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Indiana University Bloomington


General Concepts

Immunoassay techniques can be used to measure the amount of an antigen (for example, a hormone) in biological samples. There are two types of immunoassays available here at IU: enzyme immunoassays (EIA) and radioimmunoassays (RIA). Some examples of hormones measured using EIA or RIA in collaboration with the CISAB lab include estradiol, testosterone, progesterone, cortisol and oxytocin.

These assays are all based on a similar concept, where labeled antigen (called the ‘tracer’) competes with unlabeled antigen (present in your sample) for binding sites on an antibody (Ab) in limited availability. Unlabeled antigen with known hormone concentrations (your ‘standards’) also are incubated with a labeled antigen in a similar manner.

The components above are allowed to incubate together in a test tube or microtiter plate well, and then unbound (excess) antigen (Ag) is removed so you are left only with the Ab-Ag complexes. If you have a very high concentration of hormone in your sample or standard, most of the resulting Ab-Ag complexes will be unlabeled. If you have a very low concentration of hormone, most of the Ab-Ag complexes will be labeled. These labeled Ab-Ag complexes are what you are actually measuring when you perform the assay. It is an inverse relationship, where the higher the number of labeled Ab-Ag complexes you measure, the lower the concentration of hormone in your sample or standard.

The major difference between EIA and RIA is the manner in which the ‘tracer’ antigen is labeled. EIA tracers are labeled with an enzyme that undergoes a colorimetric reaction under the appropriate conditions. This color reaction is quantified using a device that can measure optical density (OD). Thus, a sample with little or no color after the enzyme reaction has a high hormone concentration. RIA tracers are radiolabeled, usually with tritium ( 3H) or iodine ( 125I). The amount of radioactivity in each sample after the assay is complete is measured in a beta or gamma counter. Thus, a sample with low radioactivity counts also has a high hormone concentration.

Remember the standards of known hormone concentrations mentioned previously? By obtaining OD or radioactivity readings for each of these standard concentrations, these values can be plotted on a graph to produce a standard curve. This standard curve is then used as a reference to calculate the concentration of hormone in each of your samples (based on the OD or radioactivity reading for each sample).

Please note that there are many variations to the basic immunoassay technique outlined above. Some assays employ two antibodies instead of one- and in this case an Ab-Ab-Ag complex is formed. Your primary antibody is what recognizes the antigen, and then a secondary antibody is used to recognize the primary antibody. Some people refer to this as a “sandwich” assay, and it is often used to increase the stability of the assay. Also, in most assays the antibody is adsorbed directly to the tube or well, but sometimes it is free-floating, as is the case in single antibody RIAs using a charcoal-dextran separation method.

It is important to understand that these immunoassay concepts can also be used to measure antibody titers, instead of antigen concentrations. This is often referred to as an ELISA (enzyme-linked immunosorbent assay). However, many people now use the terms EIA and ELISA interchangeably to describe any enzyme-based immunoassay.

EIA versus RIA

When deciding whether to employ an EIA or RIA for your research project, it is important to consider the following: (1) what methods (if any) have been used previously for your species and sample type and worked; (2) do you need your assay to be “field-friendly” and portable; (3) what sample volume or mass are you dealing with; and (4) what is your budget for the project?

Generally speaking, RIAs are more cost-effective compared to EIAs when conducted here at IU. However, RIAs must be performed in specific licensed laboratories and more regulatory practices must be followed. The CISAB lab is not licensed, but a nearby RIA lab is available for use. Also, many people find that EIA reagents are easier to use and prepare. The sensitivity of RIA versus EIA is dependent on the hormone and specific assay in question; however, most RIAs and EIAs are equally sensitive. Choosing which technique to use must be decided on a case-by-case basis, and the CISAB lab director is available to advise in this decision when needed.

Assay Validation

Whenever an assay is being used for a new species or sample type, validation of that assay must be performed to ensure that: (1) the assay is sensitive enough to detect hormone levels in your sample; (2) there are no substances in your sample that interfere with Ab-Ag binding; and (3) the antigen in your sample is immunologically similar to the antigen in your standard (and can be measured proportionately). Additional validation is required if the sample is extracted prior to analysis to ensure that you are not “losing” hormone during the extraction process and that the extraction process is consistent across samples.

A variety of different techniques can be used to validate and optimize your assay. Sometimes validation is relatively straightforward, and other times it can take months. But in the end, you want to make sure you are confident in your results, which is why validation is so important.

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