Systematic Optimisation of Microtiter Plate Lectin Assay to Improve
Sialic Acid Linkage Detection

Nur   Hanina Izzati   Khairol Mokhtar; Ainulkhir      Hussin; Aidil   Abdul   Hamid; Shahrul   Hisham   Zainal Ariffin; Muhammad   Ashraf    Shahidan

doi:10.2174/1386207324666210802122538

Abstract

Aims: We aimed to develop a high-throughput lectin assay with minimized background signals to investigate the interactions of lectins and sialic acid glycans, focusing on Prostate- Specific Antigen (PSA).

Background: High background signals resulting from nonspecific binding are a significant concern for microtiter plate-based Enzyme-Linked Lectin Sorbent Assays (ELISAs), as they can mask specific binding signals and cause false-positive results.

Methods: In this study, we constructed an ELISA based on different washing step parameters, including the number of washing cycles, NaCl and Tween-20 concentrations, and the type of blocking agent and evaluated the effects on both specific and nonspecific binding signals. Furthermore, we performed a PSA binding assay using the optimized ELISA.

Results: The optimal washing parameters based on the highest specific binding signal proposed four cycles of washing steps using a washing buffer containing a high salt concentration (0.5 M NaCl) and mild detergent (0.05% Tween-20). The utilization of the optimized washing parameters in this assay was shown to be sufficient to obtain the optimal binding signals without the use of any blocking agent. Binding assays performed using the optimized ELISA revealed that the glycan of the PSA sample used in this study mainly consists of terminal α2,6-linked sialic acid, as strongly recognized by Sambucus nigra agglutinin (SNA) with a KD value of 12.38 nM.

Conclusion: The ELISA reported in this study provides a simple yet sensitive assay for sialic acid linkage recognition.

Keywords: Sialic acid, glycans, lectins, prostate-specific antigen, enzyme-linked lectin sorbent assay, high-throughput.

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Graphical Abstract

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DOI https://dx.doi.org/10.2174/1386207324666210802122538	Print ISSN 1386-2073
Publisher Name Bentham Science Publisher	Online ISSN 1875-5402

Combinatorial Chemistry & High Throughput Screening

Systematic Optimisation of Microtiter Plate Lectin Assay to Improve Sialic Acid Linkage Detection

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