Dissecting Antibodies with Regards to Linear and Conformational Epitopes
نویسندگان
چکیده
An important issue for the performance and specificity of an antibody is the nature of the binding to its protein target, including if the recognition involves linear or conformational epitopes. Here, we dissect polyclonal sera by creating epitope-specific antibody fractions using a combination of epitope mapping and an affinity capture approach involving both synthesized peptides and recombinant protein fragments. This allowed us to study the relative amounts of antibodies to linear and conformational epitopes in the polyclonal sera as well as the ability of each antibody-fraction to detect its target protein in Western blot assays. The majority of the analyzed polyclonal sera were found to have most of the target-specific antibodies directed towards linear epitopes and these were in many cases giving Western blot bands of correct molecular weight. In contrast, many of the antibodies towards conformational epitopes did not bind their target proteins in the Western blot assays. The results from this work have given us insights regarding the nature of the antibody response generated by immunization with recombinant protein fragments and has demonstrated the advantage of using antibodies recognizing linear epitopes for immunoassay involving wholly or partially denatured protein targets.
منابع مشابه
Production and Characterization of Murine Monoclonal Antibodies Recognizing Conformational and Linear Epitopes Localized on Human IgA2 Molecules
Background: There are two subclasses of human IgA (IgA1 and IgA2) that differ in antigenic properties and in chemical composition. The constant domains of α1 and α2 heavy chains have >95% sequence homology though major structural differences exist in the hinge region. Quantitation of IgA subclass levels depends on the availability of monoclonal antibodies (MAbs) specific for conserved conformat...
متن کاملIn silico prediction of B cell epitopes of the extracellular domain of insulin-like growth factor-1 receptor
The insulin-like growth factor-1 receptor (IGF-1R) is a transmembrane receptor with tyrosine kinase activity. The receptor plays a critical role in cancer. Using monoclonal antibodies (MAbs) against the IGF-1R, typically blocks ligand binding and enhances down-regulation of the cell-surface IGF-1R. Some MAbs such as cixutumumab are under clinical trial investigation. Targeting multiple distinct...
متن کاملDissecting linear and conformational epitopes on the native thyrotropin receptor.
The TSH receptor (TSHR) is the primary antigen in Graves' disease. In this condition, autoantibodies to the TSHR that have intrinsic thyroid-stimulating activity develop. We studied the epitopes on the native TSHR using polyclonal antisera and monoclonal antibodies (mAbs) derived from an Armenian hamster model of Graves' disease. Of 14 hamster mAbs analyzed, five were shown to bind to conformat...
متن کاملتعیین اپی توپ های ناپیوسته زنجیره سبک ایمونوگلوبولین انسان توسط ایمونولوژی محاسبه ای
Background: Immunoglobulins are a group of proteins that have important role in defense against microorganisms. Immunoglobulins consist of heavy and light chains. In human, immunoglobulin light chain comprises of two isotypes: Kappa (K) and lambda (λ) based on amino acid differences in carboxylic end of their constant region. Marked changes in the K to λ ratio can happen in monocl...
متن کاملIdentification of conformational epitopes on fragment crystallizable region of human Immunoglobulin G by immunoinformatic
Background: Immunoglobulins are a group of proteins have important role in defense against microorganisms. Human immunoglobulins are divided into five classes: IgA, IgM, IgD, IgE and IgG. Immunoglobulin G (IgG) is the highest abundant antibody in serum and extravascular fluids. The extent of serum IgG is related to severity of several diseases such as infections, so IgG has great diagnostic wor...
متن کامل