Development of Recombinant Secondary Antibody Mimics as Signal Amplifiers in Immunoassays

Abstract

Immunoassay refers to a biochemical analysis that measures the presence and/or concentration of specific target molecules by using antibodies which are principally based on strong and selective interactions, and thus is widely used techniques in biomedical tests such as disease diagnoses, virus or pathogen detection, and pregnancy test. In general, the immunoassay utilizes two different types of antibodies, primary and secondary antibodies, to satisfy both specificity and sensitivity. Primary antibody selectively recognizes and tightly binds to its target analytes to give sample specificity, whereas secondary antibody binds to the Fc region of target analyte-bound primary antibodies to amplify signals. To amplify signals, a conventional secondary antibody is covalently linked with active enzymes such as horseradish peroxidase (HRP) that convert multiple copies of inactive substrates into signal-generating active forms, leading to the efficient detection of low-abundant target analytes. Although enzyme-linked secondary antibodies have been widely used in immunoassays, antibodies themselves should be obtained from animals and further modified chemically in a lengthy and complicated ways. To circumvent these issues, we have developed a couple of recombinant secondary antibody mimics, produced them at large quantity using bacterial overexpression system and simple purification methods, and demonstrated their outstanding signal amplifying capability regardless of target molecules. Each recombinant secondary antibody mimic would be selected for its own purpose and applied to various types of target analytes in combination with a variety of target-specific primary antibodies, effectively minimizing the use of animals as well as reducing cost and time for production.