Magnetic bead-based fluorometric detection of lectin-glycoprotein interactions.
A sandwich-type bioassay was developed to detect the lectin-glycoprotein interactions using fluorescein isothiocyanate (FITC) as the label. The bioassay was optimized using the well-described lectins and their target glycoprotein. The biotinylated lectins from Triticum vulgaris (wheat germ, WGA) and Arachis hypogaea (peanut, PNA) were immobilized on streptavidin-coated superparamagnetic iron oxide microparticles (beads) and utilized for the isolation of target biomolecules (Invertase from Saccharomyces cerevisiae) from complex matrices. The secondary lectin layer was labeled with biotin, which allowed the binding of ExtrAvidin-conjugated FITC. The fluorescence signal of FITC was measured at an emission wavelength of 519nm. By optimizing the experimental conditions, changes in the fluorescence signals displayed linear concentration dependence until 50microg/mL invertase. The fluorescence intensity peaked, when the WGA and PNA concentrations were at 50microg/mL. The proof-of-concept study shows that the versatile and simple bioanalytical technique is a promising candidate for the rapid screening of lectin-glycoprotein interactions.
Rambihar
Christopher
C
Department of Physical and Environmental Sciences, University of Toronto at Scarborough, 1265 Military Trail, Ontario, Canada.
Kerman
Kagan
K
D000373:Agglutinins / Q000737:chemistry; D001681:Biological Assay; D019921:Biotinylation; D002623:Chemistry Techniques, Analytical; D005290:Ferric Compounds / Q000737:chemistry; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005453:Fluorescence; D005470:Fluorometry / Q000379:methods*; D006023:Glycoproteins / Q000737:chemistry*; D037102:Lectins / Q000737:chemistry*; D008280:Magnetics; D053758:Nanoparticles / Q000737:chemistry; D019887:Peanut Agglutinin / Q000737:chemistry; D019809:Streptavidin / Q000737:chemistry; D014908:Triticum / Q000378:metabolism
D000373:Agglutinins; D005290:Ferric Compounds; D006023:Glycoproteins; D037102:Lectins; D019887:Peanut Agglutinin; C000499:ferric oxide; D019809:Streptavidin; D016650:Fluorescein-5-isothiocyanate