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 assays. Their high specificity results from the use of antibodies and purified antigens as reagents. An antibody is a protein (immunoglobulin) produced by B lymphocytes in response to stimulation by an antigen. Immunoassays measure the formation of antibody-antigen complexes and detect them via an indicator reaction. This may be done by precipitation of the immune complexes and measurement of turbidity or light scattering or by labeling either the antibody or antigen with a radioactive tag, enzyme, fluorescent, or chemiluminescent molecule. 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 or quantitative. 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. In a typical pregnancy test, two antibodies are used. The hCG molecule, a protein hormone produced by the trophoblast, is the antigen. One antibody is directed against the alpha polypeptide chain of hCG and the other against the beta polypeptide chain. The sample is added to a support medium containing immobilized antibody to the alpha subunit of hCG. If hCG is present in the sample, it will bind to the antibody. The support is washed to remove all unbound molecules, and an antibody to the beta subunit is added. This second antibody is conjugated to an enzyme. After washing away any unbound antibody-conjugate, a substrate is added that changes color when acted on by the enzyme. Therefore, the presence of color at the end of the test indicates that hCG was present in the sample. With the use of highly purified antibodies and the enzyme indicator system, pregnancy can be detected within two days after 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 using a spectrophotometer. A calibration curve is produced by measuring several standards of known hCG concentration, and the curve is used to calculate the concentration of hCG in the sample after measuring the amount of product formed.
Purpose
The purpose of an immunoassay is to measure (or in a qualitative assay detect) an analyte. Immunoassay is the method of choice for measuring analytes normally present at very low concentrations which cannot be determined accurately by less expensive colorimetric 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 produced against them. For example, immunoassays are used to detect antigens on Hemophilus, Cryptococcus, and Streptococcus organisms in the cerebrospinal fluid 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.
Immunoprecipitation
The reaction of antibodies with protein antigens is a two-phase reaction. The first phase results in the formation of an antibody-antigen complex and takes place within seconds. This is followed by cross linking of individual immune complexes to form a macromolecular aggregate which precipitates out of the solution or gel. This second reaction is slow and often requires overnight incubation to reach completion. The simplest immunoassay method measures the quantity of precipitate which forms after the reagent antibody (precipitin) has incubated with the sample and reacted with its respective antigen to form an insoluble aggregate. Immunoprecipitation reactions may be qualitative or quantitative. In quantitative assays, the immune complexes can be measured by turbidimetry or by performing the reaction in an agarose gel. In gel assays, an excess of the specific antibody is usually poured into the gel. The sample is placed in a well cut into the gel and is allowed to diffuse into the gel. The result will be a ring of precipitated immune complexes which grows larger with time until the endpoint is reached. At the endpoint, the diameter of the ring is proportional to antigen concentration. There will be insufficient antigen beyond the ring to form a visible reaction. Inside the ring, antigen concentration is in excess resulting in small invisible antibody-antigen complexes. The ring position represents the equivalence point or optimal molar ratio of antibody to antigen for the visible reaction. An alternative and more rapid immunoprecipitation method is the Laurel rocket electrophoresis or electroimmunoassay method. In this procedure the antigen is added to wells on one side of the gel which contains a specific antibody throughout. The gel is electrophoresed, and the antigen migrates toward the anodal side of the gel (see Electrophoresis tests). This results in an immunoprecipitation reaction in the shape of a rocket (peak). The height of the peak is logarithmically proportional to antigen concentration.
Particle immunoassays
Immunoprecipitation reactions can be direct or indirect. In direct assays the union of antibody with antigen occurs without attaching the antibody or antigen to a solid phase. In passive or indirect assays, the visible phase of the reaction is enhanced by binding one of the reactants to a solid phase such as latex, red blood cells, or suspension of colloidal gold particles. Particle immunoassays add sensitivity by enhancing surface area and visibility. By linking several antibodies to the particle, the particle is able to bind many antigen molecules simultaneously. This greatly accelerates the speed of the visible reaction. Particle assays may also be performed using antigens bound to the particle. This allows rapid and sensitive detection of antibodies that are markers of diseases such as infectious mononucleosis and rheumatoid arthritis.
Immunonephelometry
The immediate union of antibody and antigen forms immune complexes that are too small to precipitate. However, these complexes will scatter incident light and can be measured using an instrument called a nephelometer. These instruments measure the rate at which the immune complexes form. Incident light from a high intensity monochromatic light source is passed through the reaction cuvet. A photomultiplier tube is placed at an angle (e.g. 70 degrees) to the incident light beam. The amount of light striking the tube is proportional to antigen concentration. The antigen concentration can be determined within minutes of the reaction. A calibration curve based on a nonlinear model such as a cubic spline plot is used to calculate the antigen concentration from the reaction rate.
