Abstract
Both botulinum neurotoxins (BoNTs) and anthrax lethal factor, a component of anthrax toxin, exhibit zinc metalloprotease activity. The assay detailed here is capable of quantitatively detecting these proteins by measuring their enzymatic functions with high sensitivity. The detection method encompasses two steps: (1) specific target capture and enrichment and (2) cleavage of a fluorogenic substrate by the immobilized active target, the extent of which is quantitatively determined by differential fluorometry. Because a critical ingredient for the target enrichment is an immobilization matrix made out of hundreds of thousands of microscopic, antibody-coated beads, we have termed this detection method an assay with a large immuno-sorbent surface area (ALISSA). The binding and reaction surface area in the ALISSA is approximately 30-fold larger than in most microtiter plate-based enzyme-linked immunosorbent assays (ELISAs). ALISSA reaches atto (10–18) to femto (10–15) molar sensitivities for the detection of BoNT serotypes A and E and anthrax lethal factor. In addition, ALISSA provides high specificity in complex biological matrices, such as serum and liquid foods, which may contain various other proteases and hydrolytic enzymes. This methodology can potentially be expanded to many other enzyme targets by selecting appropriate fluorogenic substrates and capture antibodies. Important requirements are that the enzyme remains active after being immobilized by the capture antibody and that the substrate is specifically converted by the immobilized enzyme target at a fast conversion rate.
A detailed protocol to conduct ALISSA for the detection and quantification of BoNT serotypes A and E and anthrax lethal factor is described.
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Acknowledgments
We would like to thank Dr. Larry Stanker of the US Department of Agriculture for his monoclonal mouse antibodies and Dr. Bruce Kaplan of City of Hope for the synthesis of fluorogenic peptide substrates. This work was supported by the National Institutes of Health Grants U54 AI065359 (Pacific Southwest Regional Center of Excellence).
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Bagramyan, K., Kalkum, M. (2011). Ultrasensitive Detection of Botulinum Neurotoxins and Anthrax Lethal Factor in Biological Samples by ALISSA. In: Holst, O. (eds) Microbial Toxins. Methods in Molecular Biology, vol 739. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-102-4_3
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DOI: https://doi.org/10.1007/978-1-61779-102-4_3
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