Protein Binders Specific for Immunoglobulin G from Different Species for Immunoassays and Multiplex Imaging

An immunoassay is the most widely used method for analyzing a variety of analytes based on antigen-antibody interactions in the biological and medical sciences. However, the use of secondary antibodies has certain shortcomings, such as a high cost, cross-reactivity, and loss of binding affinity during labeling. Herein, we present the development of repebodies specifically binding to immunoglobulin G with a different origin, which is a small-sized nonantibody scaffold composed of leucine-rich repeat (LRR) modules, for use in immunoassays and imaging. Repebodies specific for IgG from different species (i.e., mouse, human, and rabbit) were selected through a phage display, and their affinities were matured using a modular engineering approach. The respective repebodies were labeled with various signal generators such as horseradish peroxidase (HRP), a fluorescent dye, and quantum dots, and the resulting repebodies were used as alternatives to conventional secondary antibodies in typical immunoassays and imaging. The labeled repebodies enabled the detection of diverse target analytes with high sensitivity and specificity, showing a negligible cross-reactivity. Moreover, the repebodies labeled with different color-emitting quantum dots allowed the imaging of cell-surface receptors and proteins in a multiplex manner. The developed repebodies can be effectively used for sensitive immunoassays and multiplex imaging.