Tools for the site-specific labeling and immobilization of antibodies for immunoassays - Antibodies, most commonly Immunoglobulin Gs (IgGs), are widely used in research and diagnostic assays due
to their wide array of targets, high specificity and proven efficacy. In nearly all “immunoassays”, antibodies
serve multiple functions including capturing antigens onto a solid support and associating a reporter system
with the captured antigen, so that it can be detected. In both contexts, the functionality of the antibody is highly
dependent on how it is immobilized and/or labeled. For example, the sensitivity, stability and longevity of
immobilized antibodies is highly dependent on their orienation. Similarly, the labeling of antibodies with reporter
enzymes and fluorescent dyes is highly dependent on the placement of these labels. Imprecise conjugation
methods can result in heteregeneous samples, poor reproducibility, and sub-optimal performance. Therefore,
there has been a movement towards the development of site-specific bioconjugation techniques, which allow
for precise labeling of antibodies at pre-defined locations.
Despite the enormous benefit of using site-specific antibody labeling techniques, they are rarely adopted in
commercial immunoassays due to the complexity and economic hurdles associated with these techniques.
Recently, we developed a simple, rapid, and efficient approach to site-specifically and covalently immobilize
native IgG on surfaces and/or to site-specifically label antibodies with enzymes, fluorescent dyes, or other
chemical moieties. Our approach relies on photoreactive antibody-binding domains (pAbBDs). The use of
pAbBDs is cost-effective, easily scalable, amenable to high-throughput processes, and utilizes protein
production techniques that are commercially viable. A primary goal of this SBIR Phase II application is to
expand the array of pAbBD products for immunoassay applications.
While current immunoassay formats have proven invaluable in research and in clinical diagnostics, they remain
quite laborious and time consuming, involving multiple washing and incubation steps. This prompted us to
utilize our pAbBDs to create a homogeneous assay for sensitive antigen detection that can be completed in a
single step, with no washing - just mix and read. As part of this application, we also plan to further expand this
product line. The specific aims for the proposal are as follows: Aim 1: Create pAbBDs that can be used to site-
specifically immobilize antibodies on various surfaces; Aim 2: Combine pAbBDs with various bioluminescent,
colorimetric and fluorscent reporter systems; and Aim 3: Create additional pAbBD constructs for use in
homogeneous antigen detection assays.
