Immunoassay Formats

Immunoassays can be divided into two types, heterogeneous and homogeneous immunoassays. Heterogeneous immunoassays involve a solid phase (microwell, bead) and require washing steps to remove unbound antigens or antibodies. Heterogeneous immunoassays can have a competitive or noncompetitive format.

Homogeneous immunoassays consist only of a liquid phase and do not require washing steps. Homogeneous immunoassays are faster and easier to automate than heterogeneous immunoassays. In addition, homogeneous immunoassays have competitive formats.

Types of Labels

The principles and applications of enzyme immunoassays, chemiluminescence, and fluorescent substances as labels are presented in this chapter (Table 12-1).

The original technique of using antigen-coated cells or particles in agglutination techniques may be considered as the earliest method for labeling components in immunoassays. Ideal characteristics of a label include the quality of being measurable by several methods, including visual inspection. The properties of a label used in an immunoassay determine how detection is possible. For example, coated latex particles can be detected by various methods—visual inspection, light scattering (nephelometry), and particle counting. The conversion of a colorless substrate into a colored product in enzyme immunoassay allows for two methods of detection, colorimetry and visual inspection.

Yalow and Berson developed the radioimmunoassay (RIA) method in 1959 using a radioactive label that could identify an immunocomponent at very low concentrations. In the 1960s, researchers began to search for a substitute for the successful RIA method because of the inherent drawbacks of using radioactive isotopes as labels (e.g., radioactive waste, short shelf life). Currently, chemiluminescent reactions have replaced most RIAs in the clinical laboratory. Relatively simple and cost-effective, chemiluminescence technology has sensitivity at least as good as that of an RIA.

Enzyme Immunoassay

There are two general approaches to diagnosing condition, diseases or conditions by immunoassay, testing for specific antigens or for antigen-specific antibodies. The enzyme-linked immunosorbent assay (ELISA), also known as an enzyme immunoassay (EIA), is designed to detect antigens or antibodies by producing an enzyme-triggered color change.

The EIA method uses a nonisotopic label that offers the advantage of safety. EIA is usually an objective measurement that provides numerical results. Some EIA procedures provide diagnostic information and measure immune status (e.g., detect total antibody IgM or IgG).

The EIA method uses the catalytic properties of enzymes to detect and quantitate immunologic reactions. An enzyme-labeled antibody or enzyme-labeled antigen conjugate is used in immunologic assays (Box 12-1). The enzyme, with its substrate, detects the presence and quantity of antigen or antibody in a patient specimen. In some tissues, an enzyme-labeled antibody can identify antigenic locations.

Various enzymes are used in enzyme immunoassay (Table 12-2). Common enzyme labels are horseradish peroxidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase, and beta-galactosidase. To be used in an EIA, an enzyme must fulfill the following criteria:

In a representative EIA test, a plastic bead or plastic plate is coated with antigen (e.g., virus; Fig. 12-1). The antigen reacts with antibody in the patient’s serum. The bead or plate is then incubated with an enzyme-labeled antibody conjugate. If antibody is present, the conjugate reacts with the antigen-antibody complex on the bead or plate. The enzyme activity is measured spectrophotometrically after the addition of the specific chromogenic substrate. For example, peroxidase cleaves its substrate, o-dianisidine, causing a color change. In some cases, the test can be read subjectively.

The results of a typical test are calculated by comparing the spectrophotometric reading of the patient’s serum to that of a control or reference serum. The advantage of an objective enzyme test is that results are not dependent on a technician’s interpretations. In general, the EIA procedure is faster and requires less laboratory work than comparable methods.

Chemiluminescence

Chemiluminescence refers to light emission produced during a chemical reaction; it is used extensively in automated immunoassays (see Chapter 13). This methodology has excellent sensitivity and dynamic range. It does not require sample radiation and nonselective excitation and source instability are eliminated. Most chemiluminescent reagents and conjugates are stable and relatively nontoxic.

