Principles of Immunochemical Methods Used for Organism Detection

Numerous immunologic methods are used for the rapid detection of bacteria, fungi, parasites, and viruses in patient specimens, and many of the same reagents often can be used to identify these organisms grown in culture. The techniques fall into four categories: precipitation tests, particle agglutination tests, immunofluorescence assays, and enzyme immunoassays.

Antibody molecules can be bound in random alignment to the surface of latex (polystyrene) beads (Figure 9-4). The number of antibody molecules bound to each latex particle is large, resulting in a high number of exposed potential antigen binding sites. Antigen present in a specimen binds to the combining sites of the antibody exposed on the surfaces of the latex beads, forming cross-linked aggregates of latex beads and antigen. The size of the latex bead (0.8 µm or larger) enhances the ease with which the agglutination reaction is visualized. Levels of bacterial polysaccharides detected by latex agglutination have been shown to be as low as 0.1 ng/mL.

Figure 9-4 Alignment of antibody molecules bound to the surface of a latex particle and latex agglutination reaction.

Because the pH, osmolarity, and ionic concentration of the solution influence the amount of binding that occurs, conditions under which latex agglutination procedures are carried out must be carefully standardized. Additionally, some constituents of body fluids, such as rheumatoid factor, have been found to cause false-positive reactions in the latex agglutination systems available. To counteract this problem, some agglutination methods require specimens to be pretreated by heating at 56°C or with ethylenediaminetetraacetic acid (EDTA) before testing. Commercial test systems are usually performed on cardboard cards or glass slides; manufacturer’s recommendations should be followed precisely to ensure accurate results.

Depending on the procedure, some reactions are reported as positive or negative and other reactions are graded on a 1+ to 4+ scale, with 2+ usually the minimum amount of agglutination visible in a positive sample without the aid of a microscope. Control latex (coated with antibody from the same animal species from which the specific antibody was made) is tested alongside the test latex. If the patient specimen or the culture isolate reacts with both the test and control latex, the test is considered nonspecific and the results therefore are invalid.

Latex tests are very popular in clinical laboratories for detecting antigen to Cryptococcus neoformans in cerebrospinal fluid or serum (Figure 9-5) and to confirm the presence of beta-hemolytic Streptococcus from culture plates (Figure 9-6). Latex tests are continually being developed for a variety of organisms. Some examples of additional latex tests are available for the detection of Clostridium difficile toxins A and B, rotavirus, and Escherichia coli 0157:H7 from suspect colonies of E coli.

Figure 9-5 Cryptococcal Antigen Latex Agglutination System (CALAS) (Meridian Diagnostics, Inc., Cincinnati, Ohio.) Patient 1 shows positive agglutination; patient 2 is negative.

Figure 9-6 Streptex (Remel, Inc., Lenexa, Kan.) Colony of beta-hemolytic *Streptococcus* agglutinates with group B *Streptococcus (Streptococcus agalactiae)* latex suspension.

Coagglutination

Similar to latex agglutination, coagglutination uses antibody bound to a particle to enhance the visibility of the agglutination reaction between antigen and antibody. In this case the particles are killed and treated S. aureus organisms (Cowan I strain), which contain a large amount of an antibody-binding protein, protein A, in their cell walls. In contrast to latex particles, these staphylococci bind only the base of the heavy chain portion of the antibody, leaving both antigen-binding ends free to form complexes with specific antigen (Figure 9-7). Several commercial suppliers have prepared coagglutination reagents for identification of streptococci, including Lancefield groups A, B, C, D, F, G, and N; Streptococcus pneumoniae; Neisseria meningitidis; and Haemophilus influenzae types A to F grown in culture. The coagglutination reaction is highly specific and demonstrates reduced sensitivity in comparison to commercially prepared latex agglutination systems. Therefore, coagglutination is not usually used for direct antigen detection.

Immunofluorescent Assays

Immunofluorescent assays are frequently used for detecting bacterial and viral antigens in clinical laboratories. In these tests, antigens in the patient specimens are immobilized and fixed onto glass slides with formalin, methanol, ethanol, or acetone. Monoclonal or polyclonal antibodies conjugated (attached) to fluorescent dyes are applied to the specimen. After appropriate incubation, washing, and counterstaining (staining of the background with a nonspecific fluorescent stain such as rhodamine or Evan’s blue), the slide is viewed using a microscope equipped with a high-intensity light source (usually halogen) and filters to excite the fluorescent tag. Most kits used in clinical microbiology laboratories use fluorescein isothiocyanate (FITC) as the fluorescent dye. FITC fluoresces a bright apple-green (Figure 9-8).

