Validation of a Toll-like receptor (TLR)2/TLR1 activation assay for biological standardization of Arthrospira/Limnospira immune-enhancing potency

Authors: HARON, MONA - University Of Mississippi; ZHANG, JIN - University Of Mississippi; CHITTIBOYINA, AMAR - University Of Mississippi; KHAN, IKHLAS - University Of Mississippi; PUGH, NIRMAL - University Of Mississippi

Submitted to: Journal of Dietary Supplements
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/4/2023
Publication Date: 10/4/2023
Citation: Haron, M.H., Zhang, J., Chittiboyina, A.G., Khan, I.A., Pugh, N.D. 2023. Validation of a Toll-like receptor (TLR)2/TLR1 activation assay for biological standardization of Arthrospira/Limnospira immune-enhancing potency. Journal of Dietary Supplements. https://doi.org/10.1080/19390211.2023.2263566.
DOI: https://doi.org/10.1080/19390211.2023.2263566

Interpretive Summary: The objective of this study was to establish and validate a bioassay for ImmulinaTM immune enhancing activity quantification using the U.S. FDA guidance document for botanical drug development and U.S. Pharmacopeia recommendations. Braun-type lipoproteins, have been identified as the major immune enhancing component within Immulina. It is a class of macromolecules that activate the Toll-like receptor (TLR)2/TLR1 signaling pathway. Based on the mechanism of action of the Braun-type lipoproteins, a bioassay was established using the HEK-Blue hTLR2/ TLR1 cell line to quantitate Immulina immune-enhancing potency. The validation process included establishing system suitability, reference standards and defining potency units. While validation of the assay performance parameters included precision, specificity, accuracy, linearity, and range. Validating this bioassay for Immulina activity quantification provides a tool for ensuring consistent product potency for use in future research as well as material in the consumer market.

Technical Abstract: Arthrospira/Limnospira is a popular botanical dietary supplement throughout the world and has been consumed as a food product for hundreds of years. Ongoing efforts from our research group is focused on evaluating the utility of a Limnospira-derived oral supplement (Immulina) in promoting resilience against influenza viral infection. Like other botanical extracts, Immulina is inherently a complex matrix with variation in the levels of its chemical constituents. Therefore, to ensure therapeutic consistency for future scientific research and clinical studies, we are developing standardization technology using a bioassay and chemical markers. Braun-type lipoproteins, a class of macromolecules that activate the Toll-like receptor (TLR)2/TLR1 signaling pathway, have been identified as a major active component within Immulina. Based on the mechanism of action of the Braun-type lipoproteins, an in vitro bioassay was established using the HEK-Blue hTLR2/TLR1 cell line to quantitate the immune-enhancing potency of Immulina. The objective of the current research was to validate that bioassay for Immulina activity quantification using the U.S. FDA guidance document for botanical drug development and U.S. Pharmacopeia recommendations. System suitability, reference standards and defining potency units were established. Validation of performance parameters included precision, specificity, accuracy, linearity, and range. Validating this bioassay for Immulina activity provides a tool for ensuring product consistency and quantifying the potency of this botanical for use in future research as well as material in the consumer market.