TY - JOUR A1 - Carl, Peter A1 - Ramos, InĂªs I. A1 - Segundo, Marcela A. A1 - Schneider, Rudolf T1 - Antibody conjugation to carboxyl-modified microspheres through N-hydroxysuccinimide chemistry for automated immunoassay applications: A general procedure JF - PLoS ONE N2 - Immunochemical techniques are the workhorse for sample enrichment and detection of a large variety of analytes. In contrast to classical microtiter plate-based assays, microparticles are a next generation solid support, as they promote automation of immunoassays using flow-based techniques. Antibody immobilization is a crucial step, as these reagentsmare expensive, and inefficient coupling can result in low sensitivities. This paper proposes a general procedure for efficient immobilization of antibodies onto TentaGel particles, via Nhydroxysuccinimide chemistry. The goal was the preparation of solid supports with optimum immunorecognition, while increasing the sustainability of the process. The influence of buffer composition, activation and coupling time, as well as the amount of antibody on the immobilization efficiency was investigated, resorting to fluorophore-labeled proteins and fluorescence imaging. Buffer pH and activation time are the most important parameters for efficient coupling. It is demonstrated, that the hydrolysis of N-hydroxysuccinimide esters occurs at similar rates as in solution, limiting the utilizable time for coupling. Finally, applicability of the generated material for automated affinity extraction is demonstrated on the mesofluidic platform lab-on-valve. KW - Mesofluidics KW - Bead injections KW - ELISA KW - Carbamazepine KW - Biomarkers PY - 2019 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-483919 DO - https://doi.org/10.1371/journal.pone.0218686 SN - 1932-6203 VL - 14 IS - 6 SP - e0218686, 1 EP - 18 PB - Public Library of Science CY - San Francisco, California, USA AN - OPUS4-48391 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -