Lectin-binding assay by polyethylene glycol 8000.
A quantitative lectin-binding assay using a precipitation technique and polyethylene glycol 8000 (PEG) as a precipitating agent has been described. Carcinoscorpin, a sialic acid-binding lectin isolated from the hemolymph of Indian horseshoe crab, Carcinoscorpius rotunda cauda, and iodinated fetuin, a sialoglycoprotein, were appropriately incubated as the components of the binding assay. The specific interaction between these two components developed the lectin-glycoprotein-bound complex. This was subsequently precipitated by the addition of PEG together with a coprecipitant gamma-globulin. Radioactivity of the precipitated bound complex was estimated to quantify the binding. The formation of the bound complex was effectively inhibited by a specific sialodisaccharide, O-(N-acetylneuraminyl)-(2----6)-2-acetamido-2-deoxygalactitol, implying the specific interaction for such precipitation. The probable effect of PEG was to stabilize the bound complex, precipitating it along with added gamma-globulin. This was further evident from the prevention of dissociation of the bound complex and increased binding of glycoprotein to the immobilized lectin in the presence of PEG. The assay was also applicable to other sialoglycoproteins such as alpha 1-acid glycoprotein and human chorionic gonadotropin. Moreover, the method yielded a saturation plateau with a characteristic hyperbolic binding curve. The assay was simple, quick, safe, economic, and highly sensitive.
D004355:Drug Stability; D006863:Hydrogen-Ion Concentration; D007457:Iodine Radioisotopes; D037102:Lectins / Q000032:analysis* / Q000302:isolation & purification; D011092:Polyethylene Glycols / Q000032:analysis*; D063265:Sialic Acid Binding Immunoglobulin-like Lectins; D012795:Sialoglycoproteins / Q000032:analysis; D000509:alpha-Fetoproteins / Q000032:analysis; D005719:gamma-Globulins
D007457:Iodine Radioisotopes; D037102:Lectins; D063265:Sialic Acid Binding Immunoglobulin-like Lectins; D012795:Sialoglycoproteins; D000509:alpha-Fetoproteins; D005719:gamma-Globulins; D011092:Polyethylene Glycols