title: Standardized serum GM-CSF autoantibody testing for the routine clinical diagnosis of autoimmune pulmonary alveolar proteinosis.
abstract: 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 0.78ng/ml. Receiver operating characteristic curve analysis identified a serum GMAb level of 5μg/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 1μg/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.
authors.affiliation: Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA; Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. Electronic address: uchidak-ane@h.u-tokyo.ac.jp.
authors.forename: Kanji
authors.initials: K
authors.lastname: Uchida
authors.affiliation: Niigata University Medical & Dental Hospital, Niigata, Japan. Electronic address: radical@med.niigata-u.ac.jp.
authors.forename: Koh
authors.initials: K
authors.lastname: Nakata
authors.affiliation: Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA. Electronic address: Brenna.Carey@cchmc.org.
authors.forename: Brenna
authors.initials: B
authors.lastname: Carey
authors.affiliation: Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA. Electronic address: Claudia.Chalk@cchmc.org.
authors.forename: Claudia
authors.initials: C
authors.lastname: Chalk
authors.affiliation: Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA. Electronic address: Takuji.Suzuki@cchmc.org.
authors.forename: Takuji
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA. Electronic address: stakuro@med.niigata-u.ac.mp.
authors.forename: Takuro
authors.initials: T
authors.lastname: Sakagami
authors.affiliation: Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA. Electronic address: kochde83@gmail.com.
authors.forename: Diana E
authors.initials: DE
authors.lastname: Koch
authors.affiliation: Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA. Electronic address: Carrie.Stevens@cchmc.org.
authors.forename: Carrie
authors.initials: C
authors.lastname: Stevens
authors.affiliation: National Hospital Organization, Kinki-Chuo Chest Medical Center, Sakai, Japan. Electronic address: giichi@kch.hosp.go.jp.
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Inoue
authors.affiliation: Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. Electronic address: yamaday-ane@h.u-tokyo.ac.jp.
authors.forename: Yoshitsugu
authors.initials: Y
authors.lastname: Yamada
authors.affiliation: Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA; University of Cincinnati College of Medicine, Cincinnati, OH, USA. Electronic address: Bruce.Trapnell@cchmc.org.
authors.forename: Bruce C
authors.initials: BC
authors.lastname: Trapnell
mesh_terms: D000328:Adult; D001323:Autoantibodies / Q000097:blood*; D001327:Autoimmune Diseases / Q000097:blood / Q000175:diagnosis / Q000276:immunology*; D015415:Biomarkers / Q000097:blood; D002138:Calibration; D016022:Case-Control Studies; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards*; D005260:Female; D005615:Freezing; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000276:immunology*; D006801:Humans; D057230:Limit of Detection; D008297:Male; D008875:Middle Aged; D015588:Observer Variation; D011237:Predictive Value of Tests; D055550:Protein Stability; D011649:Pulmonary Alveolar Proteinosis / Q000097:blood / Q000175:diagnosis / Q000276:immunology*; D012372:ROC Curve; D012015:Reference Standards; D015203:Reproducibility of Results; D013696:Temperature; D013997:Time Factors; D055815:Young Adult
chemical_list: D001323:Autoantibodies; D015415:Biomarkers; D016178:Granulocyte-Macrophage Colony-Stimulating Factor
keywords: Autoantibodies; Autoimmune disease; Diagnosis; Enzyme linked immunosorbent assay; Granulocyte/macrophage-colony stimulating factor; MCRS; PAP; PCRS; Pulmonary alveolar proteinosis; monoclonal reference standard; polyclonal reference standard; pulmonary alveolar proteinosis; recombinant, human GM-CSF; rhGM-CSF