Comparison of blocking reagents for antibody microarray-based immunoassays on glass and paper membrane substrates.
Background noise
Blocking strategy
Complex biological fluids
Fluorescent intensity
Substrate surface chemistry
Journal
Analytical and bioanalytical chemistry
ISSN: 1618-2650
Titre abrégé: Anal Bioanal Chem
Pays: Germany
ID NLM: 101134327
Informations de publication
Date de publication:
Apr 2023
Apr 2023
Historique:
received:
17
01
2023
accepted:
17
02
2023
revised:
14
02
2023
medline:
30
3
2023
pubmed:
25
2
2023
entrez:
24
2
2023
Statut:
ppublish
Résumé
Background noise due to nonspecific binding of biomolecules on the assay substrates is one of the most common challenges that limits the sensitivity of microarray-based immunoassays. Background signal intensity usually increases when complex biological fluids are used because they have a combination of molecules and vesicles that can adsorb onto substrate surfaces. Blocking strategies coupled with surface chemistries can reduce such nonspecific binding and improve assay sensitivity. In this paper, we conducted a systematic optimization of blocking strategies on a variety of commonly used substrates for protein measurement in complex biofluids. Four blocking strategies (BSA, non-fat milk, PEG, and a protein-free solution) coupled with four surface chemistries (3-glycidoxypropyltrimethoxysilane (GPS), poly-L-lysine (PLL), aminoalkylsilane (AAS), and nitrocellulose (NC)) were studied for their effect on background, microspot, and net signal intensities. We have also explored the effect that these blocking strategies have when proteins in complex samples (plasma, serum, cell culture media, and EV lysate) are measured. Irregular spot morphology could affect signal extraction using automated software. We found that the microspots with the best morphology were the ones printed on GPS glass surfaces for all immunoassays. On NC membrane, the protein-based blocking strategies yielded the highest net fluorescent intensity with the antigen contained in PBS, plasma, serum, and serum-free cell culture media. Differently, with EV lysate samples, Pierce™ protein-free blocker yielded the best net signal intensity on both GPS and NC surfaces. The choice of blocking strategies highly depends on the substrate. Moreover, the findings discovered in this study are not limited to microarray-based immunoassays but can provide insights for other assay formats.
Identifiants
pubmed: 36829042
doi: 10.1007/s00216-023-04614-w
pii: 10.1007/s00216-023-04614-w
doi:
Substances chimiques
Indicators and Reagents
0
Antibodies
0
Proteins
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
1967-1977Informations de copyright
© 2023. Springer-Verlag GmbH Germany, part of Springer Nature.
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