Project protocol — Contents
Workflow and sampling
Reagents, supplies, and solutions
Procedure for measuring plasma IGF-1 (radioimmunoassay)
Test Procedure performed Ages (mo) Data collected 1 body weight measurement 6, 12, 18 bw 2 blood collection 6, 12, 18 - 3 plasma collection 6, 12, 18 - 4 insulin-like growth factor 1 (IGF-I) measurement 6, 12, 18 plasma IGF-I
- Gamma Counter model C5410 Packard RIAStar
- Refrigerated centrifuge for precipitation of immunocomplexes
- Vortex mixer
- Device for aspiration of liquid supernatant (i.e. connected to a water pump)
• IGF-I (Direct, IGFBP-blocked) RIA, catalog no. 22-IGF-R20, American Laboratory Products Company (ALPCO, Windham, NH)
The reagents listed below are sufficient for 200 tubes including the standard curve:
AB Acidification Buffer - (1 bottle, 12.5 mL, ready for use, colored)
DB Dilution Buffer - (1 bottle, 125 mL, ready for use, colored)
A Assay Buffer - (1 bottle, 30 mL, ready for use)
B 1st Antibody (anti-hIGF-I) containing rabbit IgG and recombinant hIGF-II - (1 bottle, 11 mL, lyophilized)
C Tracer: 125I-IGF-I; < 1.6 μCi or < 55 kBq - (1 bottle, 11 mL, lyophilized, red colored) Radionuclide Iodine (I125 tracer), T1/2 = 60 days, emits 35.5 keV gamma radiation, 27 – 32 keV x-rays and no beta radiation. Lead shielding is effective. D Rabbit immunoglobulin for non-specific binding (NSB) - (1 vial, 500 µL, lyophilized).
E - L Standards: Concentrations given on vial-labels in ng/mL - (8 vials, 500 µL each, lyophilized).
M+N Controls (human serum): Conc. given on vial-labels in ng/mL - (2 vials, 100 µL each, lyophilized)
O 2nd Antibody (anti-rabbit immunoglobulin) - (1 vial, 1 mL, lyophilized)
P Precipitation Reagent - (1 bottle, 55 mL, ready for use after adding O)Ice-cold deionized water
• Pipettes: 10 mL, 1 mL, 500 µL, 250 µL, 100 µL, 10 µL; 100 µL, 250 µL and 1 mL repeating pipettes are recommended.
• Disposable polystyrene or polypropylene tubes. Conical tubes are highly recommended because of the small immunoprecipitates. The use of round-bottom tubes may cause formation of insufficiently compact pellets.
Acclimation to test conditions
Mice are allowed to acclimate to testing room over the weekend.
Procedure for measuring plasma IGF-I (radioimmunoassay)
I. Blood collection and plasma isolation
a. Mice at 6-7.5, 12-13.5 and 18-19.5 months of age are fasted for 4 hrs, from 7:30 am to 11:30 am, before they are bled.
b. Bodyweight measurements are recorded just before blood samples are drawn.
c. A minimum volume of 250 µL (~ 13 drops) blood is obtained via peri-orbital vein using heparin-coated microhematocrit tubes and collected into microtainer tubes.
d. Plasma is collected after the blood sample in microtainer tubes is centrifuged at 14,000 rpm, for 10 min, at 4°C.
e. An aliquot of 30 µL per sample is saved in Eppendorf tubes for IGF-I assay. The rest of the plasma (65 µL) is used for other hormone assay (i.e. insulin, leptin).
f. Extended storage of plasma is done at -70°.
II. Measurement of plasma insulin-like growth factor 1 concentration
When using the radioimmunoassay technique the IGF-I is dissociated from the binding proteins (IGFBP's) by dilution in an acidic solution. This acidic solution is then neutralized with the addition of a buffer solution, containing antibody against IGH-I plus an excess IGF-II. Because IGF-II is presented in excess, it (competitively) occupies the IGF-binding sites and the displaced IGF-I is measured through the addition of an I125 tracer. Separation of the bound I125 (or IGF-I bound to IGFBP's) and free I125 tracer (or competitively displaced IGF-I) is carried out by the addition of a second antibody (see figure).
a. Frozen specimens are thawed and prepared for analysis by acidifying and diluting 10 µl of plasma with 1 ml of Dilution Buffer (DB) to achieve a 1:101 dilution.
b. Control samples (M and N) provided in the kit by ALPCO and an in-house B6 strain mouse pool control are prepared and analyzed simultaneously with unknown samples to verify the effectiveness of the assay.
