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

Author(s):

Nur Hanina Izzati Khairol Mokhtar, Ainulkhir Hussin, Aidil Abdul Hamid, Shahrul Hisham Zainal Ariffin and Muhammad Ashraf Shahidan*   Pages 1 - 11 ( 11 )

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 (ELLSAs), as they can mask specific binding signals and cause false-positive results.

Methods: In this study, we constructed an ELLSA 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 ELLSA.

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 ELLSA 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 ELLSA 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.

Affiliation:

School of Biological Science and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, Bangi, Selangor, Department of Pathology, Queen Elizabeth Hospital, Ministry of Health, Kota Kinabalu, Sabah, School of Biological Science and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, Bangi, Selangor, School of Biological Science and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, Bangi, Selangor, School of Biological Science and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, Bangi, Selangor



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