title: Pitfalls of immunoassay and sample for IGF-I: comparison of different assay methodologies using various fresh and stored serum samples.
abstract: OBJECTIVE
  Determination of insulin-like growth factor (IGF)-I is now a routine adjunct to multiple research and clinical investigations. Evidence has associated higher IGF-I levels with various human pathologies, but the reported associations have not been invariably confirmed. We examined the potential for post-sampling proteolysis and evaluated the impact of such events on IGF-I immunoassays.
  
  
  METHODS
  We compared IGF-I in different sets of fresh and frozen old samples using four different and commonly used immunoassays. The potential for post-sampling proteolysis was further examined by assaying fresh samples stored for 4 weeks at various temperatures in the absence or presence of protease inhibitors.
  
  
  RESULTS
  IGF-I levels in fresh serum samples from adult males, females, and pregnant subjects by all methods were similar and were highly correlated (r=0.85-0.97). The same was true for levels in frozen ( approximately 2 years at --80 degrees C) samples from diabetic patients, which are reportedly associated with enhanced proteolytic activity. In contrast, in another set of frozen adult male and female samples ( approximately 8 years at --20 degrees C), the inter-method median IGF-I levels varied by approximately 3- to 4-fold and the values poorly correlated. Similar variability in the inter-method response was also observed when IGF-I in the replicates of fresh samples stored at 4 degrees C for 4 weeks was measured. However, the 4 degrees C storage effect could be completely blocked by the addition of protease inhibitors, allowing for all assays to detect 92--101% of the expected mean levels.
  
  
  CONCLUSIONS
  The data indicate susceptibility of IGF-I to significant post-sampling proteolysis and suggest the importance of immunoassays for the intact molecule. Immunoassays that lack specificity for intact IGF-I may mask the potential pathophysiological effects of proteolysis and generate misleading results, particularly in studies involving inappropriately stored and/or proteolyzed samples. In such cases, underestimation of the in vivo levels by the intact assays would occur, but the findings of low IGF-I levels may be indicative of questionable sample quality.
authors.affiliation: Diagnostic Systems Laboratories (Canada) Inc., Mount Sinai Hospital, Toronto, Ontario, Canada M5G 1X5. jkhosravi@mtsinai.on.ca
authors.forename: Javad
authors.initials: J
authors.lastname: Khosravi
authors.forename: Anastasia
authors.initials: A
authors.lastname: Diamandi
authors.forename: Umesh
authors.initials: U
authors.lastname: Bodani
authors.forename: Najmuddin
authors.initials: N
authors.lastname: Khaja
authors.forename: Radha G
authors.initials: RG
authors.lastname: Krishna
mesh_terms: D000328:Adult; D000368:Aged; D007611:Aprotinin / Q000494:pharmacology; D003924:Diabetes Mellitus, Type 2 / Q000097:blood; D010450:Endopeptidases / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D005615:Freezing; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000032:analysis*; D008297:Male; D008875:Middle Aged; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011247:Pregnancy; D011480:Protease Inhibitors / Q000494:pharmacology; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods
chemical_list: D018972:Insulin-Like Growth Factor Binding Protein 3; D011480:Protease Inhibitors; D010664:Phenylmethylsulfonyl Fluoride; D007334:Insulin-Like Growth Factor I; D007611:Aprotinin; D010450:Endopeptidases; C080473:insulin-like growth factor binding protein-3 protease