Scope of Microbial Transglutaminase for Site-Specific and Oriented Immobilization of Native Antibodies from Various...

Modification of antibodies to chemically couple labels or immobilization reagents is essential for developing biosensors. Typically, conjugation occurs through chemical methods that leverage reactive amines and thiols on native antibodies; however, this nonspecific approach can interfere with antibody function. Microbial transglutaminase (mTG) is an enzyme that has been used for site-specific conjugation of chemical modifiers to the F_c region of native antibodies, but thus far mTG-mediated conjugation has been limited to production of antibody-drug conjugates with human IgGs. Here, we assessed the scope and versatility of mTG to target IgGs, with the goal of site-specific conjugation to facilitate oriented immobilization. A fluorescently labeled peptide was conjugated to several IgG host species and subclasses commonly used to produce monoclonal (e.g., mouse IgG1 and rat IgG1) and polyclonal (e.g., rabbit IgG and goat IgG) antibodies. SDS-PAGE confirmed site-specific conjugation of the peptide to each of these IgG subclasses. In addition, NH₂-PEG₄-biotin was chemo-enzymatically installed on the F_c region of each tested IgG, as confirmed by Western blot analysis. Site-specific biotinylated antibody was immobilized on a streptavidin-coated substrate to evaluate antigen binding activity in a functional assay. The site-specific conjugation of biotin enabled the formation of an oriented capture antibody layer to enhance antigen binding when compared to the performance of a functional assay constructed by immobilizing a randomly biotinylated antibody prepared by conventional chemical conjugation. These results highlight the broad scope of mTG to site-specifically conjugate native antibodies to improve analytical performance of biosensing platforms.