In immunoassays, chemiluminescent labels can be attached to an antigen or antibody. Acridinium esters are highly specific activity labels that can be used to label antibodies and haptens. Chemiluminescent labels are used to detect proteins, viruses, oligonucleotides, and genomic nucleic acid sequences in an immunoassay. Two formats are used, competitive and sandwich immunoassays.

In a competitive immunoassay, a fixed amount of labeled antigen competes with unlabeled antigen from a patient specimen for a limited number of antibody-binding sites (Fig. 12-2). The amount of light emitted is inversely proportional to the amount of analyte (antigen) measured.

In a sandwich immunoassay, the sample antigen binds to an antibody fixed onto a solid phase; a second antibody, labeled with a chemiluminescent label, binds to the antigen-antibody complex on the solid phase (Fig. 12-3). In the sandwich assay, the emitted light is directly proportional to the analyte concentration. The detection device for analyses is a simple photomultiplier tube used to detect the emitted light.

Chemiluminescent labels can be divided into five major groups: (1) luminol; (2) acridinium esters; (3) peroxyoxalates; (4) dioxetanes; and (5) tris(2,2′−bipyridyl)-ruthenium (II). Direct labels include luminol, acridinium ester, and electrogenerated luminescent chelate from ruthenium and tripropylamine (TPA) complex [Ru(bpy)³⁺]. These labels are attached directly to antigens, antibodies, or deoxyribonucleic acid (DNA) probes, depending on the assay format.

Oxidation of isoluminol by hydrogen peroxide (H₂O₂) in the presence of a catalyst (e.g., microperoxidase) produces a relatively long-lived emission at 425 nm. Oxidation of acridinium ester by alkaline H₂O₂ in the presence of detergent produces a rapid flash of light lasting from 1 to 5 seconds at 429 nm. Peak intensity can be used for the measurement. An alternate method is to use an integrator to measure the entire light output for greater sensitivity.

Enzymes are typically used for indirect labels. Indirect labels are attached to antibodies, antigens, and DNA probes, depending on the assay format. Enzyme labels often used in indirect procedures include the following:

An interesting label is native or recombinant apoaequorin (from the bioluminescent jellyfish, Aequorea). It is activated by reaction with coelenterazine. Light emission at 469 nm is triggered by reaction with calcium chloride.

Immunofluorescence

Fluorescent labeling is another method used to demonstrate the complexing of antigens and antibodies (Fig. 12-4). Fluorescent molecules are used as substitutes for radioisotope or enzyme labels. The fluorescent antibody technique consists of labeling antibody with fluorescein isothiocyanate (FITC), a fluorescent compound with an affinity for proteins, to form a complex (conjugate). This conjugate is able to react with antibody-specific antigen.

Fluorescent techniques are extremely specific and sensitive. Antibodies may be conjugated to other markers in addition to fluorescent dyes; the use of these markers is called colorimetric immunologic probe detection. The use of enzyme-substrate marker systems has been expanded. HRP, ALP, and avidin-biotin conjugated enzyme labels have all been used as visual tags for the presence of antibody. These reagents have the advantage of requiring only a standard light microscope.

Fluorescent conjugates are used in the following basic methods, which are widely used:

Direct Immunofluorescent Assay

In the direct fluorescent antibody (DFA) technique, a conjugated antibody is used to detect antigen-antibody reactions at a microscopic level (Fig. 12-5). DFA can be applied to tissue sections or in smears for microorganisms (see Color Plate 2).

Fluorescein-conjugated antibodies bound to the fluorochrome FITC are used to visualize many bacteria in direct specimens (see later, “Direct Fluorescent Antibody Test for Neisseria gonorrhoeae”). HRP conjugated to antibody, the immunoperoxidase stain, can be used to detect CMV, other viruses, or nucleic acids in cells. In biotin-avidin, enzyme-conjugated methods, single-stranded nucleic acid probes, antimicrobial antibodies, or antibiotin antibodies can be bound to the small biotin molecule. These molecules have a strong affinity for the protein avidin, which has four binding sites. Biotin bound to avidin or antibody can be complexed to fluorescent dyes or to color-producing enzymes to form specific detector systems. This system can be applied to the detection of nucleic acids in organisms such as CMV, hepatitis B virus (HBV), Epstein-Barr virus (EBV), and Chlamydia.

