Serum assays for total IGF-I, free IGF-I, intact IGFBP-3, GH, and insulin

Serum samples for insulin and glucose measurement were collected from WT and Pappa2 KI mice (15 weeks of age) via cheek punch after a 6-hour fast (0900 to 1500 hours). Blood samples for free IGF-I, total IGF-I, and intact IGFBP-3 measurements were collected from WT and Pappa2 KI mice at 16 weeks of age. Blood samples were placed on ice for 30 minutes to allow for clotting. Serum was separated via centrifugation at 8000 rpm for 10 minutes at 4°C and stored at −80°C until analysis. Serum insulin levels were measured by ELISA (catalog no. 80-INSHU-E01.1) from ALPCO (Windham, NH). Serum GH values were determined using a rat/mouse GH ELISA kit (EZRMGH-45K) from EMD Millipore (St. Louis, MO). Serum mouse free IGF-I, mouse total IGF-I, and intact IGFBP-3 values were determined using commercially available ELISA kits (free rat/mouse IGF-I, catalog no. AL-136; total rat/mouse IGF-I, catalog no. AL-137; intact IGFBP-3, catalog no. AL-149) from Ansh Laboratories (Webster, TX). The total and free IGF-I assays use the same pair of monoclonal antibodies. In the two-step sandwich assay configuration, the capture antibody preferentially binds free IGF-I whereas the detection antibody binds either free IGF-I or IGF-I bound to IGFBP-3. Thus, in the absence of preanalytical treatments to release IGF-I from IGFBP-3, only free IGF-I is quantitated using the Ansh Free IGF-I kit (AL-136). Quantitative measurement of total IGF-I requires preanalytical release of IGF-I bound to IGFBP-3 by acidification using reagents supplied as a separate test kit (AL-137). Neither assay, free or total IGF-I, demonstrates cross-reactivity (<0.01%) with IGFBP-2, IGFBP-3, IGFBP-4, of IGFBP-5. The limit of detection for free IGF-I was 0.155 ng/mL and for total IGF-I was 1.60 ng/mL. The coefficient of variation of replicate determinations was <9% for mouse serum specimens with total IGF-I concentrations between 5.20 and 270 ng/mL and <6% for specimens with free IGF-I concentrations between 0.160 and 12 ng/mL. To validate that the free IGF-I ELISA kit (AL-136) is in fact measuring the free hormone level and not IGF-1 bound to IGFBP-3, we added recombinant mouse IGFBP-3 (catalog no. 775-B3-025; R&D Systems, Minneapolis, MN) in excess (100-fold molar ratio) to serum samples from two WT mouse strains. This resulted in complete elimination of measurable free IGF-I as expected (28). The intact IGFBP-3 assay (AL-149) is a double monoclonal antibody sandwich-type assay and uses an acidification and neutralization preanalytical procedure to dissociate intact IGFBP-3 from all of the binding subunits. The assay demonstrates no significant (<0.1%) cross-reactivity with IGF-I, IGF-2, IGFBP-2, IGFBP-4, or IGFBP-5. IGFBP-3 calibrators in intact IGFBP-3 ELISA are traceable to Non WHO Reference Material IGFBP-3 (National Institute for Biological Standards and Control code 93/560; calibrators, 0.34). The antibodies used in the assay were mapped using six IGFBP-3/IGFBP-5 chimeras. The capture antibody mapped to the C-terminal region whereas the detection antibody mapped to the mid-region. Therefore, this assay does not detect cleaved IGFBP-3 fragments. The lower limit of quantitation was 46.5 ng/mL, and the lower limit of detection was 27 ng/mL. The coefficient of variation of replicate measurements was <8% for mouse serum specimens with IGFBP-3 concentrations between 65 and 2018 ng/mL. All assays were performed according to the manufacturers’ instructions.