The biotin/avidin-mediated microtiter plate lectin assay with the use of chemically modified glycoprotein ligand.
The conditions of a simple and sensitive microtiter plate lectin assay with biotinylated TF- and/or Tn-reactive lectins and ExtrAvidin/alkaline phosphatase conjugate are described. As ligand for lectin binding, chemically modified glycophorin A from human erythrocytes was used. The TF and Tn receptors present in glycophorin A in cryptic form were exposed by desialylation under mild acidic conditions (TF) and by removing galactose residues from asialoglycophorin by Smith degradation (Tn). These modifications can be performed either in solution or on the plate coated with untreated glycophorin. It was demonstrated with six lectins that their biotinylation via lectin amino groups gave products of higher binding activity than biotinylation via periodate-oxidized carbohydrate residues of lectins. The first step in the binding assay requires the selection of the proper concentration of the glycoprotein used for coating the plate, since the lectins tested showed a maximal binding at an optimal glycophorin concentration, and in many cases the binding was distinctly lower when a higher ligand concentration was used for coating. The inhibition of binding of Tn-reactive lectins to plates coated with asialo-agalactoglycophorin (Tn antigen) was performed using low- and high-molecular-weight inhibitors (1-2 micrograms lectin was used for each inhibition curve) and concentrations of inhibitors required for 50% inhibition of lectin binding were compared. The results were in agreement with the known specificity of the lectins tested. In conclusion, the method described is simple, sensitive, and versatile, enabling the characterization of lectin specificity with a broad spectrum of inhibitors using microgram quantities of lectin only.
Duk
M
M
Department of Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw.
Lisowska
E
E
Wu
J H
JH
Wu
A M
AM
D000116:Acetylgalactosamine; D000818:Animals; D001667:Binding, Competitive; D001710:Biotin; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005690:Galactose; D006021:Glycophorins / Q000737:chemistry; D006023:Glycoproteins; D006801:Humans; D037102:Lectins / Q000032:analysis*; D008024:Ligands; D008969:Molecular Sequence Data; D009077:Mucins; D012680:Sensitivity and Specificity; D012756:Sheep
D006021:Glycophorins; D006023:Glycoproteins; D037102:Lectins; D008024:Ligands; D009077:Mucins; D001710:Biotin; D000116:Acetylgalactosamine; D005690:Galactose