Characterization of methacrylate chromatographic monoliths bearing affinity ligands

Černigoj, Urh; Vidic, Urška; Němec, B.; Gašperšič, Jernej; Vidič, Jana; Krajnc, Nika Lendero; Štrancar, Aleš; Podgornik, Aleš

doi:10.1016/j.chroma.2016.08.014

Cited by 33 publications

(26 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Large‐pore monoliths are more amenable to HTP applications due to their higher permeability and decreased possibility of clogging, therefore we used 2.1 μm average pore size diameter monoliths for preparation of CIMac‐@FIB 8‐well plates, as compared to 1.3 μm average pore size diameter monoliths which were used for CIMac‐@FIB columns. On the other hand, large pores decrease the surface area of the monolith resulting in approximately 1/3 decreased binding capacity for the antigen , so we used monoliths of larger volume (200 μL) for the CIMac‐@FIB 8‐well plate preparation, compared to the monoliths used for the column format (100 μL). @FIB was successfully bound in each well and the CIMac‐@FIB 8‐well plate was characterized for pure FIB binding capacity with the same reagents as with the CIMac‐@FIB columns.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2017

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Resultsmentioning

confidence: 99%

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

et al. 2017

Self Cite

View full text Add to dashboard Cite

show abstract

“…In this work, carbonyldiimidazole (CDI)‐modified CIM columns were used for the immobilization of recombinant protein G (Reprokine, Rehovot, Israel) and recombinant protein L (Acro Biosystems, Beijing, China) following similar procedure, as described by Černigoj et al. for protein A immobilization . Measured dynamic binding capacity for protein G column was ≥9.0 and ˂15 mg/mL, with a recovery of ≥90%.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2017

Self Cite

View full text Add to dashboard Cite

Posttranslational modifications of immunoglobulins have been a topic of great interest and have been repeatedly reported as a major factor in disease pathology. Cost-effective, reproducible, and high-throughput (HTP) isolation of immunoglobulins from human serum is vital for studying the changes in protein structure and the following understanding of disease development. Although there are many methods for the isolation of specific immunoglobulin classes, only a few of them are applicable for isolation of all subtypes and variants. Here, we present the development of a scheme for fast and simultaneous affinity purification of α (A), γ (G), and μ (M) immunoglobulins from human serum through affinity monolith chromatography. Affinity-based monolithic columns with immobilized protein A, G, or L were used for antibody isolation. Monolithic stationary phases have a high surface accessibility of binding sites, large flow-through channels, and can be operated at high flow rates, making them the ideal supports for HTP isolation of biopolymers. The presented method can be used for HTP screening of human serum in order to simultaneously isolate all three above-mentioned immunoglobulins and determine their concentration and changes in their glycosylation pattern as potential prognostic and diagnostic disease biomarkers.

show abstract

“…Two types of immunoglobulin‐binding proteins that have been used in AMC are protein A and protein G . These are bacterial cell wall proteins that can bind to the F c regions of many types of immunoglobulins and antibodies, making them versatile agents for the purification, analysis, or biospecific adsorption of such targets .…”

Section: Binding Agents and Applications Of Amcmentioning

confidence: 99%

Affinity monolith chromatography: A review of general principles and applications

et al. 2017

View full text Add to dashboard Cite

Affinity monolith chromatography, or AMC, is a liquid chromatographic method in which the support is a monolith and the stationary phase is a biological binding agent or related mimic. AMC has become popular for the isolation of biochemicals, for the measurement of various analytes, and for studying biological interactions. This review will examine the principles and applications of AMC. The materials that have been used to prepare AMC columns will be discussed, which have included various organic polymers, silica, agarose and cryogels. Immobilization schemes that have been used in AMC will also be considered. Various binding agents and applications that have been reported for AMC will then be described. These applications will include the use of AMC for bioaffinity chromatography, immunoaffinity chromatography, dye-ligand affinity chromatography, and immobilized metal-ion affinity chromatography. The use of AMC with chiral stationary phases and as a tool to characterize biological interactions will also be examined.

show abstract

Characterization of methacrylate chromatographic monoliths bearing affinity ligands

Cited by 33 publications

References 27 publications

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

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

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

Affinity monolith chromatography: A review of general principles and applications

Contact Info

Product

Resources

About