Immunoassays are chemical tests used to detect or quantify a specific substance, the analyte, in a blood or body fluid sample, using an immunological reaction. Immunoassays are highly sensitive and specific. Their high specificity results from the use of antibodies and purified antigens as reagents. An antibody is a protein (immunoglobulin) produced by B-lymphocytes (immune cells) in response to stimulation by an antigen. Immunoassays measure the formation of antibody-antigen complexes and detect them via an indicator reaction. High sensitivity is achieved by using an indicator system (e.g., enzyme label) that results in amplification of the measured product.
Immunoassays may be qualitative (positive or negative) or quantitative (amount measured). An example of a qualitative assay is an immunoassay test for pregnancy. Pregnancy tests detect the presence of human chorionic gonadotropin (hCG) in urine or serum. Highly purified antibodies can detect pregnancy within two days of fertilization. Quantitative immunoassays are performed by measuring the signal produced by the indicator reaction. This same test for pregnancy can be made into a quantitative assay of hCG by measuring the concentration of product formed.
The purpose of an immunoassay is to measure (or, in a qualitative assay, to detect) an analyte. Immunoassay is the method of choice for measuring analytes normally present at very low concentrations that cannot be determined accurately by other less expensive tests. Common uses include measurement of drugs, hormones, specific proteins, tumor markers, and markers of cardiac injury. Qualitative immunoassays are often used to detect antigens on infectious agents and antibodies that the body produces to fight them. For example, immunoassays are used to detect antigens on Hemophilus, Cryptococcus , and Streptococcus organisms in the cerebrospinal fluid (CSF) of meningitis patients. They are also used to detect antigens associated with organisms that are difficult to culture, such as hepatitis B virus and Chlamydia trichomatis . Immunoassays for antibodies produced in viral hepatitis, HIV, and Lyme disease are commonly used to identify patients with these diseases.
There are several different methods used in immunoassay tests.
Blood samples are collected by vein puncture with a needle. It is not necessary to restrict fluids or food prior to collection. Blood should be collected in tubes containing no additive. Risks of vein puncture include bruising of the skin or bleeding into the skin. Random urine samples are acceptable for drug assays; however, 24-hour urine samples are preferred for hormones and other substances which show diurnal or pulse variation.
Special safety precautions must be observed when performing RIA methods. Radioactive isotopes are used by RIA tests to label antigens or antibodies. Pregnant females should not work in an area where RIA tests are being performed. Personnel handling isotope reagents must wear badges which monitor their exposure to radiation. Special sinks and waste disposal containers are required for disposal of radioactive waste. The amount of radioisotope discarded must be documented for both liquid and solid waste. Leakage or spills of radioactive reagents must be measured for radioactivity; the amount of radiation and containment and disposal processes must be documented.
Immunoassays which are qualitative are reported as positive or negative. Quantitative immunoassays are reported in mass units, along with reference intervals (normal ranges) for the test. Normal ranges may be age- and gender-dependent. Positive immunoassay test results for HIV and drugs of abuse generally require confirmatory testing.
Although immunoassays are both highly sensitive and specific, false positive and negative results may occur. False-negative results may be caused by improper sample storage or treatment, reagent deterioration, or improper washing technique. False-positive results are sometimes seen in persons who have certain antibodies, especially to mouse immunoglobulins (immune cells) that may be used in the test. False-positive results have been reported for samples containing small fibrin strands that adhere to the solid phase matrix. False-positives may also be caused by substances in the blood or urine that cross-react or bind to the antibody used in the test.
Generally, no special instructions need be given to patients for immunoassay testing. Some assays require a timed specimen collection, while others may have special dietary restrictions.
When blood testing is used for the immunoassay, the vein puncture site will require a bandage or light dressing to accomplish blood clotting.
Immunoassay is an in vitro procedure, and is therefore not associated with complications. When blood is collected, slight bleeding into the skin and subsequent bruising may occur. The patient may become lightheaded or queasy from the sight of blood.
Bishop, M. L., J. L. Duben-Engelkirk, and E. P. Fody. Clinical Chemistry Principles, Procedures, Correlations. 4th ed. Lippincott, Williams, and Wilkins, 2001.
Burtis, C. A., and E. R. Ashwood, eds. Tietz Fundamentals of Clinical Chemistry. 5th ed. Philadelphia: W.B. Saunders, 2001.
Henry, J. B., ed. Clinical Diagnosis and Management by Laboratory Methods. 20th ed. Philadelphia: W. B. Saunders, 2001.
Wallach, Jacques. Interpretation of Diagnostic Tests. 7th ed. Philadelphia: Lippincott Williams & Wilkens, 2000.
Wild, D., ed. Immunoassay Handbook. 2nd ed. London: Nature Publishing Group, 2000.
Mark A. Best