title: Alkaline phosphatase-immunoglobulin conjugate binds to lipids in vitro, independent of antibody selectivity.
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.
authors.affiliation: Department of Integrative Biology, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada N1G 2W1. lingwood@mpi-cbg.de
authors.forename: D
authors.initials: D
authors.lastname: Lingwood
authors.forename: J S
authors.initials: JS
authors.lastname: Ballantyne
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry / Q000378:metabolism*; D000918:Antibody Specificity; D002855:Chromatography, Thin Layer; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000378:metabolism; D018796:Immunoconjugates / Q000737:chemistry / Q000378:metabolism*; D007124:Immunoenzyme Techniques / Q000379:methods*; D007136:Immunoglobulins / Q000737:chemistry / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D011136:Polysorbates / Q000494:pharmacology; D013107:Sphingolipids / Q000378:metabolism*
chemical_list: D018796:Immunoconjugates; D007136:Immunoglobulins; D010743:Phospholipids; D011136:Polysorbates; D013107:Sphingolipids; D006735:Horseradish Peroxidase; D000469:Alkaline Phosphatase