Radioimmunoassay (RIA)
RIA is a method employing radioactive isotopes to label either the antigen or antibody. Isotopes are atoms that have unstable nuclei and emit radiation in order to transform into stable atoms. Most RIA methods employ 125 iodine (125I) as the radiolabel. This isotope emits gamma rays. It has a high specific activity so that a very small mass of isotope is needed, and a short half-life (60 days). These properties result in minimal disposal problems with leftover or spent reagents. Gamma rays emitted by the immune complexes are usually measured following removal of unbound (free) radiolabel. Since background radiation is very low and the counting time can be extended if needed to generate more counts, RIA is the most sensitive of all immunoassay methods.
There are two types of RIA, competitive and immunoradiometric (sandwich) assays. Competitive assays use radiolabeled antigen. The labeled antigen "competes" with non-radioactive antigen in the sample for a limited number of binding sites on the reagent antibody. Following incubation, the free radiolabeled antigens are removed by decanting or washing and the radioactivity of the antibody-bound antigens is measured. The radioactivity of the antibody-antigen complexes is inversely proportional to antigen concentration. In the immunoradiometric (IRMA) or sandwich assay, two antibodies are used and one is radiolabeled. In the test system, the sample is incubated with a specific antibody usually attached to a solid phase such as a plastic bead or the wall of a plastic test tube. After washing to remove unbound sample components, a radioactively labeled antibody is added. The second antibody may be directed against a different part of the antigen molecule, or it may be directed against the first antibody (e.g. anti-human immunoglobulin). The second antibody binds to the immune complexes making an antibody-antigen-antibody "sandwich." After washing to remove the unbound radiolabeled antibody, the radioactivity is measured. The amount of radioactivity is directly proportional to antigen concentration.
As with immunonephelometric assays, the calibration curve for RIA is nonlinear. The reagent antibodies reacting with different parts of the antigen have different binding affinities causing the curve to be hyperbolic. Various methods are used to transform the plot so that result can be more accurately determined. Concentration is plotted on the x-axis and radioactivity on the y-axis. In competitive assays, radioactivity is usually expressed as %B/Bo where B is the count per minute of the sample and Bo is the count per minute of the zero calibrator. This keeps the slope of the curve from changing each day as the amount of radioactivity of the reagent decreases naturally. The most common plotting method converts the concentration (x-axis) to log10 and the %B/Bo to logit B/Bo (the natural log of B/Bo divided by 1-B/Bo). This produces a linear plot from which the concentration of unknown can be easily determined.
The major advantages of RIA when compared to other immunoassays are higher sensitivity, easy signal detection, and well-established, rapid assays. The major disadvantages are the health and safety risks posed by use of radiation and the time and expense associated with maintaining a licensed radiation safety and disposal program. For this reason, RIA has been largely replaced in routine clinical laboratory practice by enzyme immunoassay. It is still the gold standard to which other immunochemical methods are compared and is still performed in reference laboratories for analytes such as 11-deoxycortisol, which are not available by other methods.
Enzyme immunoassay (EIA)
Enzyme immunoassay was developed as an alternative to RIA. These methods use an enzyme to label either the antibody or antigen. As for RIA, the EIA methods may be divided into competitive or sandwich type assays. Competitive assays use enzyme labeled antigen, and sandwich assays use an enzyme labeled antibody. The steps performed in RIA and EIA are similar. However, EIA requires an additional step, the addition of substrate which follows the immunological reaction. The sensitivity of EIA approaches that for RIA because a single enzyme molecule can catalyze the conversion of many molecules of substrate to product. Therefore, the enzyme label amplifies the reaction by producing many colored, fluorescent, or chemiluminescent molecules for each antibody-antigen reaction. As with RIA, the relationship between enzyme activity and concentration is nonlinear, and curve-fitting methods such as the cubic spline plot or the four-parameter logistic curve are used to calculate concentration. Many EIA assays use monoclonal antibodies as reagents to increase the sensitivity and lot to lot reproducibility of the assay. Monoclonal antibodies are made by fusing the immunoglobulin genes from a B lymphocyte, which produces the desired antibody specificity with a malignant plasmacytoma cell line. This results in a malignant cell line called a hybridoma that secretes large quantities of the desired antibody. Since the antibodies are derived from identical cells, the antibody molecules are identical and all have the same binding affinity for the antigen.
In addition to safety, another advantage of EIA is that light (versus) radiation is measured. This obviates the need for a scintillation counter that is more expensive than a light measuring instrument. In addition, some competitive EIA methods do not require the separation of antibody-bound and free antigen. These methods are called homogenous assays. Examples of homogenous EIAs are the enzyme multiplied immunoassay technique (EMIT), fluorescence polarization immunoassay (FPIA), and cloned enzyme donor immunoassay (CEDIA).