The chemical manipulation in labeling antibodies with fluorescent dyes to permit detection by direct microscopic examination does not seriously impair antibody activity, the ability of fluorescent antibody conjugate to react specifically with its homologous antigen. Monoclonal antibodies (MAbs) have also been successfully conjugated to fluorescein for the detection of chlamydiae, rabies virus, and other pathogens in directly stained specimens.

When absorbing light of one wavelength, a fluorescent substance emits light of another (longer) wavelength. In fluorescent antibody (FA) microscopy, the incident or exciting light is often blue-green to ultraviolet. The light is provided by a high-pressure mercury arc lamp with a primary (e.g., blue-violet) filter between the lamp and the object that passes only fluorescein-exciting wavelengths. The color of the emitted light depends on the nature of the substance. Fluorescein gives off yellow-green light and the rhodamines fluoresce in the red portion of the spectrum. The color observed in the fluorescence microscope depends on the secondary or barrier filter used in the eyepiece. A yellow filter absorbs the green fluorescence of fluorescein and transmits only yellow. Fluorescein fluoresces an intense apple-green color when excited.

An advanced labeling technique, quantum dots are semiconductor nanocrystals used as fluorescent labeling reagents for biological imaging. A valuable property of Q dots is that different sizes of crystals produce different signals with a single laser excitation. This seemingly simple physical property implies that different-sized Q dots could be directed against different analyte targets, and the Q dots would fluoresce with different colors in a size-dependent manner. This allows for the detection of multiple analytes with a single assay. Q dots are the next step in the evolution of luminescence-based assays.

Magnetic Labeling Technology

Magnetic labeling technology is an application of the high-resolution magnetic recording technology developed for the computer disk drive industry. Increased density of microscopic, magnetically labeled biological samples (e.g., nucleic acid on a biochip) translates directly into reduced sample-processing times. Magnetic labeling can be applied to automated DNA sequences, DNA probe technology, and gel electrophoresis (Fig. 12-6). Compared with other nonradioactive labeling systems, magnetic labels are inherently safe, instrumentation is less expensive, signals are almost permanent, and spatial resolution is increased.

In a magnetic label–based gel electrophoresis application sphere, DNA is analyzed. DNA is separated into bands using electrophoresis and magnetic labels are bound to the DNA in each band. By applying and then removing a magnetic field, the magnetic domains in each label are oriented in the same direction, resulting in a net magnetic field near the bands in the direction of the applied field (Fig. 12-7).

Pregnancy Testing

Immunofluorescence is a reliable, simple, rapid test used extensively in the clinical laboratory. The demonstration of microbial antigens is one of the many applications of the direct immunofluorescence procedure; the microbes are incubated with fluorescent-labeled antibodies. Under appropriate conditions, the labeled antibodies bind to specific antigens. Any unbound antibodies are washed off and the bound antibodies are visualized with a fluorescence microscope.

N. gonorrhoeae is a gram-negative diplococcus that causes urogenital infections. The Syva Microtrak N. gonorrhoeae culture confirmation test is a direct fluorescent antibody (FA) assay that uses fluorescein-labeled MAbs that react specifically with N. gonorrhoeae . The test is performed on primary culture isolates and requires only a small inoculum. Culture isolates presumptively identified as N. gonorrhoeae are transferred to a slide well and stained with fluorescein-labeled anti–N. gonorrhoeae reagent antibody (anti-GC/FITC). The antibodies bind specifically to gonococcal antigen. Unbound antibodies are then removed by a rinse step. Under a fluorescence microscope, cultures positive for N. gonorrhoeae show apple-green fluorescent staining of the kidney-shaped diplococci.

See for procedural protocol.

Chapter Highlights