Pitfalls of immunoassay and sample for IGF-I: comparison of different assay methodologies using various fresh and stored serum samples

Clin Biochem. 2005 Jul;38(7):659-66. doi: 10.1016/j.clinbiochem.2005.04.001.

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.

Design and 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.

Publication types

  • Comparative Study

MeSH terms

  • Adult
  • Aged
  • Aprotinin / pharmacology
  • Diabetes Mellitus, Type 2 / blood
  • Endopeptidases / metabolism
  • Enzyme-Linked Immunosorbent Assay* / methods
  • Female
  • Freezing
  • Humans
  • Insulin-Like Growth Factor Binding Protein 3 / metabolism
  • Insulin-Like Growth Factor I / analysis*
  • Male
  • Middle Aged
  • Phenylmethylsulfonyl Fluoride / pharmacology
  • Pregnancy
  • Protease Inhibitors / pharmacology
  • Sensitivity and Specificity
  • Specimen Handling / methods

Substances

  • Insulin-Like Growth Factor Binding Protein 3
  • Protease Inhibitors
  • Phenylmethylsulfonyl Fluoride
  • Insulin-Like Growth Factor I
  • Aprotinin
  • Endopeptidases
  • insulin-like growth factor binding protein-3 protease