A Lectin-Binding Assay for the Rapid Characterization of the Glycosylation of Purified Glycoproteins

Goodarzi, Mohammad Taghi; Fotinopoulou, Angeliki; Turner, G. A.

doi:10.1385/1-59259-169-8:795

Cited by 6 publications

(5 citation statements)

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“…The lectin/ELISA was performed as described previously [9]. Briefly, antibody (0.5 μg/ml) in 125 mM Tris, pH 7.5, containing 100 mM NaCl [Tris‐buffered saline (TBS)] was coated on to the surface of a multiwell plate for 2 h at 37 °C and unbound antibody was removed by washing with 0.1% (v/v) Tween 20/TBS (Tween/TBS).…”

Section: Methodsmentioning

confidence: 99%

“…Some samples that were probed with SNA and all samples that were probed with PNA were pre‐treated with neuraminidase [9]. PNA will only bind to O‐linked glycans containing galactose/ N ‐acetylgalactosamine if terminal N ‐acetylneuraminic acid (Neu5Ac) is removed.…”

Section: Methodsmentioning

confidence: 99%

“…In the lectin/ELISA [9], purified antibody is absorbed on to the plastic surface of a well in a microtitre plate. A lectin labelled with digoxigenin (DIG) is added and allowed to interact with the carbohydrate on the absorbed glycoprotein.…”

Section: Introductionmentioning

confidence: 99%

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Screening for glycosylation changes on recombinant human IgG using lectin methods

Fotinopoulou

Meyers

Varley

et al. 2003

Biotech and App Biochem

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Two lectin-binding methods were investigated as possible ways of monitoring the glycosylation of human monoclonal antibodies during their development and production. Carbohydrate composition was assessed in various preparations that were produced in different host cell types, cell sublines or batches of the same cells. The lectin binding was measured with ELISA and surface plasmon resonance (SPR). For comparative purposes, the monosaccharide content of many of the preparations was also measured by high-pressure anion exchange chromatography (HPAEC)/pulsed amperometric detection (PAD). Both lectin methods detected modifications in glycosylation when antibodies were produced in different ways; SPR was more sensitive than ELISA for some lectins and vice versa. Generally, the lectin results agreed with those obtained by the monosaccharide analysis; however, the former were much better for assessing N -acetylneuraminic acid changes. The latter were impossible to assess by HPAEC/PAD because of their low levels. The lectin-based methods also had the advantages that they were quicker to perform and required less expertise and could quickly identify structures that monosaccharide analysis might miss. It is suggested that, in the development of therapeutic proteins, monosaccharide analysis and/or oligosaccharide profiling is initially performed but later routine batches of the glycoprotein are screened with a lectin method. Of the two lectin methods used, SPR is much quicker when performing a screen, whereas ELISA is particularly useful for comparing a particular carbohydrate feature on different samples of the same glycoprotein.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Screening for glycosylation changes on recombinant human IgG using lectin methods

Fotinopoulou

Meyers

Varley

et al. 2003

Biotech and App Biochem

Self Cite

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“…Other approaches utilized more conventional constituents for saccharide recognition and binding such as lectins (section 2.2) for assaying glycoprotein composition and glycosylation. Several reviews summarize lectin overlay assays in which the sugar moieties were initially detached from the protein residues by enzyme digestion procedures. , The effectiveness and clinical potential of lectin-based assays for studying subtle changes in serum protein glycosylation, particularly associated with disease onset, have been reviewed …”

Section: 3 Glycoprotein and Glycosylation Biosensorsmentioning

confidence: 99%

Carbohydrate Biosensors

2004

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“…Lectin-based methods are used to study the glycosylation alterations of glycoproteins [14,15]. McDowell et al, by using an antibody-lectin sandwich assay, measured the asialylated LDL in the blood of patients with CAD [9].…”

Section: Introductionmentioning

confidence: 99%

Changes in sialylation of low‐density lipoprotein in coronary artery disease

Goodarzi

Rezaei

Moadel

et al. 2008

Euro J Lipid Sci & Tech

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Reduction of LDL sialylation may correlate with coronary artery disease (CAD), but the details of this modification and its effect on CAD are not well studied. This study was aimed to show desialylation of LDL and to reveal more details of this modification. Blood samples were collected from 16 patients with CAD and 25 healthy individuals. Serum sialic acid was determined. LDL was extracted from all samples, and the interaction of the extracted materials with lectins (MAA, SNA, and DSA) was studied using the lectin blotting method. Serum total sialic acid (TSA) concentrations in CAD patients and healthy individuals were 71.9 6 2.66 and 60.76 6 2.34 mg/dL, respectively, and the difference between the two groups was statistically significant (p ,0.001). The intensity of interaction of extracted LDL with SNA and MAA lectins was lower in CAD patients compared to that in normal subjects (p ,0.001). The intensity of interaction of LDL with DSA was higher in CAD (p ,0.001). There was a reverse correlation between TSA and intensity of LDL interaction with SNA and MAA in both groups, but in the case of DSA this correlation was direct and positive. These findings indicated an increase in desialylation of LDL in CAD. It was concluded that LDL was subjected to glycosylation changes in CAD and that there was a positive correlation between TSA and the desialylated form of LDL. This modification may contribute to the pathogenesis of CAD.

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A Lectin-Binding Assay for the Rapid Characterization of the Glycosylation of Purified Glycoproteins

Cited by 6 publications

References 0 publications

Screening for glycosylation changes on recombinant human IgG using lectin methods

Screening for glycosylation changes on recombinant human IgG using lectin methods

Carbohydrate Biosensors

Changes in sialylation of low‐density lipoprotein in coronary artery disease

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