SBIR-STTR Award

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 many of these “immunoassays” (e.g. ELISA's, biosensors, etc.), antibodies must be immobilized onto surfaces. The sensitivity, stability and longevity of antibodies used in this context are highly dependent on orienation of these molecules.1 Numerous work have shown that correct orienation can result in tens to even a 1000-fold increase in sensitivity.2-5 Despite the enormous benefit of using site-specific immobilization techniques, they are rarely adopted in commercial immunoassays. This is largely due to the complexity, shortcomings and economic hurdles associated with site- specific immobilization techniques.1 Recently, we developed a simple, rapid, and efficient approach to site- specifically and covalently immobilize native IgG on surfaces. Our approach relies on the use of low-molecular weight, monovalent subdomains of Protein A and G that possess a UV-active non-natural amino acid, benzoylphenyalanine (BPA), within the Fc-binding domains. Upon exposure to innocuous long wavelength UV light, the BPA is activated and forms a covalent link between the antibody-binding domain and the antibody. The recombinant production of photoreactive antibody-binding domains (pAbBDs) has been combined with expressed protein ligation (EPL) to allow for the introduction of nearly any desirable chemical handle onto the C-terminus of the pAbBDs during protein purification. This enables the site-specific attachment of the pAbBDs to a wide range of surfaces. Overall, the use of pAbBDs for the site-specific immobilization of antibodies is cost-effective, easily scalable, amenable to high-throughput processes, and utilizes protein production techniques that are commercially viable. The primary goal of this proposal is to acquire data that directly demonstrates the value of our technology in immunoassay applications. We believe that this will open up new opportunities for collaboration and commercial partnerships in the immunoassay industry. The specific aims for the proposal are: Aim 1: Optimize the conditions for the site-specific immobilization of antibodies in a microplate; Aim 2: Quantify the effect of site-specific IgG immobilization on immunoassay sensitivity, limit of detection, and dynamic range.

Public Health Relevance Statement:
We have developed a new bioconjugation technology that allows for the rapid, efficient, and site-specific immobilization of full-length antibodies onto solid surfaces. The primary goal of this proposal is to acquire data that directly demonstrates the value of our technology in immunoassay applications.

Project Terms:
Adopted; Aldehydes; Antibodies; antigen binding; Binding; Binding Sites; Biosensor; Chemicals; Collaborations; commercialization; cost; cost effective; Data; density; Detection; diagnostic assay; Diagnostics Research; Economics; Enzyme-Linked Immunosorbent Assay; Exposure to; Goals; Immobilization; Immunoassay; Immunoglobulin G; Immunoglobulins; Industry; Length; Ligand Binding Domain; Ligation; Link; Longevity; Maleic Anhydride; Maleimides; Molecular Weight; Process; Production; Prostate-Specific Antigen; protein purification; Proteins; Randomized; Recombinants; Site; Solid; Specificity; Surface; Techniques; Technology; tool; Ultraviolet Rays; Work

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.

Thesaurus Terms:
Adopted; Alkaline Phosphatase; Alkynes; Amino Acids; Antibodies; Antibody Affinity; Antigens; Azides; Base; Beta-Lactamase; Binding; Biological Assay; Biological Sciences; Biosensor; Biotin; Cells; Chemicals; Chemiluminescence Assay; Chemistry; Clinical Diagnostics; Collaborations; Complement System; Cost; Cost Effective; Crosslink; Detection; Development; Diagnostic Assay; Economics; Enzyme Activity; Enzyme-Linked Immunosorbent Assay; Enzymes; Exposure To; Firefly Luciferases; Fluorescence; Fluorescent Dyes; Fluorophore; Glass; Goals; Gold; Haptens; Horseradish Peroxidase; Immobilization; Immunoassay; Immunoglobulins; Industry; Label; Latex Bead; Ligand Binding Domain; Ligation; Link; Location; Longevity; Luciferases; Magnetism; Methods; Molecular Weight; Movement; Optics; Performance; Phase; Process; Production; Property; Prostate-Specific Antigen; Protein Fragment; Protein Purification; Proteins; Pyroxylin; Reaction; Reader; Reagent; Recombinants; Renilla Luciferases; Reporter; Reproducibility; Research; Robotics; Sampling; Sepharose; Signal Transduction; Site; Small Business Innovation Research Grant; Solid; Specificity; Surface; System; Techniques; Time; Tool; Ultraviolet Rays; Variant;