Immunoassay

Immunology is a laboratory science that studies the body's immunity to disease. The basic mechanism of immunity is the binding of drugs or other chemical compounds to antibodies (large proteins produced by the body's immune system). An assay is a general term for an analytical laboratory procedure designed to detect the presence of and/or the quantity of a drug in a biological fluid such as urine or serum (the fluid component of the blood obtained after removal of the blood cells and fibrin clot). An immunoassay, therefore, is an analytical procedure which has as its basis the principles of immunology—specifically the binding of drugs to antibodies.

Several different types of immunoassay are routinely performed in the laboratory. Although they differ in the types of reagents and instrumentation used, they are all based on the same scientific principle (the binding of drugs to antibodies). The three types of immunoassay that are commonly used for drug testing are the radioimmunoassay (RIA), enzyme multiplied immunoassay (EMIT), and fluorescence polarization immunoassay (FPIA).

It may facilitate the reader's understanding of immunoassay to envision the reactions that occur in the body following a vaccination (e.g., polio). The vaccine contains a weak or a killed solution of (polio) virus. When the vaccine is injected into the body, the immune system recognizes the presence of a foreigner (the polio virus), and it generates antibodies to that virus. These antibodies circulate in the blood, and they constitute the body's protection; if at some later date a live (polio) virus invades the body, the antibodies recognize it by its unique size and shape (similar to the fit of a lock and key); they spontaneously bind to the virus, leading to its inactivation and removal from the body.

This binding of antibodies to drugs forms the basis for immunoassay. In the development of an immunoassay, the first step is to inject an animal (host) with the drug that we ultimately wish to analyze. The host immune system, recognizing the drug as a "foreigner," generates antibodies to this drug, and these antibodies can then be harvested from the serum of the animal. In the test-tube environment of the laboratory (in vitro), these antibodies can be recombined with the appropriate drug. Just as it did inside the body (in vivo), the antibody will recognize the drug based on the lock-and-key fit and will spontaneously bind to it.

The second step in the development of an immunoassay is to synthesize a "labeled" drug. This involves the chemical addition of a "marker" to the drug. This marker can be small, such as an atom of radioactive iodine, or it can be large, such as an enzyme, which is a fairly large protein. Irrespective of its size, this marker is added in such a way that it does not interfere with the lock-and-key recognition between the antibody and the drug.

Commercially available immunoassay kits contain the antibody (which the company has prepared as described above) and the labeled drug (which has been chemically synthesized) necessary to perform the assay. In the laboratory, a fixed amount of antibody and a fixed amount of labeled drug are placed into a reaction vessel (test tube). If these were the only two ingredients, all the binding sites on the antibody would react with (bind to) the labeled drug. A third ingredient added to the assay is, however, the unlabeled drug (i.e., the urine, saliva, or serum specimen containing the drug that is being measured). Because the label on the labeled drug is placed in a position that does not interfere with binding to the antibody (i.e., it is "hidden"), the antibody cannot distinguish between the labeled and unlabeled drug.

Immunoassays are always designed so that there are fewer antibody-binding sites present in the reaction mixture than there are molecules of (labeled plus unlabeled) drug. Because the labeled and unlabeled drug appear the same to the antibody, they will compete equally for the limited number of available binding sites on the antibody. By measuring the amount of labeled drug bound to the antibody, the analyst can calculate the amount of unlabeled drug in the biological specimen.

All immunoassays work in the same basic fashion. They differ in the types of labels that are added to the labeled drug and in the analytical methods by which the amount of binding of labeled drug to the antibody is measured.

RADIOIMMUNOASSAY

Radioimmunoassay (known as RIA) was the earliest of the immunoassay techniques. It was developed during the 1950s by a pair of research immunologists in New York City, Dr. Solomon A. Berson and Dr. Rosalyn S. Yalow. Their initial RIA was designed to detect very low blood levels of insulin and they published their findings in 1959. Their development of this technique was considered of such importance to science that Dr. Yalow was awarded a Nobel prize in 1977 for their work (since Dr. Berson died in 1972 and Nobels are not awarded posthumously, Berson's contribution was remembered in Yalow's acceptance speech).

In RIA, the marker is an isotope of a radioactive element, hence the name radioimmunoassay. In most RIAs performed in the laboratory today, the radioactive isotope used as the marker is iodine 125, although tritium (hydrogen 2), carbon 14, and cobalt 57 are used in some assays. RIAs can be used in two different fashions to give information about the drug in a sample: (1) they can be used qualitatively—to determine whether a drug is present or absent (e.g., in urine drug testing); (2) they can be used quantitatively—to determine how much of a drug is present (e.g., to measure serum levels of drugs such as digoxin, a heart medication, or theophylline, an asthma medication).

RIA is an extremely powerful tool. One of its main advantages is the sensitivity that can be achieved. Drug levels in serum and urine that are as low as 10 to 100 parts per billion are routinely measured. Two of the most sensitive of the radioimmunoassays are the urine LSD assay and the serum digoxin assay, both of which can detect less than one part per billion. RIA is also an extremely versatile tool. It is used to measure a wide range of drugs of abuse in blood, serum, saliva, and urine, as well as therapeutic (physician administered) drugs in blood or serum. It is also used as a diagnostic tool to detect and quantify numerous naturally occurring chemicals in human serum and urine. Another characteristic that makes RIA such a powerful tool is the specificity of the assay. The antibodies are highly specific for the drugs analyzed and they rarely make a mistake in recognizing the lock-and-key fit between antibody and drug.

One of the major limitations of the radioimmunoassay is that it generates radioactive waste. To avoid spreading the radioactive compounds and contaminating the environment, the laboratory must conform to very strict regulations, including very elaborate procedures for waste disposal—and undergo frequent inspections. Because of a short half-life for some isotopes, another limitation is that the reagents with a radioactive label have a short shelf life. For instance, the majority are RIAs labeled with iodine 125; they have a shelf life of only approximately sixty days.