Figure 9-8 Legionella (Direct) Fluorescent Test System (Scimedx Corp., Denville, N.J.). *Legionella pneumophila* serogroup 1 in sputum.

Fluorescent antibody tests are performed using either a direct fluorescent antibody (DFA) or and indirect fluorescent antibody (IFA) technique (Figure 9-9). In the DFA technique, FITC is conjugated directly to the specific antibody. In the IFA technique, the antigen-specific antibody is unlabeled, and a second antibody (usually raised against the animal species from which the antigen-specific antibody was harvested) is conjugated to the FITC. The IFA is a two-step, or sandwich, technique. The IFA technique is more sensitive than the DFA method, although the DFA method is faster because it involves a single incubation.

Figure 9-9 Direct and indirect fluorescent antibody tests for antigen detection.

The major advantage of immunofluorescent microscopy assays is the ability to visually assess the adequacy of a specimen. This is a major factor in tests for the identification of chlamydial elementary bodies or respiratory syncytial virus (RSV) antigens. Microbiologists can discern whether the specimen was collected from the columnar epithelial cells at the opening of the cervix in the case of the Chlamydia DFA test or from the basal cells of the nasal epithelium in the case of RSV. Reading immunofluorescent assays requires extensive training and practice for laboratory personnel to become proficient. Finally, fluorescence dyes fade rapidly over time, requiring digital imaging to maintain archives of the results. For this reason, some antibodies have been conjugated to other markers instead of fluorescent dyes. These colorimetric labels use enzymes, such as horseradish peroxidase, alkaline phosphatase, and avidin-biotin, to detect the presence of antigen by converting a colorless substrate to a colored end product. The advantage of these tags is that they allow the preparation of permanent mounts, because the reactions do not fade with storage and visualization does not require a fluorescent microscope.

In clinical specimens, fluorescent antibody tests are commonly used to detect infected cells that harbor Bordetella pertussis; T. pallidum; L. pneumophila; Giardia, Cryptosporidium, Pneumocystis, and Trichomonas spp.; herpes simplex virus (HSV), cytomegalovirus, varicella-zoster virus, RSV, adenovirus, influenza virus, and parainfluenza virus.

Enzyme Immunoassays

Enzyme immunoassay (EIA), or enzyme-linked immunosorbent assay (ELISA), was developed during the 1960s. The basic method consists of antibodies bonded to enzymes; the enzymes remain able to catalyze a reaction, yielding a visually discernible end product while attached to the antibodies. Furthermore, the antibody binding sites remain free to react with their specific antigen. The use of enzymes as labels has several advantages. First, the enzyme itself is not changed during activity; it can catalyze the reaction of many substrate molecules, greatly amplifying the reaction and enhancing detection. Second, enzyme-conjugated antibodies are stable and can be stored for a relatively long time. Third, the formation of a colored end product allows direct observation of the reaction or automated spectrophotometric reading.

The use of monoclonal antibodies has helped increase the specificity of currently available ELISA systems. New ELISA systems are continually being developed for detection of etiologic agents or their products. In some instances, such as detection of RSV, human immunodeficiency virus (HIV), and certain adenoviruses, ELISA systems may even be more sensitive than culture methods.

Solid-Phase Immunoassay

Most ELISA systems developed to detect infectious agents consist of antibody firmly fixed to a solid matrix, either the inside of the wells of a microdilution tray or the outside of a spherical plastic or metal bead or some other solid matrix (Figure 9-10). Such systems are called solid-phase immunosorbent assays (SPIA). If antigen is present in the specimen, stable antigen-antibody complexes form when the sample is added to the matrix. Unbound antigen is thoroughly removed by washing, and a second antibody against the antigen is then added to the system. This antibody has been complexed to an enzyme such as alkaline phosphatase or horseradish peroxidase. If the antigen is present on the solid matrix, it binds the second antibody, forming a sandwich with antigen in the middle. After washing has removed unbound, labeled antibody, the addition and hydrolysis of the enzyme substrate causes the color change and completes the reaction. The visually detectable end point appears wherever the enzyme is present (Figure 9-11). Because of the expanding nature of the reaction, even minute amounts of antigen (greater than 1 ng/mL) can be detected. These systems require a specific enzyme-labeled antibody for each antigen tested. However, it is simpler to use an indirect assay in which a second, unlabeled antibody is used to bind to the antigen-antibody complex on the matrix. A third antibody, labeled with enzyme and directed against the nonvariable Fc portion of the unlabeled second antibody, can then be used as the detection marker for many different antigen-antibody complexes (Figure 9-12). ELISA systems are important diagnostic tools for hepatitis Bs (surface) and hepatitis Be (early) antigens and HIV p24 protein, all indicators of early, active, acute infection.