Immunoassay Tests Health Article

Definition

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.