Bioanalytical Reagent: Nanobody Fusion Protein for ELISA and Related Bioanalytical Techniques

Opportunity

Available for Licensing

IP Status

US Utility Patent Pending: US 2018/0224459 A1

Inventors

Virginia Bruce
Brian McNaughton

At a Glance

Researchers at Colorado State University have developed a simple way to produce, stable, potent, and selective reagents for ELISA, flow cytometry and Western blot assays.

 

Licensing Director

Steve Foster
Steve.Foster@colostate.edu
970-491-7100

Reference No.: 17-050

Background

Enzyme-linked immunosorbent assay (ELISA), flow cytometry, and Western blot are common bioanalytical techniques. Successful execution of these experiments traditionally requires the use of one or more commercially available antibody–small-molecule dye or antibody–reporter protein conjugates that recognize relatively short peptide tags (<15 amino acids). However, the size of antibodies and their molecular complexity (by virtue of post-translational disulfide formation and glycosylation) typically require either expression in mammalian cells or purification from immunized mammals. The preparation and purification of chemical dye– or reporter protein–antibody conjugates is often complicated and expensive.

Technology Overview

In response, researchers at Colorado State University have developed comparatively simpler protein scaffolds for macromolecular recognition, which can be expressed with relative ease in E. coli and can be evolved to bind virtually any target. Nanobodies, a minimalist scaffold generated from camelid-derived heavy-chain IgGs, are one such example. A multitude of nanobodies have been evolved to recognize a diverse array of targets, including a short peptide. Here, this peptide tag (termed BC2T) and BC2 nanobody–dye conjugates or reporter protein fusions are evaluated in ELISA, flow cytometry, and Western blot experiments and compared to analogous experiments using commercially available antibody–conjugate/peptide tag pairs. Collectively, the utility and practicality of nanobody-based reagents in bioanalytical chemistry is demonstrated.  Particularly, the BC2-nanoluciferase fusion protein is a simple to prepare, stable, potent, and selective reagent for ELISA.

Benefits
  • Antibody free detection of protein-protein interactions
  • Simple and stable reagent for ELISA and related bioanalytical methods
  • Easier to produce and purify than traditional antibodies
  • Performs at or above traditional reagents for the bioanalytical methods (e.g. ELISA, Flow Cytometry, and Western Blot)
  • Inexpensive to manufacture
  • Long shelf-life
  • Ability to be conjugated to a multitude of known reporters
Applications
  • ELISA

  • Western Blot

  • Flow Cytometry

  • Immunohistochemistry

  • Other possible techniques

Publications

Bruce, V.J.; McNaughton, B.R. “Evaluation of Nanobody Conjugates and Protein Fusions as Bioanalytical Reagents” Analytical Chemistry DOI: 1021/acs.analchem.7b00470

Last updated: March 2020