TY - JOUR A1 - Kanje, S. A1 - Hober, S. A1 - Mueller, L. A1 - Herrmann, Antje Jutta T1 - Next generation of labeling reagents for quantitative and multiplexing immunoassays by the use of LA-ICP-MS N2 - Immuno imaging by the use of Laser Ablation Inductively Coupled Mass Spectrometry (LA-ICP-MS) is a growing research field in life sciences such as biology and biomedicine. Various element labeling strategies for antibodies have been developed for the application of multiplex immunoassays analyzed by the use of LA-ICP-MS. High multiplexing capabilities, a wide linear dynamic range and the possibility of absolute quantification are the main advantages of ICP-MS. But in the context of immuno imaging by the use of LA-ICP-MS, quantification of analytes is limited due to non-controllable antibody labeling chemistry. In the presented proof-of-principle a novel antibody labeling technique has been investigated which results in a controlled labeling degree. A small affinity protein based on the C2 domain of protein G was modified with conventional metal coded tags (MeCAT) after introducing a cysteine into the C-terminus of the protein. The modified C2 domain photo-crosslinks to the Fc or Fab region of the IgG and allows specific and covalent labeling of antibodies for multiplex immunoassay analysis by the use of LA-ICP-MS. In combination with a house-made calibration membrane the amount of labeled antibody–antigen complexes in a multiplex western blot immunoassay was determined by LA-ICP-MS. KW - LA-ICP-MS based immunoassays KW - Antibody labeling PY - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:b43-384712 SN - 0003-2654 SN - 1364-5528 VL - 141 IS - 23 SP - 6374 EP - 6380 PB - The Royal Society of Chemistry AN - OPUS4-38471 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Wanka, Antje Jutta A1 - Schwab, Karima A1 - Theuring, Franz A1 - Müller, Larissa A1 - Jakubowski, Norbert A1 - Lauer, Dilyara T1 - Novel antibody tagging strategy using lanthanide loaded NHS-dota-ester for the application in highly selective LA-ICP-MS-based immunoassays N2 - Using metal tagged antibodies for multiplex immunoassays is an important aspect in the research field of bioanalytics. Various tagging strategies have been published, however the formed antibody conjugates were described only rudimentarily. We present an inexpensive, simple and fast DOTA-NHS-ester tagging strategy and the characterization of the conjugates. The monoclonal anti-beta-actin-antibody was used as model system and was modified with the bifunctional ligand DOTA-NHS-ester complexing monoisotopic lanthanide ions. In contrast to the antibody tagging with other tags based on maleimide chemistry the DOTA-NHS-ester reacts with unprotonated amino groups instead of free sulfhydryl groups. The great advantage of this is that a partially reduction of the antibody is not required and therefore the antibody remains intact. The characterization of the conjugates by different analytical techniques demonstrates that no antibody fragments are formed. A mean tagging degree of 8 tags/antibody was determined. The applicability of this tagging strategy is shown in first results of an Alzheimer’s disease study in a mouse model. We have chosen three different NHS-DOTA-ester tagged antibodies for multielement LA-ICP-MS based bioimaging of mice brain tissue. The artificial introduced lanthanides as well as naturally occurring elements were analyzed simultaneously and differences between transgenic and wild-type animals are presented. T2 - Metallomics CY - Wien, Austria DA - 13.08.2017 KW - Antibody tagging KW - LA-ICP-MS based immunoassays PY - 2017 AN - OPUS4-41877 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Herrmann, Antje Jutta A1 - Techritz, Sandra A1 - Jakubowski, Norbert A1 - Haase, A. A1 - Luch, A. A1 - Panne, Ulrich A1 - Müller, Larissa T1 - A simple metal staining procedure for identification and visualization of single cells by LA-ICP-MS N2 - High lateral resolution of metal detection in single cells by use of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) demands powerful staining methods. In this work different staining procedures for the single cell analysis with LA-ICP-MS were optimized. An iridium intercalator was utilized to stain the cell nuclei whereas the whole cell was stained by the use of maleimido-mono-amide-DOTA (mDOTA) complexing lanthanide(III) ions. The content of the artificially introduced metals per cell was quantified using a matrix matched calibration approach based on cellulose membranes onto which standards were spotted by a microarray spotter. Absolute metal stain amounts in the range of 2.34 to 9.81 femtomole per cell were determined. The metal staining procedures allow direct identification and visualization of single cells and their cell compartments by element microscopy without the use of bright field images of the sample. KW - Single Cell Analysis KW - Bioimaging KW - LA-ICP-MS based immunoassays PY - 2017 U6 - https://doi.org/10.1039/c6an02638a SN - 0003-2654 SN - 1364-5528 VL - 142 IS - 10 SP - 1703 EP - 1710 PB - The Royal Society of Chemistry AN - OPUS4-40251 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -