Tu102 - A Bioluminescent Homogeneous Immunoassay to Detect Binding and Neutralising Anti-interferon Gamma antibodies Using Nanobit® Technology

Alex Stoyanov, MBBS (Hons) MSc – Consultant Immunologist, Royal Prince Alfred Hospital, Immunology, Royal Prince Alfred Hospital; Stephen A, MBBCh FRACP FRCPA PhD – Head, Institute of Academic Medicine, Head, Clinical Immunology and Immunopathology, Immunology, Royal Prince alfred Hospital; Nicolás Urriola, MBBS PhD BMedSci FRACP FRCPA – Consultant Immunologist, Royal Prince Alfred Hospital, Immunology, Royal Prince Alfred Hospital

Abstract Text: Endogenous antibodies that neutralize interferon-γ are a cause of severe adult-onset immunodeficiency with susceptibility to mycobacteria and other opportunistic infections. The syndrome was first recognised in 2012 in a cohort of Thai and Taiwanese patients with an AIDS-like immunodeficiency without evidence of HIV infection.
A homogeneous immunoassay using NanoBiT® (Promega) was developed that demonstrates anti-interferon-γ antibody binding and neutralization of interferon-γ in vitro. NanoBiT® involves splitting the bioluminescent NanoLuc enzyme into a Large (18kDa) and Small (11 amino acids) BiT. Conjugation of Large and Small BiT to antibodies enables specific analyte detection with reconstitution of the functional enzyme when components are brought into proximity.
Serum from patients known to have the AIDS-like immunodeficiency caused by the presence of anti-interferon-γ antibodies is added to a solution of interferon-γ. Binding of these antibodies to interferon-γ is demonstrated by the addition of human anti-interferon-γ conjugated to Large BiT and anti-human IgG. Reconstitution of NanoLuc occurs in the presence of an interferon-γ/anti-interferon-γ IgG immune-complex.
The neutralising ability of these antibodies was demonstrated by measuring recovery of interferon-γ in 50μL of a 90000pg/mL solution. Interferon-γ levels were measured by detection antibodies to interferon-γ conjugated to Large and Small BiT. Patients with the AIDS-like immunodeficiency caused by the presence of anti-interferon-γ antibodies repeatedly demonstrated >99% neutralisation of interferon gamma in solution.
These assays demonstrate the presence and functional significance of autoantibodies to interferon-γ in the aqueous phase, enabling rapid assessment without cumbersome wash steps. The methodology could be utilized to demonstrate the presence of any autoantibody.