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

• Type: Article in International Scientific Journal
• Date: 2019

Authors

Ramos, II (Author) Other; Marques, SS (Author) Other; Magalhaes, LM (Author) Other; Luisa Barreiros (Author) FFUP; Salette Reis (Author) FFUP; Lima, JLFC (Author) FFUP; Marcela A Segundo (Author) FFUP

Journal

Title: Talanta

Vol. 204

Pages: 542-547

ISSN: 0039-9140

Publisher: Elsevier

Indexing

ISI Web of Knowledge - 0 Citations

Scopus - 1 Citation

Other information

Authenticus ID: P-00Q-QCK

DOI: 10.1016/j.talanta.2019.06.023

Abstract (EN): 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 mu g per ca. 5.5 mg of sorbent. The implemented detection strategy allowed the quantification of human IgG in certified human serum (ERM (R) - DA470k/IFCC) and spiked saliva, yielding recoveries of 102-108% and requiring minimal volume (1-15 mu L) from serum and saliva.

Language: English

Type (Professor's evaluation): Scientific

No. of pages: 6