Preparation of neoglycoprotein-enzyme conjugate using a heterobifunctional reagent and its use in solid-phase assays and histochemistry.
A conjugate of a neoglycoprotein (chemically lactosylated bovine serum albumin) and an enzyme (horseradish peroxidase) has been prepared in solution using a heterobifunctional reagent, N-succinimidyl-3-(2-pyridyldithio)propionate, and has been purified by gel filtration on an Ultrogel AcA-44 column. To preclude any carbohydrate-dependent binding to the sugar residues on the glycoprotein peroxidase, the enzyme has to be treated with sodium periodate and sodium cyanoborohydride prior to coupling, which results in oxidative cleavage of the carbohydrates and reduction of the aldehydes thus formed to primary alcohols. Lactosylated bovine serum albumin-peroxidase conjugate has been employed to detect plastic-bound Ricinus communis agglutinin with dependence of the concentration of the lectin and with dependence of the presence of specific inhibitors. Enzyme-labeled conjugates with unmodified bovine serum albumin are completely ineffective in this assay. Localization of beta-galactoside-specific sugar receptors in connective tissue is used to demonstrate the feasibility of application of such neoglycoprotein-enzyme conjugates in histochemistry with a minimum number of steps.
Gabius
H J
HJ
Max-Planck-Institut für experimentelle Medizin, Abteilung Chemie, Göttingen, Federal Republic of Germany.
Engelhardt
R
R
Hellmann
K P
KP
Hellmann
T
T
Ochsenfahrt
A
A
D000230:Adenocarcinoma / Q000473:pathology; D000818:Animals; D002241:Carbohydrates / Q000032:analysis*; D002850:Chromatography, Gel; D003110:Colonic Neoplasms / Q000473:pathology; D006023:Glycoproteins / Q000138:chemical synthesis*; D006652:Histological Techniques; D006735:Horseradish Peroxidase; D006801:Humans; D007202:Indicators and Reagents; D007785:Lactose; D051379:Mice; D008819:Mice, Nude; D010544:Peroxidases; D012710:Serum Albumin, Bovine
D002241:Carbohydrates; D006023:Glycoproteins; D007202:Indicators and Reagents; D012710:Serum Albumin, Bovine; D006735:Horseradish Peroxidase; D010544:Peroxidases; D007785:Lactose