Bioorthogonal Protein Conjugation: Application to the Development of a Highly Sensitive Bioluminescent Immunoassay for the Detection of Interferon-γ

Abstract

Bioorthogonal conjugation eliminates the short-comings of classical conjugation methods. The conjugation of antibodies to reporter proteins, such as bioluminescent protein, can be controlled with orthogonal conjugation methods. Here we report a bioluminescent immunoassay for the sensitive detection of interferon-γ (IFN-γ) that utilizes orthogonal conjugation of bioluminescent protein, Gaussia luciferase to anti-IFN-γ antibody. The IFN-γ is produced by the immune system and the detection of the IFN-γ is pivotal for the detection of persistent viral and bacterial infections. A bioorthogonal conjugation approach is used to conjugate an anti-IFN-γ antibody with a GLuc mutant containing the N-terminal tyrosine using formylbenzene diazonium hexafluorophosphate reagent (FBDP) in hydrophilic mild pH environment yielding high conjugation efficiency (60%). This reagent is shown to be specific for tyrosine (Tyr) residues. Therefore, conjugation through Tyr was orthogonal and not detrimental to the bioluminescence activity of GLuc. The immunoassay described in this paper is a sandwich type assay and involves a capture and a detection antibody. The assay was validated for its robustness, precision, accuracy, limit of detection, and recovery.

Design of Gaussia luciferase-based bioluminescent stem-loop probe for sensitive detection of HIV-1 nucleic acids

“…This can also be mitigated by the introduction of a non-native conjugation site, as we demonstrated previously through a Tyr introduced at the N-terminus of Gluc to achieve site-specific conjugation since Gluc lacks Tyr residues. 24…”

“…21 These properties of GLuc make it an attractive reporter in terms of bioconjugation and bioanalytical applications. Therefore, it has been widely used for in vitro and in vivo imaging of tumor cells, 22 gene expression analysis, 18 protein–protein interaction study, 23 immunoassay development, 24 and DNA hybridization. 25 We hypothesized that the small size of GLuc, high bioluminescence emission, and temperature and pH stability compared to other bioluminescent proteins would result in GLuc-based SLP with superior performance.…”

Coelenterazine-Dependent Luciferases as a Powerful Analytical Tool for Research and Biomedical Applications

“…To overcome the problem, several original approaches were developed. So, the bioorthogonal conjugation or photoconjugation methods of obtaining luciferase-antibody conjugates that would provide a high conjugate yield, control over conjugation site and stoichiometry, and retention of the luciferase activity were elaborated [ 108 , 109 ]. However, the analytical application of luciferases in most cases is based on the bioluminescence of their derivatives extended with biospecific polypeptides obtained by genetic fusing.…”

Bioluminescent Antibodies through Photoconjugation of Protein G–Luciferase Fusion Proteins

“…Genetic fusion has the advantage of generating homogeneous conjugates with a well-defined antibody–luciferase stoichiometry. − However, genetic fusion requires cloning for each new antibody–luciferase conjugate and often involves cumbersome expression optimization and access to mammalian expression systems. A second general approach is to chemically conjugate the luciferase and antibody proteins, either covalently or noncovalently. − While several approaches are available for covalent conjugation to commercially available monoclonal antibodies, these approaches do not allow precise control over the conjugation site, yielding a heterogeneous mixture of luciferase–antibody conjugates with little control over conjugation site and stoichiometry . The latter can be improved by fusing a luciferase to antibody-binding domains targeting the invariable part of antibodies such as protein A or protein G. − However, this approach results in the formation of a noncovalent complex, which can dissociate under dilute conditions or extensive washing.…”