An independent evaluation of the S1Fcs produced by these colonies using MS analytics demonstrated >80% similarity in the ranking of the individual clones (Fig. to MS for quantitative glycan analysis. Keywords:lectin, ELISA, glycoform, IVIG, sialylation == Introduction == Biologics, predominantly recombinant immunoglobulins, make up a significant share of todays pharmaceutical market. There has been an impetus to engineer or enrich for certain terminal glycan motifs, specifically sialylation, because of their effect on the stability and activity of therapeutic glycoproteins. Sialic acid has been reported to exert influence via prevention of serum proteins from degradation, masking antigenic epitopes, resistance on proteolytic degradation, and thermal stability.1For example, research on erythropoietin and tissue plasminogen activator has demonstrated the significance of sialylation for increased in vivo half-life.24More recently, not just terminal sialylation but galactosylation57and fucosylation8on recombinantly generated biologics as well as intravenous immunoglobulin (IVIG)6,9have also been shown to have important functions in determining in vivo efficacy. Hence, the need to evaluate the levels of these glycans is usually a necessary first step for understanding how structure modulates activity in preparations with nuanced differences in motif distribution. The ability to decipher the glycoform repertoire has benefited from the developments in high-resolution analytical tools such as mass spectrometry (MS), liquid chromatography (LC), capillary electrophoresis (CE), nuclear magnetic resonance (NMR), and combinations thereof.10,11However, apart from requiring significant investment in specialized expertise, materials requirements, and gear, data analysis for these analytical methods requires customization and is complex and labor intensive. Finally, obtaining topological information for terminal acidic carbohydrates via these methods entails additional derivatization, which adds to methodological complexity.12 More accessible methods for glycan analysis have been developed recently but have several limitations. Microarray technology has been adapted to immobilize lectins as probes, on glass or nitrocellulose surfaces, to exploit their innate ability to recognize and bind sugars for in situ glycoprofiling of labeled protein or cells.13,14Diverse binding specificities of lectins coupled with evanescent field-activated fluorescence and ratio metric/dual-color based detection have enabled mechanistic, organism-wide glycoprofiling and biomarker identification.13However, the weak monovalent lectin-glycan interactions demand either saturating concentrations of glycans on lectin microarrays or multivalent presentation of the carbohydrate structure. To overcome this, lectins have been presented in multimeric fashion in the in vitro assays of hemagglutinin, the influenza computer virus surface protein to glycan receptors,15and antibody-lectin sandwich assays wherein antibodies are immobilized on glass surfaces to selectively concentrate specific proteins from body fluids and multiplexed lectins added Angiotensin 1/2 (1-6) subsequently for profiling of the captured sample isolates.1619Geared toward biomarker discovery, sandwich-type assays require additional preparatory steps aimed to reduce the false positives due to lectin binding to the capture antibodies. This step specifically involves glycans around the capture antibodies to be chemically modified by the addition of a bulky dipeptide that effectively eliminates binding/recognition by lectins through an extensive routine. We report here an elegant enzyme-linked immunosorbent assay (ELISA) approach that exploits the specificity of lectin-glycan interactions to obtain quantitative information on protein glycosylation using apposing readouts from a pair of lectins. This method uses an easy approach for attaching proteins of interest on microtiter plates, providing an expeditious, low-infrastructure, RSK4 and minimal resource-requiring method of characterizing glycan epitopes on immunoglobulins. Moreover, Angiotensin 1/2 (1-6) the proposed method eliminates sample processing to allow for rapid and efficient scrutiny of multiple N-linked and O-linked glycan structures by the choice Angiotensin 1/2 (1-6) of cognate lectins. The assay not only provides linkage information on terminal glycans but has also been demonstrated to be able to distinguish single- versus dual-arm sialylation on complex N glycans on IgGs. This assay thus potentially can provide revealing insights previously considered affordable only via the use of MS/chromatographic analyses.20 A panel of five grow lectins (Table 1) was chosen to quantify sialylation, fucosylation, mannosylation, and galactosylation of therapeutic immunoglobulins. To demonstrate the practical applicability of.