c. Creation of the assay standard curve is achieved using undiluted standards (E-L) of known concentrations of recombinant human IGF-I (provided by ALPCO).
d. Diluted unknowns, diluted controls, and undiluted standards (100µl) are pipetted into duplicate RIA tubes for analysis.
e. Then the 1st antibody (100µl, reagent B) is added to all but the "total counts" and "NSB" (non-specific binding) tubes.
f. Iodine (I125) tracer (100µl, reagent C) is then added to all tubes. The tubes are allowed to incubate at 2-8 °C for 2-days.
g. Following the 2-day incubation period, 500µL of precipitation reagent P (containing the 2nd antibody) is added.
h. After incubating for 1-hr at 2-8 °C, 1 mL of ice-cold water is added.
i. The sample tubes are centrifuged at 3740 rpm for 30 min at a temperature of 2-8 °C.
j. The supernatant is decanted and the activity of the precipitate counted for 3 min using a Packard RIAStar gamma counter model C5410.
k. A standard curve is constructed (using the software provided by Packard Instruments) and the sample IGF-I concentrations and controls are read from this curve (sample results are automatically multiplied by the dilution factor).
l. The IGF-I data are then transferred to a Dell computer and set-up as a Microsoft Excel spreadsheet.
m. For additional information go to The Aging Center at The Jackson Laboratory.
Submitting investigator's notes: "Because MOLF mice were started later than other strains, their IGF1 levels at 12 and 18 months have not been measured. Most AK mice died before 12 months, so that their IGF1 levels at 12 and 18 months are absent. Five outliers (z-score > 2) of 1358 measurements were excluded from the analysis." (Yuan et. al., 2009)
Definitions & formulas
If it is preferred to express the results as nmol/L, rather than giving values in ng/mL, then dividing the results with 7.649 will give nmol/L.
For example: 203 ng/mL ÷ 7.649 = 26.5 nmol/L
Data collected by investigator
Plasma insulin-like growth factor 1 (IGF-I) concentration and body weight, blood glucose.
Adamo ML, Ma X, Ackert-Bicknell CL, Donahue LR, Beamer WG, Rosen CJ. Genetic increase in serum insulin-like growth factor-I (IGF-I) in C3H/HeJ compared with C57BL/6J mice is associated with increased transcription from the IGF-I exon 2 promoter. Endocrinology. 2006 Jun;147(6):2944-55. Epub 2006 Mar 9.
Iida K, Rosen CJ, Ackert-Bicknell C, Thorner MO. Genetic differences in the IGF-I gene among inbred strains of mice with different serum IGF-I levels. J Endocrinol. 2005 Sep;186(3):481-9.
Pratipanawatr T, Pratipanawatr W, Rosen C, Berria R, Bajaj M, Cusi K, Mandarino L, Kashyap S, Belfort R, DeFronzo RA. Effect of IGF-I on FFA and glucose metabolism in control and type 2 diabetic subjects. Am J Physiol Endocrinol Metab. 2002 Jun;282(6):E1360-8.
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Rosen CJ, Churchill GA, Donahue LR, Shultz KL, Burgess JK, Powell DR, Ackert C, Beamer WG. Mapping quantitative trait loci for serum insulin-like growth factor-1 levels in mice. Bone. 2000 Oct;27(4):521-8.
Rosen CJ, Conover C. Growth hormone/insulin-like growth factor-I axis in aging: a summary of a National Institutes of Aging-Sponsored Symposium. J Clin Endocrinol Metab. 1997 Dec;82(12):3919-22.
Rosen CJ, Dimai HP, Vereault D, Donahue LR, Beamer WG, Farley J, Linkhart S, Linkhart T, Mohan S, Baylink DJ. Circulating and skeletal insulin-like growth factor-I (IGF-I) concentrations in two inbred strains of mice with different bone mineral densities. Bone. 1997 Sep;21(3):217-23.
Rosen CJ, Donahue LR, Hunter SJ. Insulin-like growth factors and bone: the osteoporosis connection. Proc Soc Exp Biol Med. 1994 Jun;206(2):83-102.
Rosen CJ, Glowacki J, Craig W. Sex steroids, the insulin-like growth factor regulatory system, and aging: implications for the management of older postmenopausal women. J Nutr Health Aging. 1998;2(1):39-44.
Rosen CJ, Pollak M. Circulating IGF-I: New Perspectives for a New Century. Trends Endocrinol Metab. 1999 May;10(4):136-141.
Yakar S, Wu Y, Setser J, Rosen CJ. The role of circulating IGF-I: lessons from human and animal models. Endocrine. 2002 Dec;19(3):239-48.