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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 (∼2 years at −80°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 (∼8 years at −20°C), the inter-method median IGF-I levels varied by ∼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°C for 4 weeks was measured. However, the 4°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.

Objective: Inhibin circulates in various molecular weight forms. Alpha (α)-subunit-directed total inhibin immunoassays, which detect all forms of α subunits plus the α/β inhibin dimers, have been found valuable in the diagnosis and monitoring of ovarian cancer. Because of the dependency of the published methods on boiling sample pre-treatment with SDS and unavailability of a commercial assay, we developed an enzyme-linked immunosorbent assay (ELISA) for direct determination of total inhibin. Design and methods: Method development involved a pair of well-characterized inhibin α subunit-directed antibodies and determination of the effects of various assay parameters. Selection of the optimized protocol was guided by the outcome of comparative sample analysis using previously reported boiling sample pre-treatment reagents and protocols. Results: We report development of a simplified ELISA for total inhibin. Method evaluation data demonstrated acceptable analytical performance characteristics with detection limit of 2 ng/l (recombinant inhibin-A), dynamic range of 12.5–500 ng/l, and intra- and inter-assay imprecision of 2.3–4.6% and 3.3–5.1% at total inhibin concentrations of approximately 60–400 ng/l, respectively. The mean (±SD) recovery from spiked serum samples averaged 109 ± 14% and recovery in response to serial sample dilution was 99 ± 10%. Serum values by the direct method ( n = 40) correlated strongly with those obtained after sample pre-treatment by boiling with SDS ( r = 0.97). As expected, the total inhibin immunoreactivity in human follicular fluid fractionated by HPLC gel filtration in multiple immunoreactive peaks (8–250 kDa). In serum samples from postmenopausal women with ovarian cancer, the assay detected significantly higher total inhibin levels than in samples from normal postmenopausal controls. Conclusion: The development of a fast and simplified ELISA should facilitate wider investigations of pathophysiology and diagnostic potential of total inhibin measurement.

Markers of myocardial injury have been vital in the assessment of patients with coronary heart disease. Pregnancy associated plasma protein A (PAPP)-A is an insulin-like growth factor (IGF) binding protein (IGFBP)-4 protease and a potential early indicator of unstable angina. We developed an ultrasensitive enzyme-linked immunosorbent assay (ELISA) for PAPP-A and measured serum PAPP-A in patients with biochemical evidence of acute coronary syndrome.Method development was based on pair-wise evaluation of a panel of antibodies and determination of PAPP-A specificity and sensitivity relative to those of a conventional method. Association of PAPP-A with myocardial damage was assessed in serum samples classified based on serum creatine kinase (CK)-MB or cardiac troponin-T levels.Serum PAPP-A was significantly higher in samples with elevated CK-MB or troponin-T than in samples with normal CK-MB (p0.001). Marker-association studies showed strong correlation between PAPP-A and troponin-T (r = 0.59, p0.001) in a subset of troponin-T positive samples. Indications for both parallel as well as divergence in the expression of PAPP-A and troponin-T was also evident when serial timed samples available from a number of patients were analyzed.The data are consistent with the conclusion that expression of PAPP-A is enhanced in patients with biochemical evidence of acute coronary syndrome and suggest strongly that demonstration of PAPP-A association with other cardiac markers might be influenced by their relative release dynamics (timing and duration). The availability of the ultrasensitive PAPP-A ELISA should facilitate systematic investigations of PAPP-A expression in this and other pathophysiological conditions that might involve altered expression of the IGF/PAPP-A system.