Abstract
Antigen-antibody complexes are central players in an effective immune response. However, finding those interactions relevant to a particular disease state can be arduous. Nonetheless many paths to discovery have been explored since deciphering these interactions can greatly facilitate the development of new diagnostics, therapeutics, and vaccines. In silico B cell epitope mapping approaches have been widely pursued, though success has not been consistent. Antibody mixtures in immune sera have been used as handles for biologically relevant antigens, but these and other experimental approaches have proven resource intensive and time consuming. In addition, these methods are often tailored to individual diseases or a specific proteome, rather than providing a universal platform. Most of these methods are not able to identify the specific antibody’s epitopes from unknown antigens, such as un-annotated neo antigens in cancer. Alternatively, a peptide library comprised of sequences unrestricted by naturally-found protein space provides for a universal search for mimotopes of an antibody’s epitope. Here we present the utility of such a non-natural random sequence library of 10,000 peptides physically addressed on a microarray for mimotope discovery without sequence information of the specific antigen. The peptide arrays were probed with serum from an antigen-immunized rabbit, or alternatively probed with serum pre-absorbed with the same immunizing antigen. With this positive and negative screening scheme, we identified the library-peptides as the mimotopes of the antigen. The unique library peptides were successfully used to isolate antigen-specific antibodies from complete immune serum. Sequence analysis of these peptides revealed the epitopes in the immunized antigen. We present this method as an inexpensive, efficient method for identifying mimotopes of any antibody’s targets. These mimotopes should be useful in defining both components of the antigen-antibody complex.
Highlights
Characterizing the interactions between disease-specific antibodies and their cognate antigens has proven highly informative in the study of host-pathogen relationships and critical in the development of effective biomedical products
To identify non-natural sequences recognized by the immune serum, both naïve and SMCfsimmune rabbit sera were applied to the array
Sequence analysis to identify the mimic epitope to keyhole limpet hemocyanin (KLH) protein
Summary
Characterizing the interactions between disease-specific antibodies and their cognate antigens has proven highly informative in the study of host-pathogen relationships and critical in the development of effective biomedical products. Autoantigens that are recognized by patient antibodies is of growing importance in disease research and target development for diagnostics, vaccines, and therapeutics. These complexes are typically found by querying immune sera against possible ligands in lysates, or in libraries of proteins or peptides made recombinantly or synthetically. Myriad of binding assays such as immunoblots [1], ELISAs [2], phage display [3], ribosome display [4], beads [5], and microarrays [6, 7] have been employed to identify the antigen or epitope recognized by an antibody. We explore using a simple, universal system for epitope identification
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