Assessment of immunoglobulin capture in immobilized protein A through automatic bead injection

• Autor(a) principal: Ramos, Inês I.
• Data de Publicação: 2019
• Outros Autores: Marques, Sara S., Magalhães, Luís M., Barreiros, Luisa, Reis, Salette, Lima, José L.F. C., Segundo, Marcela A.
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.22/16263
• topic: Armoracia; Benzidines; Chromatography, Affinity; Chromogenic Compounds; Colorimetry; Horseradish Peroxidase; Immobilized Proteins; Immunoglobulin G; Microspheres; Oxidation-Reduction; Saliva; Sepharose; Staphylococcal Protein A

The repeatable immobilization of molecular recognition elements onto particle surfaces has a strong impact on the outcomes of affinity-based assays. In this work, an automatic method for the immobilization of immunoglobulin G (IgG) onto protein A-Sepharose microbeads was established through the flow programming features of the portable lab-on-valve platform using micro-bead injection spectroscopy. The reproducible packing of protein A-microbeads between two optic fibers was feasible, allowing on-column probing of IgG retention. The automation of solutions handling and the precise control of time of IgG interaction with the beads rendered repeatable immobilization cycles, within a short timeframe (<2 min). The proposed method featured the preparation of disposable immunosorbents for downstream analytical applications, such as immunosensing or microenrichment of target analytes. In-situ quantification of IgG@protein A-microbeads was carried out using a horseradish peroxidase-labeled detection IgG. The colorimetric oxidation of 3,3',5,5'-tetramethylbenzidine was monitored on-column. Quantitation of mouse and human IgG immobilized@protein A-microbeads was achieved for loading masses between 0.1 and 0.4 μg per ca. 5.5 mg of sorbent. The implemented detection strategy allowed the quantification of human IgG in certified human serum (ERM®- DA470k/IFCC) and spiked saliva, yielding recoveries of 102-108% and requiring minimal volume (1-15 μL) from serum and saliva.