Semi‐high‐throughput isolation and N‐glycan analysis of human fibrinogen using monolithic supports bearing monoclonal anti‐human fibrinogen antibodies

Abstract

Fibrinogen (FIB) is a secretory glycoprotein synthesized by hepatocytes that has a key role in blood clotting. Its glycosylation has not been studied in detail and little is known about the biological variability of FIB N-glycosylation, mainly due to the lack of fast, simple, and robust approaches to purify FIB from blood plasma samples. In recent years, customised chromatographic monoliths have been used for a variety of biological applications due to their unique characteristics. Here we describe development and optimisation of monolithic supports bearing monoclonal anti-human fibrinogen antibodies in a single column as well as in multi-well plate formats with high FIB specificity and binding capacity for fast immunoaffinity purification of FIB from human blood samples. The developed semi-high-throughput workflow has been successfully applied for FIB immunoaffinity isolation and subsequent ultra performance liquid chromatography N-glycosylation analysis in ten healthy human individuals, demonstrating the potential of monolithic supports in glycomics studies.

Nanoparticle Biomolecular Corona-Based Enrichment of Plasma Glycoproteins for N-Glycan Profiling and Application in Biomarker Discovery

“…Both protein precipitation and corona enrichment are not as selective as the affinity method; however, the major advantage of the corona enrichment method for glycan profiling compared to the precipitation method is that it could be utilized in different contexts, for different corona protein enrichment by changing the surface chemistry or plasma protein/NP ratio. The affinity method provided good selectivity, and its application in glycan profiling has been demonstrated . However, the cost of the antibodies and the optimization steps would need to be considered in a cohort analysis.…”

Affinity chromatography on monolithic supports for simultaneous and high‐throughput isolation of immunoglobulins from human serum

“…Since high flow rates can easily be applied and analysis time can be very short, monoliths are perfect supports for HTP screening . A semi‐HTP isolation of human fibrinogen using monolithic supports with immobilized monoclonal antihuman fibrinogen antibodies was successfully performed . Another group used monoliths for HTP isolation of transferrin from human plasma by use of antitransferrin monoclonal antibodies immobilized on monoliths .…”

Affinity monolith chromatography: A review of general principles and recent developments

“…6) [265]. Several commercial organic monoliths were employed with IAC and anti‐human fibrinogen antibodies for the isolation of fibrinogen from human blood samples [266]. CIM disks were used to immobilize protein G, which was then used to adsorb antibodies for the capture of ammodytoxins (i.e., the most toxic components of the snake venom) and their interacting protein partners from snake venom [267].…”