Demonstration of Glycosylation Variants of Human Fibrinogen, Using the New Technique of Glycoprotein Lectin Immunosorbent Assay (GLIA)

Abstract

A new highly sensitive method, incorporating the E LISA technique (enzymelinked immunosorbent assay), is described for the quantitation of the glycan residues of glycoproteins. With the aid of this "glycoproteinlectin immunosorbent assay (GLIA)", it is possible to determine the nature of the glycan residues of a single protein in a glycoprotein mixture, without prior purification. The GLIA can be used for the accurate determination of the inhibitor constant for the interaction of any monosaccharide with any lectin.Using the described technique, glycosylation of human fibrinogen from plasma and amniotic fluid were compared. In fibrinogen from amniotic fluid a "fetal" glycosylation type could be demonstrated. In addition, evidence is presented for the first time that plasma fibrinogen possesses (GlcA/Ac/31-*4Man|3) residues (bisecting Glo/VAc) and O-glycosidically bound carbohydrate units. Preliminary results were published as abstract (E. Kotigen et al. (1988) Fresenius Z. Anal Chem. 330, 448).

Evidence that the major outer membrane protein of Chlamydia trachomatis is glycosylated

“…Lectin binding assay. The technique for binding glycoprotein to lectin was adapted from Kottgen et al (10). The MAb (1:500 dilution) in HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) basic solution (HBS) (137 mM NaCl, 2.7 mM KCI, 0.9 mM CaCl2, 0.5 mM MgCl2, 30 mM HEPES [pH 7.4]) was adsorbed onto the wells of a microtiter plate at 37°C overnight.…”

The development of a sialic acid specific lectin‐immunoassay for the measurement of human chorionic gonadotrophin glycoforms in serum and its application in normal and Down's syndrome pregnancies

“…Furthermore, when expressed as a ratio in conjunction with a conventional immunoassay the average number of sialic acid residues per hCG molecule can be calculated. Lectin immunoassays for plasma glycoproteins and hormones employing solid phase antibody and labelled lectin have been previously described for the detection of CA125 (Madiyalakan et al, 1996), a 1 acid glycoprotein and transferrin (Parkkinen & Oksanen, 1989), a-fetoprotein (Kinoshita et al, 1988;Suzuki et al, 1990), and fibrinogen (Kottgen et al, 1988). An alternative approach using solid phase peanut lectin (PNA) which specifically binds to the desialylated 0-serine linked oligosaccharides of hCG and labelled antibody has been employed for the measurement of desialylated forms of hCG in urine (Imamura, 1985;Imamura et al, 1987).…”

“…A further potential problem is that it is possible that the lectin itself can bind to carbohydrate on the side chain Fc region of immunoglobulin using either configuration and give rise to even higher non specific binding. Various techniques have been proposed to overcome this problem including the use of affinity purified antibodies (Pekelharing et al, 1987;Suzuki et al, 1990) and chemical or enzymatic deglycosylation of the solid phase antibody (Kinoshita et Kottgen et al, 1988).…”

Thrombin-Antithrombin III Complexes and Antithrombin III in Amniotic Fluid

“…In normal pregnancy there is no clottable fibrino gen, though its traces could be detected by an immunocnzymatic method [1], In pregnancy complicated with anencephaly and spina bifida, fibrinogen/fibrin degrada tion products are significantly elevated [2,3], and some times even fibrinogen could be found [4], Factor V activi ty has been observed only in some samples [5] and factor VIII coagulant antigen was undetectable [6]. Concentra tions of prothrombin and factors VII and X as well as antithrombin III (AT III) have been estimated as very low, ranging from several to a dozen or so percent of the KARGER © I9 9 7 S .…”