporters based on their fluorescence decay kinetics. Here, we propose a platform of surface-functionalizable polymeric nanoparticles stained with fluorophores differing in their fluorescence lifetimes as a new multiplexing and encoding approach. Proof-of-concept measurements with different sets of lifetime-encoded polystyrene nanoparticles are presented, obtained via staining of preformed particles with visible (vis)- and near-infrared (NIR)-emissive organic dyes, which display very similar absorption and emission spectra to enable excitation and detection at the same wavelengths, yet sufficiently different fluorescence decay kinetics in suspension, thereby minimizing instrumentation costs. Data analysis was performed with a linear combination approach in the lifetime domain. Our results and first cell experiments with these reporter sets underline the suitability of our multiplexing strategy for the discrimination between and the quantification of different labels. This simple and versatile concept can be extended to all types of fluorophores, thereby expanding the accessible time scale, and can be used, e.g., for the design of labels and targeted probes for fluorescence assays and molecular imaging, cellular imaging studies, and barcoding applications, also in conjunction with spectral and intensity encoding. KW - Fluorescent label KW - Multiplexing KW - Optical KW - encoding KW - Lifetime multiplexing KW - Fluorescence lifetime imaging FLIM KW - Nanoparticles PY - 2014 DO - https://doi.org/10.1007/s00216-013-7597-3 SN - 1618-2642 SN - 1618-2650 VL - 406 IS - 14 SP - 3315 EP - 3322 PB - Springer CY - Berlin AN - OPUS4-30077 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -