title: Pitfalls in the determination of human acylated ghrelin plasma concentrations using a double antibody enzyme immunometric assay.
abstract: OBJECTIVE
  To investigate the effect of hemolysis and protease inhibitors on acylated ghrelin (AG) concentrations measured with a double antibody enzyme immunometric assay that uses an acetylcholinesterase (AChE)-Fab' conjugate.
  
  
  METHODS
  Samples were hemolysed or treated with PHMB (p-hydroxymercuribenzoate), PMSF (phenylmethanesulfonylfluoride) or AEBSF (4-(2-Aminoethyl) benzenesulfonyl fluoride) to prevent AG degradation.
  
  
  RESULTS
  Hemolysis decreased AG concentrations. PHMB or PMSF did not affect the assay. The standard curve was abolished by AEBSF but rescued by addition of a washing step prior to the AChE-Fab' conjugate.
  
  
  CONCLUSIONS
  Hemolysis and AEBSF may affect AG determination.
authors.affiliation: Department of Pediatrics, British Columbia Children's Hospital and Child and Family Research Institute, University of British Columbia, Vancouver, BC, Canada, V6H 3V4. atrivedi@interchange.ubc.ca
authors.forename: Arjun
authors.initials: A
authors.lastname: Trivedi
authors.forename: Sandra
authors.initials: S
authors.lastname: Babic
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Chanoine
mesh_terms: D000110:Acetylcholinesterase / Q000737:chemistry; D000215:Acylation; D000906:Antibodies / Q000737:chemistry*; D019411:Clinical Laboratory Techniques; D057075:Enzyme Assays / Q000379:methods*; D054439:Ghrelin / Q000097:blood* / Q000737:chemistry; D006461:Hemolysis; D006801:Humans; D006902:Hydroxymercuribenzoates / Q000494:pharmacology; D007128:Immunoglobulin Fragments / Q000737:chemistry; D011480:Protease Inhibitors / Q000494:pharmacology; D015203:Reproducibility of Results; D013450:Sulfones / Q000494:pharmacology
chemical_list: D000906:Antibodies; D054439:Ghrelin; D006902:Hydroxymercuribenzoates; D007128:Immunoglobulin Fragments; D011480:Protease Inhibitors; D013450:Sulfones; C030601:4-hydroxymercuribenzoate; C002010:4-(2-aminoethyl)benzenesulfonylfluoride; D000110:Acetylcholinesterase