title: Factors influencing the measurement of plasma/serum surfactant protein D levels by ELISA.
abstract: BACKGROUND
  Extensive variations in human surfactant protein D (SP-D) levels in circulation as measured by ELISA exist in the published literature. In order to determine the source of these variations, factors influencing the measurement by ELISA were explored.
  
  
  METHODS
  Peripheral blood from healthy individuals was collected into various vacutainers during the same blood draw. Recombinant SP-D was diluted into different matrices and used for a standard curve. Samples were analyzed by capture ELISA using one of two distinct detection antibodies.
  
  
  RESULTS
  The type of matrix had some effects on detection of recombinant SP-D. The type of anticoagulant used and dilution factor had very little effect, except for in plasma collected in EDTA vacutainers. The extent of variation in published values seemed to be due to the ELISA configuration employed, and, in agreement with this, we found that by switching the detection antibody, there was a 50% decrease in the extrapolated SP-D value of serum and plasma samples. Storage of samples resulted in slight changes in measured SP-D levels.
  
  
  CONCLUSIONS
  The ELISA configuration employed to measure circulating levels of SP-D has a significant effect on the extrapolated values. In both configurations tested, the use of EDTA as a coagulant resulted in inconsistent values, and we, therefore, suggest the avoidance of this anticoagulant when assaying for SP-D by ELISA. While the demonstrated effects of several factors on measurement of SP-D may not account for all the disparities amongst the previous studies, they stress that variations in methodologies for measuring the same protein can result in very inconsistent results.
authors.affiliation: Department of Medicine and Division of Pulmonary, Allergy, and Critical Care Medicine, University of Alabama at Birmingham, Birmingham, Alabama, United States of America; Program in Protease and Matrix Biology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Preston E
authors.initials: PE
authors.lastname: Bratcher
authors.affiliation: Department of Medicine and Division of Pulmonary, Allergy, and Critical Care Medicine, University of Alabama at Birmingham, Birmingham, Alabama, United States of America; Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama, United States of America; University of Alabama at Birmingham, UAB Lung Health Center, Birmingham, Alabama, United States of America; Medicine Service, United States Department of Veterans Affairs Medical Center, Birmingham, Alabama, United States of America; Program in Protease and Matrix Biology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Amit
authors.initials: A
authors.lastname: Gaggar
mesh_terms: D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards; D064368:Healthy Volunteers; D006801:Humans; D037663:Pulmonary Surfactant-Associated Protein D / Q000097:blood*; D012016:Reference Values; D015203:Reproducibility of Results
chemical_list: D037663:Pulmonary Surfactant-Associated Protein D