Development of IGF-1 ELISA Assays to Measure Free and Total Circulating IGF-1
Presentation Number: SUN-0642
Date of Presentation: June 22nd, 2014
Ajay Kumar*1, Bhanu Kalra1, Koushik Chowdavarapu1, Shivani Shah1, Gopal Savjani1 and Claus Oxvig2
1Ansh Labs, Webster, TX, 2Aarhus University, Aarhus, Denmark
Relevance: Insulin-like growth factor-1 (IGF-1) is a 7.6kDa, 70aa residue peptide. IGF-1 is primarily produced by the liver and circulates in the blood as an endocrine hormone. It is also produced locally by various tissues as a paracrine/ autocrine factor. IGF-1 found in circulation is primarily in a high molecular weight tertiary complex with IGF-binding protein-3 and acid-labile subunit. The unbound or free portion of IGF-1 in circulation is considered as the bioactive part as it is not inhibited by any binding proteins. Beyond its well-established mitogenic and metabolic functions, studies have shown the importance of IGF-1 and its use as a biomarker in detecting acromegaly and gigantism. Epidemiological studies have also linked elevated IGF-1 levels to increased cancer risk.
Methodology: Bioactive and Total IGF-1 ELISAs were developed to measure unbound, total (acidified and neutralized) IGF in serum in 1.5 hours. Multiple mAbs were selected based on their high affinity and specificity to unbound full-length IGF-1 and no binding affinity to IGFBP-complexes. The antibody pair used in the bioactive IGF-1 assay measures >99% of unbound IGF-1 and does not cross-react with IGF-II and IGF-1-BPs. The same antibody pair measures Total IGF-1 levels by way of acidification and neutralization of samples, which dissociates the various IGF-1 and IGF-1-BP complexes, prior to assaying. In addition, a Mouse/Rat IGF-1 kit has been developed using a different pair of mAbs and measures Total IGF-1.
Validation: The analytical sensitivity of the Total and Bioactive assays, as calculated by the interpolation of mean plus two standard deviations of 20 replicates of zero calibrator and calibrator B (0.48 ng/mL) was 0.025 ng/mL. Total imprecision calculated on four samples, run in duplicates over six runs was 6.35% at 2.087 ng/mL, 5.97% at 8.191 ng/mL, 6.39% at 1.708 ng/mL and 5.17% at 4.794 ng/mL Inhibition up to 97% was observed when IGFBP-3 was spiked into IGF-1 in excess. The Bioactive IGF-1 and Total IGF-1 ELISAs did not correlate when measured in serum (R² = 0.186) or ascites from ovarian cancer (R² = 0.026) indicating to be an independent biomarker. Potential interferents hemoglobin (1.35mg/mL), triglycerides (5mg/mL) and bilirubin (0.5mg/mL) yielded average interference of 8.0%, 5.2%, and 12.1% respectively in the Bioactive IGF-1 assay and -4.8%, 10.5%, and -3.0%, respectively, in the Total IGF-1 assay.
Conclusion: Highly reproducible Bioactive and Total IGF-1 ELISAs have been developed to measure total and free IGF-1 in serum and other biological fluids. Use of same antibody pair and identical calibrators (standardized to WHO code 91/554) enables researchers to measure the true ratio of unbound IGF-1 and total IGF-1 levels in circulation by means of differential sample treatment. Mouse/Rat IGF-1 ELISA provides researchers an additional tool to study circulating IGF-1 in mouse/rat model.
Nothing to Disclose: AK, BK, KC, SS, GS, CO