Lectin ELISA for Analysis of α1-Acid Glycoprotein Fucosylation in the Acute Phase Response

Abstract

In recent years, increasing attention has been directed to the specific changes in glycosylation of glycoproteins that occurs in response to certain physiologic and pathologic conditions (1)(2)(3)(4)(5)(6). α1-Acid glycoprotein (AGP; orosomucoid) is a heavily glycosylated acute phase protein with five glycosylation sites carrying N-linked, complex-type oligosaccharides (N-glycans). The function of AGP is not well understood, but immunomodulating properties have been suggested that may be dependent on the expression of the sialyl LewisX (SLeX) epitope, in which fucose is a necessary component (7). However, methods for analysis of the glycosylation of acute phase proteins have been time-consuming and not suitable for routine analysis in a clinical laboratory (3)(6)(8). A lectin ELISA has been used previously for analysis of a tumor marker (9). The Aleuria aurantia lectin (AAL) was used in an ELISA for analysis of fucosylation of serum cholinesterase in liver disease (10). We developed a lectin ELISA that uses biotinylated AAL and a capture antibody specific for AGP to study the fucosylation on AGP in the acute phase response. As a model for acute inflammation, we monitored the daily changes in AGP fucosylation in patients with severe burns. The plasma concentration of AGP and C-reactive protein (CRP) was analyzed on a Cobas Integra 700 (Roche). AAL was purified from locally picked mushrooms as described previously (7). AAL fractions were pooled, lyophilized, and stored at −20 °C until biotinylation was performed according to the manufacturer’s instructions (ImmunoprobeTM Biotinylation Kit; Sigma). Biotinylated AAL was stored at 4 °C. Microtiter plates (Nunc-ImmunoTM Maxisorp) were coated with polyclonal antibodies directed against human AGP (anti-human orosomucoid, cat. no. A0011; Dako), diluted 1:100 in coating buffer (15 mmol/L Na2CO3, 35 mmol/L …