Performance benchmarking microplate-immunoassays for quantifying target-specific cysteine oxidation reveals their potential for understanding redox-regulation and oxidative stress

Tuncay, Ahmet; Crabtree, Daniel R.; Muggeridge, David J.; Husi, Holger; Cobley, James N.

Abstract

The antibody-linked oxi-state assay (ALISA) for quantifying target-specific cysteine oxidation can benefit specialist and non-specialist users. Specialists can benefit from time-efficient analysis and high-throughput target and/or sample n-plex capacities. The simple and accessible “off-the-shelf” nature of ALISA brings the benefits of oxidative damage assays to non-specialists studying redox-regulation. Until performance benchmarking establishes confidence in the “unseen” microplate results, ALISA is unlikely to be widely adopted. Here, we implemented pre-set pass/fail criteria to benchmark ALISA by robustly evaluating immunoassay performance in diverse biological contexts. ELISA-mode ALISA assays were accurate, reliable, and sensitive. For example, the average inter-assay CV for detecting 20%- and 40%-oxidised PRDX2 or GAPDH standards was 4.6% (range: 3.6–7.4%). ALISA displayed target-specificity. Immunodepleting the target decreased the signal by ∼75%. Single-antibody formatted ALISA failed to quantify the matrix-facing alpha subunit of the mitochondrial ATP synthase. However, RedoxiFluor quantified the alpha subunit displaying exceptional performance in the single-antibody format. ALISA discovered that (1) monocyte-to-macrophage differentiation amplified PRDX2-specific cysteine oxidation in THP-1 cells and (2) exercise increased GAPDH-specific cysteine oxidation in human erythrocytes. The “unseen” microplate data were “seen-to-be-believed” via orthogonal visually displayed immunoassays like the dimer method. Finally, we established target (n = 3) and sample (n = 100) n-plex capacities in ∼4 h with 50–70 min hands-on time. Our work showcases the potential of ALISA to advance our understanding of redox-regulation and oxidative stress.

Citation

Tuncay, A., Crabtree, D. R., Muggeridge, D. J., Husi, H., & Cobley, J. N. (2023). Performance benchmarking microplate-immunoassays for quantifying target-specific cysteine oxidation reveals their potential for understanding redox-regulation and oxidative stress. Free Radical Biology and Medicine, 204, 252-265. https://doi.org/10.1016/j.freeradbiomed.2023.05.006

• Journal Article Type: Article
• Acceptance Date: May 5, 2023
• Online Publication Date: May 14, 2023
• Publication Date: 2023-08
• Deposit Date: May 23, 2023
• Publicly Available Date: May 23, 2023
• Print ISSN: 0891-5849
• Electronic ISSN: 1873-4596
• Publisher: Elsevier
• Peer Reviewed: Peer Reviewed
• Volume: 204
• Pages: 252-265
• DOI: https://doi.org/10.1016/j.freeradbiomed.2023.05.006
• Keywords: Cysteine, ALISA, Redox regulation, Oxidative stress, Immunology, Development, Exercise