Toshiro Niki1,2, Koji Fujita3, Hugo Rosen4, Mitsuomi Hirashima5,3, Tsutomu Masaki3, Toshio Hattori6, Katsuaki Hoshino7. 1. Department of Immunology, Faculty of Medicine, Kagawa University, Kita-gun, Japanniki@med.kagawa-u.ac.jp. 2. GalPharma Co., Ltd., Takamatsu, Japanniki@med.kagawa-u.ac.jp. 3. Department of Gastroenterology and Neurology, Faculty of Medicine, Kagawa University, Kita-gun, Japan. 4. Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, California, USA. 5. GalPharma Co., Ltd., Takamatsu, Japan. 6. Graduate School of Health Science, Kibi International University, Okayama, Japan. 7. Department of Immunology, Faculty of Medicine, Kagawa University, Kita-gun, Japan.
Abstract
BACKGROUND/AIMS: Galectin-9 is a soluble immune modulator with versatile functions, including a role as an immune checkpoint molecule. Therefore, the amount of galectin-9 in the blood may reflect an individual's immunological balance. Many studies have conducted galectin-9 measurements; however, the reported galectin-9 concentration in the blood varies greatly, even within healthy controls. This study investigates the variation between the reported and actual concentrations of galectin-9. METHODS: A GalPharma ELISA and an R&D Systems ELISA kit were directly compared using the same set of plasma and a series of recombinant galectins, including degraded galectin-9. Furthermore, galectin-9 in plasma was concentrated using anti-galectin-9 antibody-conjugated beads, and subjected to western blotting to estimate the quantity and integrity of galectin-9 and assess the consistency of ELISA measurements. RESULTS: The R&D Systems' ELISA indicated a 50-fold higher median concentration of plasma galectin-9 than that indicated by the GalPharma ELISA. This variation is due to aberrantly enhanced reactivity of the R&D Systems' ELISA to degraded galectin-9 present in small quantities in the plasma. The GalPharma ELISA could detect only intact galectin-9 and its results correlated well with the plasma galectin-9 level obtained by western blotting. CONCLUSION: ELISA kits from R&D Systems reacts aberrantly higher against degraded galectin-9 than the intact galectin-9. Therefore, the existence of a small amount of degraded galectin-9 in a test sample hinders the quantification. As galectin-9 is a fragile protein, this is a serious concern when using this kit. Based on quantifications from the GalPharma ELISA, the median (25th-75th percentiles) galectin-9 concentration in healthy subjects in the current study cohort was calculated as 110 pg/mL (67 -154 pg/mL).
BACKGROUND/AIMS: Galectin-9 is a soluble immune modulator with versatile functions, including a role as an immune checkpoint molecule. Therefore, the amount of galectin-9 in the blood may reflect an individual's immunological balance. Many studies have conducted galectin-9 measurements; however, the reported galectin-9 concentration in the blood varies greatly, even within healthy controls. This study investigates the variation between the reported and actual concentrations of galectin-9. METHODS: A GalPharma ELISA and an R&D Systems ELISA kit were directly compared using the same set of plasma and a series of recombinant galectins, including degraded galectin-9. Furthermore, galectin-9 in plasma was concentrated using anti-galectin-9 antibody-conjugated beads, and subjected to western blotting to estimate the quantity and integrity of galectin-9 and assess the consistency of ELISA measurements. RESULTS: The R&D Systems' ELISA indicated a 50-fold higher median concentration of plasma galectin-9 than that indicated by the GalPharma ELISA. This variation is due to aberrantly enhanced reactivity of the R&D Systems' ELISA to degraded galectin-9 present in small quantities in the plasma. The GalPharma ELISA could detect only intact galectin-9 and its results correlated well with the plasma galectin-9 level obtained by western blotting. CONCLUSION: ELISA kits from R&D Systems reacts aberrantly higher against degraded galectin-9 than the intact galectin-9. Therefore, the existence of a small amount of degraded galectin-9 in a test sample hinders the quantification. As galectin-9 is a fragile protein, this is a serious concern when using this kit. Based on quantifications from the GalPharma ELISA, the median (25th-75th percentiles) galectin-9 concentration in healthy subjects in the current study cohort was calculated as 110 pg/mL (67 -154 pg/mL).
Authors: Valerie R Wiersma; Alex Clarke; Simon D Pouwels; Elizabeth Perry; Trefa M Abdullah; Clive Kelly; Anthony De Soyza; David Hutchinson; Paul Eggleton; Edwin Bremer Journal: Int J Mol Sci Date: 2019-08-19 Impact factor: 5.923
Authors: Thomas A Premeaux; Carlee B Moser; Ashley McKhann; Martin Hoenigl; Elizabeth I Laws; Draven L Aquino; Michael M Lederman; Alan L Landay; Sara Gianella; Lishomwa C Ndhlovu Journal: AIDS Date: 2021-12-01 Impact factor: 4.632