Standardized serum GM-CSF autoantibody testing for the routine clinical diagnosis of autoimmune pulmonary alveolar proteinosis

Autoantibodies against granulocyte/macrophage colony-stimulating factor (GMAbs) cause autoimmune pulmonary alveolar proteinosis (PAP) and measurement of the GMAb level in serum is now commonly used to identify this disease, albeit, in a clinical research setting. The present study was undertaken to optimize and standardize serum GMAb concentration testing using a GMAb enzyme-linked immunosorbent assay (GMAb ELISA) to prepare for its introduction into routine clinical use. The GMAb ELISA was evaluated using serum specimens from autoimmune PAP patients, healthy people, and GMAb-spiked serum from healthy people. After optimizing assay components and procedures, its accuracy, precision, reliability, sensitivity, specificity, and ruggedness were evaluated. The coefficient of variation in repeated measurements was acceptable (< 15%) for well-to-well, plate-to-plate, day-to-day, and inter-operator variation, and was not affected by repeated freeze–thaw cycles of serum specimens or the reference standards, or by storage of serum samples at − 80 °C. The lower limit of quantification (LLOQ) of the PAP patient-derived polyclonal GMAb reference standard (PCRS) was 0.78 ng/ml. Receiver operating characteristic curve analysis identified a serum GMAb level of 5 μg/ml (based on PCRS) as the optimal cut off value for distinguishing autoimmune PAP serum from normal serum. A pharmaceutical-grade, monoclonal GMAb reference standard (MCRS) was developed as the basis of a new unit of measure for GMAb concentration: one International Unit (IU) of GMAb is equivalent to 1 μg/ml of MCRS. The median [interquartile range] serum GMAb level was markedly higher in autoimmune PAP patients than in healthy people (21.54 [12.83–36.38] versus 0.08 [0.05–0.14] IU; n = 56, 38; respectively; P < 0.0001). Results demonstrate that serum GMAb measurement using the GMAb ELISA was accurate, precise, reliable, had an acceptable LLOQ, and could be accurately expressed in standardized units. These findings support the use of this GMAb ELISA for the routine clinical diagnosis of autoimmune PAP and introduce a new unit of measure to enable standardized reporting of serum GMAb data from different laboratories.