Alkaline phosphatase-immunoglobulin conjugate binds to lipids in vitro, independent of antibody selectivity

Journal of Immunological Methods
D Lingwood, J S Ballantyne

Abstract

At present, alkaline phosphatase (AP) conjugates are major workhorses of immunological detection. However, APs are membrane bound enzymes, and therefore have the potential to interact with lipids. Using TLC overlay, we screened AP-conjugated immunoglobulins (IgGs), and AP-conjugated streptavidin, for their ability to bind sphingolipids and phospholipids non-specifically. Horseradish peroxidase (HRP)-conjugated IgG was tested as a negative control. AP-conjugates bound to all sphingolipids and phospholipids assayed, whereas no HRP-IgG binding was observed. AP conjugate-lipid binding could be reduced by pretreatment of chromatograms with polyisobutylmethacrylate. Addition of Tween 20 also abolished AP-lipid binding, except to lactosyl ceramide, suggesting a degree of specificity. This study serves to prevent spurious interpretation of AP-conjugate based binding assays, be they against purified lipids/lipid mixtures or tissue samples from which lipids have not been removed.

Citations

Jul 11, 2012·Biotechnology Letters·Kosuke MinamihataNoriho Kamiya
Nov 27, 2019·Jornal brasileiro de nefrologia : ʹorgão oficial de Sociedades Brasileira e Latino-Americana de Nefrologia·Hanna Karla Andrade Guapyassú MachadoRosa Maria Affonso Moysés

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