IGF-1 assay protocol
Protocol for: Measurement of IGF-I concentrations
Prepared by: Rong Yuan
Reviewed by: David Harrison
Last revision date: 05/01/2006
Measurement of IGF-I concentrations
1. – Assay principle:
1.a – Serum (or plasma) IGF-I levels are determined using the IGF-I (IGFBP-blocked) RIA distributed by American Laboratory Products Company (ALPCO, Windham, NH). The calculated sensitivity of the assay is 0.02 ng/mL. The cross reactivity with IGF-II is small (<0.05%). Using this radioimmunoassay technique, IGF-I is dissociated from the binding proteins (IGFBP’s) by dilution in an acidic buffer. An antibody solution containing excess IGF-II is added to neutralize the samples. The excess IGF-II then occupies the IGF-binding sites and IGF-I is measured through addition of an 125I tracer. Separation of the bound and free tracer is carried out by the addition of a second antibody.
2. – Assay procedure:
2.a – Frozen specimens are thawed and prepared for analysis by diluting 10 µl of serum with 1 ml of Dilution Buffer (DB) to achieve a 1:101 dilution.
2.b – Kit control samples (M and N) provided 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.
Creation of the assay standard curve is achieved using undiluted standards (E-L) of known concentrations of recombinant human IGF-I (provided by ALPCO).
Diluted unknowns, diluted controls and undiluted standards (100µl) are pipetted into duplicate RIA tubes for analysis.
2.c – The first antibody (100µl, reagent B) is added to all but the “total counts” and “NSB” (non-specific binding) tubes.
125I tracer (100µl, reagent C) is then added to all tubes. The tubes are allowed to incubate at 2-8 oC for two days.
2.d – Following the incubation, 500µl of precipitation reagent P (containing the 2 nd antibody) is added.
After incubating for one hour at 2-8 oC, 1 mL of ice-cold water is added.
2.e – The assay tubes are centrifuged at 3740 rpm for 30 minutes at a temperature of 2-8 oC.
The supernatant is decanted and the activity of the precipitate counted for 3 minutes using a Packard RIAStar Gamma Counter model C5410.
2.f – Using software provided by Packard Instruments, a standard curve is constructed and the IGF-I concentrations of the unknowns and controls read from this curve (sample results are automatically multiplied by the dilution factor).
2.g – The IGF-I data are transferred to a Dell computer and set-up as a Microsoft Excel spreadsheet.