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

TL;DR

This paper explores how microbial transglutaminase can be used to precisely attach molecules to antibodies from different species, improving biosensor performance.

Contribution

The study demonstrates the broad applicability of microbial transglutaminase for site-specific antibody conjugation across multiple host species and subclasses.

Findings

Microbial transglutaminase enabled site-specific conjugation of a fluorescent peptide to various IgG subclasses.

Biotin was successfully site-specifically attached to the Fc region of multiple IgG types using a chemo-enzymatic method.

Oriented immobilization of biotinylated antibodies improved antigen binding compared to conventional random conjugation.

Abstract

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 Fc 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 Fc 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.

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