Label Free Screening of mAb Libraries against a 96 Spot Peptide Array
SPRi can be used for antibody epitope discovery at the amino acid level by immobilizing a panel of up to 96 biotinylated peptides with overlapping sequences onto the SPR chip. A library of mAbs is then injected across the surface. The results can be processed with the Binning Software Tool to generate a heatmap and sort it by like behaved antibodies.
Alternatively, enzymatic digestion can be used to generate different sequence contexts for epitope discovery, which can be immobilized on the array via the CFM and probed with antibody libraries or patient sera.
In this white paper, a new method is presented for the antibody epitope discovery at the amino acid level. Antigens contain many undetermined epitopes which all can be a target for antibodies.
In this application note, a new method is presented for discovery and mapping of the immunogenic epitopes of auto-antigens. Antigens containing undetermined antigenic epitopes were subjected to tryptic digestion and then separated using strong cation exchange liquid chromatography.
PUBLICATION: Identification of Autoantibody Profiles by Monitoring Autoantibody Biomarkers in Rheumatoid Arthritis with Microarray Surface Plasmon Resonance Imaging
Arthritis & Rheumatism, Volume 63, November 2011 Abstract Supplement
Van Beers1, Joyce J.B.C., Segbers-Lokate1, Angelique M.C., Egberts1, Wilma T.M. Vree, Schasfoor2, Richard B.M., Pruijn1, Ger J.M.
1Radboud University Nijmegen, Nijmegen, Netherlands
2University of Twente and IBIS Technologies BV, Enschede, Netherlands
Autoantibodies against citrullinated proteins (ACPA) are specifically found in approximately 75% of rheumatoid arthritis (RA) patients. Citrullination is the post-translational conversion of peptidylarginine into peptidylcitrulline, which is catalyzed by peptidylarginine deiminase (PAD) in a calcium-dependent manner. Several citrullinated antigens have been identified in the inflamed joints of RA patients. These include fibrinogen, alpha-enolase, vimentin and collagen type II. Accumulating evidence suggests a role of citrullinated proteins and ACPA in the pathophysiology of RA. The results of many studies indicate that the ACPA response is highly heterogeneous with diverse patterns of reactivity to distinct citrullinated epitopes. This study aimed to identify clinically meaningful ACPA profiles in RA patients using a microarray containing different citrullinated peptides in combination with surface plasmon resonance imaging (iSPR).
PUBLICATION: Mapping of citrullinated fibrinogen B-cell epitopes in rheumatoid arthritis by imaging surface plasmon resonance
Arthritis Res Ther. 2010;12(6):R219.
van Beers JJ1, Raijmakers R, Alexander LE, Stammen-Vogelzangs J, Lokate AM, Heck AJ, Schasfoort RB, Pruijn GJ.
Department of Biomolecular Chemistry, Nijmegen Center for Molecular Life Sciences, Institute for Molecules and Materials, Radboud University, PO Box 9101, NL-6500 HB Nijmegen, The Netherlands.
Rheumatoid arthritis (RA) frequently involves the loss of tolerance to citrullinated antigens, which may play a role in pathogenicity. Citrullinated fibrinogen is commonly found in inflamed synovial tissue and is a frequent target of autoantibodies in RA patients. To obtain insight into the B-cell response to citrullinated fibrinogen in RA, its autoepitopes were systematically mapped using a new methodology.