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

Bioorthogonal conjugation eliminates the shortcomings 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, <i>Gaussia</i> 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