In this article we try to elucidate the genesis of Immunoassays that are now commonplace. Immunoassay is an assay that quantifies antigen (can be a simple substance such as a drug or a protein or virus) or antibody using Immunochemical means.

Supposing you want to measure the presence of a hormone like insulin in blood the laboratory based biochemical test called an immunoassay would be used. The principle behind this test is that an antibody will specifically bind to its antigen, and in this case the antigen is the insulin. If it is a question of detecting an infection, the presence of the antibodies against the pathogen that is specific to the disease is measured. So these tests can measure both antigen and antibodies present in serum and urine. Immunoassay as a biochemical tool first appeared in the 1960s, and can now be used as tumor markers.

As regards the antibodies that are used for these tests, the rule is that it must have high affinity for the antigen if present, or in other words a large number of the antigens must bind to the antibody. Two types of antibodies are used namely monoclonal antibodies and polyclonal antibodies. Use of monoclonal antibodies gives accurate and specific results that don't get influenced if other molecules are present, although polyclonal antibodies are also used often.

Most hormones and proteins as well as many drugs are currently determined by immunoassay. The most common immunoassay is the ELISA test (perfected by Peter Perlmann and Eva Engvall) that can detect antigen and antibody quantities. ELISA is an acronym for Enzyme-linked immunosorbent assay which is a diagnostic blood test to validate patients for viruses like that of AIDS. If an antigen or antibody is labeled with an enzyme it is called enzyme immunoassay or EIA (first developed by Anton Schuurs and Bauke van Weemen at the Research Laboratories of NV Organon), and if it is labeled with a radioisotope it is called radioimmunoassay or RIA. For example, screening blood for hepatitis B antigen until the 1970s was done by hemagglutination. Now it is done by radioimmunoassay. RIA was first developed by Berson and Yalow for which the latter was awarded a Nobel Prize.

Small labs will find it difficult to use RIA as an analytical method because it is a labor intensive batch process that's beyond automation and more importantly raises several safety issues. However the radioactive problems associated with RIA have taken a backseat after iodine-125 preparations with high specificity and weak radiation became available.

Types of Immunoassays

Immunoassay methods could be either heterogenous (radioimmunoassay) or homogenous. In heterogenous immunoassay the bound (the tracer that binds) and free fractions of the tracer have to be separated physically, which is also the reason why it is difficult to automate a heterogenous assay.

As for the difference between heterogenous and homogenous immunoassay systems, the former best suits detection of high or low molecular weight molecules. On the contrary, homogenous assays best suit small molecule measurement (as for example used in drugs monitoring)that does not need physical separation of free and bound factions of tracers and therefore more amenable to automation as well.

However the question that can be asked when enzyme labels are used in immunoassay is will not the large enzyme molecule when sticking to the antigen or antibody impact the immunochemical reactions between them (between antigen and antibody)?

Why the need for Immunoassays?

Malignant tumor cells release certain biochemicals albeit in small quantities that cannot be measured by normal chemical analysis. The strong and specific interaction of an antibody with an antigen (the analyte could be either antibody or antigen) is monitored with a tracer. The tracer (either antigen or antibody as the case may be) is tagged with a lebel which may be enzyme or a nuclide that breaks down with radiation. The result is obtained by detecting and quantifying the signal given by the tracer, as for example fluorescence.

Future trends in Immunoassay

Identifying proteins that are in the blood or in the cells to serve as biomarkers for diagnosing and monitoring treatments for diabetes, cardiac, and cancer are areas of current and future biotech research for which biomarker immunoassays are a required tool. Furthermore, the need for multiple biomarkers to ensure specificity means that in future biomarker validation will be key area to work on.

Now the identification of certain reliable markers present in low concentrations such as myoglobin protein which is moderately cardiac specific using an ultrasensitive immunoassay technique called plasmonic technology helps in clinical diagnostics to detect cardiac problems.

Although partially automated immunoassay systems are in use today, in the not too distant future, we can hope to see fully automated immunoassay systems. However, despite the advances in immunoassays, more research needs to be done in developing assays suitable for the oncology and stem cell research specialities.

In the final analysis, diagnostic immunoassays whether they are RIA, EIA, or ELISA, has had a profound impact on patients and clinicians alike, and the development of innovative recombinant proteins assays and antibodies is a continuing effort directed at combating both metabolic and neurological diseases.

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