Kits or protocols that use competitive binding of antibodies to a target antigen versus a radioactively labeled quantity of the same antigen are referred to as radioimmunoassays (RIAs). A variety of kits are commercially available through various companies. MP Biomedicals (formerly ICN Biomedicals) has an extensive variety, including the ImmuChem Double Antibody Progesterone and Corticosterone
125I RIA Kits.
These kits use small quantities of rat or mouse plasma (10ul for the corticosterone kit and 100ul for the progesterone) to determine the levels of circulating steroids. The kits come with steroid specific standards (0 – 1000 ng/ml for corticosterone and 0 - 50 ng/ml for progesterone) and in the case of the corticosterone kit, controls are included. These controls are treated as a sample unknown and the results should fall within the High or Low range to which they belong. After addition of the 125I radiolabeled steroid and antibody solutions to the sample, incubation and centrifugation, a precipitant solution is added to precipitate bound anitgens. The supernatant is removed and the remaining 125I radiolabeled steroid is counted using a gamma counter. Using the counts from known standards, the unknown sample concentration can be calculated using formulas included in the protocols. There are also samples and examples included to assist with calculations if necessary.
Our lab has used these kits many times with a high degree of repeatability across experiments and correlation between samples tested in duplicate. The protocols are very easy to follow, with detailed instructions for each step of the assay. The entire assay (for either of the above) can easily be completed in one day, and allowed to count overnight. Some disadvantages include the use of radioactivity, the relatively short expiration dates of the kits and the expense of the kits. However, as all reagents are not usually consumed and have different expiration dates, it is possible to order the kit in parts.
Overall, the MP Biomedicals ImmuChem Double Antibody Progesterone and Corticosterone 125I RIA Kits are very reliable and easy to use methods of quantification of the respective steroids.
Todd O’Buckley
Research Technician/Lab Manager
Bowles Center for Alcohol Studies
UNC, Chapel Hill