A bioluminescent inhibition immunoassay for detecting GM-CSF inhibitory activity in serum

Autoantibodies against granulocyte-macrophage colony-stimulating factor (GM-CSF) are the cause of autoimmune pulmonary alveolar proteinosis (aPAP). They are also found in the serum/plasma of previously immunocompetent patients with disseminated Cryptococcus spp. infection (typically spp. gattii) and disseminated Nocardia spp. infection. Detection of this autoantibody is generally performed through an antibody binding assay, followed by a live-cell-based pSTAT5 neutralization to confirm functional significance of detected autoantibodies. The requirement for a live-cell-based assay increases the technical difficulty of diagnosis and means that this testing is generally only offered in specialist research centres. We report a bioluminescent immunoassay (BLIA) platform, which accurately detects inhibition of GM-CSF detection in serum. The BLIA method has previously been described in detail to detect anti-cytokine autoantibodies against IFNγ. The BLIA platform was able to distinguish 24 blinded disease control samples with 100% accuracy when compared with gold standard testing. We sought referrals for patients with aPAP, Cryptococcus spp. Infection, and Nocardia spp. infection to test the performance of this assay. Positive results were seen in 100% of patients with clinically confirmed aPAP and a proportion of patients with disseminated Cryptococcus or Nocardia spp. infection. We conclude that the BLIA is technically simple, rapid, and easily automatable and could be easily employed in a diagnostic laboratory. The performance characteristics of the BLIA result in rapid and accurate screening of patients with these uncommon autoantibody-mediated conditions, allowing for quick diagnosis and referral for specialist management.