Percorrer por autor "Afshari, Ali R."
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- Clinical Europium fluorescent based lectin assays for mucin O-glycomicsPublication . Afshari, Ali R.; Bello-Gonzalez, Marysol; Andersen, Ida E.; Nogueira, Rita Martins; Karlsson, Niclas G.; Malaker, Stacy A.In this chapter, we are presenting two assay formats for glycomic discovery and validation: Fluorescent Immune-Lectin Assay (FILA) and Fluorescent Lectin Assay (FLA). Both methods rely on conjugation of lectins to highly sensitive Europium nanoparticles (Eu-NPs) that provide specific detection of glycans. The FILA method is based on antibody immobilization, where biotinylated antibodies capture target mucin onto microplate wells before lectin-Eu-NP binding. In contrast, the FLA method is based on hydrophobic interactions that allow direct attachment of mucins or mucin-type glycoproteins to the plate surface without requiring a capturing antibody. The FLA method also uses the same lectin-Eu-NP detection strategy. Each method is described in a detailed workflow from buffer and sample preparation, detection and obtaining signals, to concentration measurement. Evaluation protocols for both methods are provided at the end of this chapter. These assays provide effective and flexible strategies for screening of glycoproteins and mucin-type molecules and their glycosylation in complex biological samples such as plasma (e.g., lubricin in plasma) or mucosal surfaces (e.g., mucins in tear fluid). Orthogonal tests using mass spectrometry (MS) and western blot analysis are recommended to enhance certainty in glycosylation profiles obtained from either the FILA or FLA method. Ultimately, these methods support researchers in selecting and implementing practical approaches for clinical and diagnostic applications in glycomics.