One of the most widely used EIA methods for detection of infectious diseases is the enzyme-linked immunosorbent assay (ELISA). The ELISA method is a heterogenous sandwich immunoassay, which means that separation of bound and free enzyme label is required. The term ELISA refers to the use of a solid phase to which the antibody (or antigen) is bound in order to facilitate the separation. ELISA methods are usually performed using a microtiter plate containing 96 wells rather than in test tubes. Incubation, washing, and signal reading steps are performed as with EIA sandwich assays described above.
Fluorescent immunoassay (FIA)
FIA refers to immunoassays that utilize a fluorescent label or an enzyme label that acts on the substrate to form a fluorescent product. In fluorescence measurements short wavelength light (usually near ultraviolet light) is used to excite the molecules. Fluorescent molecules stabilize by losing part of the absorbed light energy as heat and part as longer wavelength (visible) light. Fluorescent measurements are inherently more sensitive than colorimetric (spectrophotometric) measurements. Therefore, FIA methods have greater analytical sensitivity that EIA methods which employ absorbance (optical density) measurement.
Chemiluminescent immunossay
Chemiluminescent immunosassays utilize a chemiluminescent label. Chemiluminescent molecules produce light when they are excited by chemical energy. The energy usually comes from an oxidation-reduction reaction. These molecules can be conjugated directly to antigens, or they can be used as substrates for enzyme labels. The most commonly used chemiluminescent labels are acrodinium, luminol, and dioxetane. Acrodinium and luminol are excited by peroxidase enzyme reactions and can be used with EIAs that employ a horseradish peroxidase label. Dioxetane-phosphate can be excited by hydrolysis of the phosphate bond using the enzyme alkaline phosphatase as the label. Consider this example of a competitive binding assay for thyroxine (T4) based on chemiluminescence. The sample is mixed with T4 labeled with alkaline phosphatase (ALP) in a plastic tube containing anti-T4 conjugated to the tube wall. T4 in the sample competes with the ALP-labeled T4 for the antibody. After the reaction, the tube is washed to remove any unbound T4 and dioxetane-phosphate is added. The enzyme hydrolyzes the phosphate ester bond exciting the dioxetane which releases flashes of light. These emissions are measured by a light detector and are inversely proportional to the T4 concentration of the sample.
Precautions
Blood samples are collected by venipuncture using standard precautions for reducing exposure to blood-borne pathogens. It is not necessary to restrict fluids or food prior to collection. Blood should be collected in tubes containing no additive. Risks of venipuncture 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 radioimmunoassay (RIA) methods. RIA tests use radioactive isotopes to label antigens or antibodies. Pregnant females should not work in the 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.
Results
Immunoassays that 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. Immunoassays that measure antibody concentration may be reported as an antibody titre. The titre is the reciprocal of the highest dilution of sample that gives a positive (detectable) result. 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 heterophile antibodies, especially to mouse immunoglobulins 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.
Preparation
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.
Aftercare
When blood testing is used for the immunoassay, the venipuncture site will require a bandage or light dressing to accomplish hemostasis.
Complications
Immunoassay is an in vitro procedure, and 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.
Health care team roles
Immunoassay tests are ordered by physicians and samples may be collected by a physician, physician assistant, nurse, or phlebotomist. Simple immunoassay tests (e.g. pregnancy tests and rapid Strep tests) may be performed by medical personnel without special laboratory training. More complex testing is preformed by clinical laboratory scientists CLS(NCA) or medical technologists, MT(ASCP) or by clinical laboratory technicians, CLT(NCA) or medical laboratory technicians, MLT(ASCP).
KEY TERMS
Antibody—A protein produced by B lymphocytes in response to stimulation by an antigen.
Chemiluminescent immunoassay—An immunoassay in which the label is a molecule that emits light when excited by a chemical reaction.
Enzyme immunoassay—An immunoassay using an enzyme as the label. The enzymatic reaction product is measured to determine the concentration of the analyte in the sample.
Fluorescent immunoassay—An immunoassay that uses a fluorescent label or produces a fluorescent product.
Immunoassay—A method that measures antibody-antigen complexes formed by reacting purified antibody or antigen with the sample.
Nephelometry—A method for measuring the light scattering properties of a sample.
Radioimmunoassay—A method that uses a radioisotope label in an immunoassay.
BOOKS
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.
Kaplan, L.A. and A.J. Pesce. Clinical Chemistry Theory, Analysis, Correlation, 3rd ed. St. Louis: Mosby, 1996.
Law, B. Immunoassay: A Practical Guide. London: Taylor and Francis, 1996.