Some very sophisticated automated equipment is available for performing RIA or, if need be, the assays can be performed manually. All RIAs require the use of an instrument called a gamma counter, which measures the amount of gamma radiation given off by the radioactive drug bound to the antibody. In the 1990s, gamma counters can be purchased for as little as a few thousand dollars; but the reagents are moderately expensive (costing from less than fifty cents/test to two to three dollars/test, depending on the specific assay and the volume of reagents purchased).

ENZYME MULTIPLIED IMMUNOASSAY

The enzyme multiplied immunoassay technique, also known as EMIT™, is a variation of the general immunoassay technique, in which the marker used to prepare the labeled drug is an enzyme, rather than a radioactive isotope. EMIT is a two-stage assay. As in the other immunoassays, the sample, which contains some amount of the drug being measured, is combined with the antibody plus a fixed amount of the enzyme-labeled drug. In the first reaction, the labeled and the unlabeled drug compete for the available binding sites on the antibody (standard immunoassay reaction). A secondary reaction is then performed, which involves only the enzyme portion of the labeled drug. The results of this secondary reaction are used to calculate the amount of enzyme-labeled drug that is bound to the antibody and thus how much (un-labeled) drug there was in the original urine or serum specimen.

As with other forms of immunoassay, the EMIT can be used either qualitatively or quantitatively. In urine specimens, it is used to detect the presence of drugs, such as THC (MARIJUANA), COCAINE, PCP, OPIATES (HEROIN), AMPHETAMINES, and BARBITURATES. In serum specimens, EMIT is used to determine the amount present of drugs used for therapeutic (medical) purposes. Such drugs include acetaminophen (Tylenol), salicylate (aspirin), theophylline (widely used to treat asthma), several drugs used to treat epilepsy, and several drugs used to treat heart abnormalities.

Advantages that the EMIT technology has over the RIA are (1) that no radioactivity is involved, so the waste is more readily disposable; (2) the re-agents are relatively stable, which may be particularly attractive to a small laboratory, which runs only a few specimens. The EMIT reagents are also less costly than the RIA reagents. The basic instrumentation requires less capital outlay than does the RIA, however the expense grows as more sophisticated automation is acquired.

Some limitations of the EMIT technique are (1) that it is somewhat less sensitive than the RIA (in particular, the LSD assay requires detection of such minute levels of the drug in urine that it can only be done by RIA); (2) also, EMIT is less specific than RIA and is subject to some interferences that do not affect the RIA—for example, the EMIT assay for amphetamines in urine gives a positive response with several other drugs that are similar in structure to amphetamines.

FLUORESCENCE POLARIZATION IMMUNOASSAY

Fluorescence polarization immunoassay (known as FPIA) is a technique that was developed by Abbott laboratories and marketed under the trade name TDX. As the name FPIA implies, the marker for the labeled drug is a molecule of a naturally fluorescent compound called fluorescein. The amount of labeled drug that binds to the antibody is measured by a sophisticated instrument called a spectrofluorometer. As with the other immunoassays, this measurement is used to calculate the amount of labeled drug bound to the antibody and thus the amount of drug in the original urine or serum specimen.

The instrumentation necessary to perform the FPIA is only made by Abbott. It is expensive to purchase (upwards of $50,000) but can be leased from the manufacturer. The reagents are more expensive than EMIT reagents, being roughly comparable in cost to RIA reagents. They come in a liquid form and have a more limited shelf life than those for EMIT, but they tend to be more stable than RIA reagents.

The attractiveness of FPIA is in the speed and ease of operation of the instrument. The reagents come in a kit that is bar coded and is placed right into the instrument. All the operator has to do is fill the sample cups with serum or urine, place the reagent pack inside the instrument, and push a button marked "run." The instrument reads the bar code, enters the necessary programs into its memory, performs the assay, and prints out the results. For the routine hospital lab or small drug-testing lab, it is as fast or faster than EMIT or RIA and a lot easier; however, the instrument can only run twenty specimens at a time. For the large drug-testing laboratory, more rapid results can be achieved with the automated instrumentation available for the EMIT or RIA techniques.

FPIA is nearly as sensitive as RIA; digoxin can be run by FPIA, although LSD is still not available. The specificity of FPIA is also comparable to that of RIA.

(SEE ALSO: ; Hair Analysis as a Test for Drug Use)

BIBLIOGRAPHY

BLAKE, C. C. F. (1975). Antibody structure and antigen binding. Nature(London), 253, 158.

CHARD, T. (1990). Laboratory techniques in biochemistry and molecular biology: An introduction to radio-immunoassay and related techniques, 4th ed. Amsterdam: Elsevier.

EKINS, R. (1989). A shadow over immunoassay. Nature (London), 340, 599.

HOWANITZ, J. H., & HOWANITZ, P. J. (1979). Radioimmunoassay and related techniques. In J. B. Henry (Ed.), Clinical diagnosis and management by laboratory methods, 16th ed. Philadelphia: W. B. Saunders.

NAKAMURA, R. M., TUCKER, E. S., III & CARLSON, I. H. (1991). Immunoassays in the clinical laboratory. In J. B. Henry (Ed.), Clinical diagnosis and management by laboratory methods, 18th ed. Philadelphia: W. B. Saunders.

STEWART, M. J. (1985). Immunoassays. In A. C. Moffat (Ed.), Clark's isolation and identification of drugs. London: Pharmaceutical Press.

TIETZ, N. W. (1982). Radioimmunoassay. In Fundamentals of clinical chemistry. Philadelphia: W. B. Saunders.

JEFFREY A. GERE