Immunochemical Methods Used for Organism Detection

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Immunochemical Methods Used for Organism Detection

The diagnosis of an infectious disease by culture and biochemical techniques can be hindered by several factors. These factors include the inability to cultivate an organism on artificial media, such as Treponema pallidum, the agent that causes syphilis, or the fragility of an organism and its subsequent failure to survive transport to the laboratory, such as with respiratory syncytial virus and varicella-zoster virus. Another factor, the fastidious nature of some organisms (e.g., Leptospira or Bartonella spp.) can result in long incubation periods before growth is evident. In addition, administration of antimicrobial therapy before specimen collection, such as with a patient who has received partial treatment, can impede diagnosis. In these cases, detecting a specific product of the infectious agent in clinical specimens is very important, because this product would not be present in the specimen in the absence of the agent. This chapter discusses the direct detection of microorganisms in patient specimens using immunochemical methods and the identification of microorganisms by these methods once they have been isolated on laboratory media. Chapter 10 discusses the diagnosis of infectious diseases using serological methods.

Production of Antibodies for Use in Laboratory Testing

Immunochemical methods use antigens and antibodies as tools to detect microorganisms. Antigens are substances recognized as “foreign” in the human body. Antigens are usually high-molecular-weight proteins or carbohydrates that elicit the production of other proteins, called antibodies, in a human or animal host (see Chapter 3). Antibodies attach to the antigens and aid the host in removing the infectious agent (see Chapters 3 and 10). Antigens may be part of the physical structure of the pathogen, such as the bacterial cell wall, or they may be a chemical produced and released by the pathogen, such as an enzyme or a toxin. Each antigen contains a region that is recognized by the immune system. These regions are referred to as antigenic determinants or epitopes. Figure 9-1 shows the multiple molecules within group A Streptococcus (Streptococcus pyogenes) that are recognized by the immune system as antigenic.

Polyclonal Antibodies

Because an organism contains many different antigens, the host response produces many different antibodies to these antigens; these antibodies are heterogenous and are called polyclonal antibodies. Polyclonal antibodies used in immunodiagnosis are prepared by immunizing animals (usually rabbits, sheep, or goats) with an infectious agent and then isolating and purifying the resulting antibodies from the animal’s serum. Antibody idiotype variation is due to alterations in the nucleotide sequence during antibody production. Individual animals are able to produce different antibodies with different idiotypes (antigen binding sites). This variation in antigen binding sites creates a lack of uniformity in polyclonal antibody reagents and requires continual monitoring and comparisons of different antibody reagent lots for specificity and avidity (strength of binding) in any given immunochemical test system.

Monoclonal Antibodies

Monoclonal antibodies are antibodies that are completely characterized and highly specific. The ability to create an immortal cell line that produces large quantities of a monoclonal antibody has revolutionized immunologic testing. Monoclonal antibodies are produced by the fusion of a malignant single antibody-producing myeloma cell with an antibody-producing plasma B cell, forming a hybridoma cell. Clones of the hybridoma cells continuously produce specific monoclonal antibodies. One technique for the production of a clone of cells is illustrated in Figure 9-2.

The process starts with immunization of a mouse with the antigen for which an antibody is to be produced. The animal responds by producing many antibodies to the epitope (antigenic determinant) injected. The mouse’s spleen, which contains antibody-producing plasma cells, is removed and emulsified to separate antibody-producing cells. The cells are then placed into individual wells of a microdilution tray. Viability of cells is maintained by fusing them with cells capable of continuously propagating, or immortal cells of the multiple myeloma. A multiple myeloma is a disease that produces a malignant tumor containing antibody-producing plasma cells. Myeloma tumor cells used for hybridoma production are deficient in the enzyme hypoxanthine phosphoribosyl transferase. This defect leads to their inability to survive in a medium containing hypoxanthine, aminopterin, and thymidine (HAT medium). Antibody-producing spleen cells, however, contain the enzyme. Thus, fused hybridoma cells survive in the selective medium and can be recognized by their ability to grow indefinitely in the medium. Unfused antibody-producing lymphoid cells die after several multiplications in vitro because they are not immortal, and unfused myeloma cells die in the presence of the toxic enzyme substrates. The only surviving cells are true hybrids.

The growth medium supernatant from the microdilution tray wells in which the hybridoma cells are growing is then tested for the presence of the desired antibody. Many such cell lines are usually examined before a suitable antibody is identified. The antibody must be specific enough to bind the individual antigenic determinant to which the animal was exposed, but not so specific that it binds only the antigen from the particular strain of organism with which the mouse was first immunized. When a good candidate antibody-producing cell is found, the hybridoma cells are either grown in cell culture in vitro or are reinjected into the peritoneal cavities of many mice, where the cells multiply and produce large quantities of antibody in the ascitic (peritoneal) fluid. Ascitic fluid can be removed from mice many times during the animals’ lifetime, providing a continual supply of antibody formed to the originally injected antigen. Polyclonal and monoclonal antibodies are both used in commercial systems to detect infectious agents.

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.

Precipitation Tests

The classic method of detecting soluble antigen (i.e., antigen in solution) is the Ouchterlony method, a double immunodiffusion precipitation method.

Double Immunodiffusion

In the double immunodiffusion method, small circular wells are cut in an agarose gel, a gelatin-like matrix derived from agar, which is a chemical purified from the cell walls of brown algae. The agarose forms a porous material through which molecules can readily diffuse. The patient specimen containing antigen is placed in a well, and antibody directed against the antigen is placed in the adjacent well. Over 18 to 24 hours, the antigen and antibody diffuse toward each other, producing a visible precipitin band (a lattice structure or visible band) at the point in the gel where the antigen and antibody are in equal proportion (zone of equivalence). If the concentration of antibody is significantly higher than that of the antigen, no lattice forms and no precipitation reaction occurs; this is known as prozone effect. Conversely, if excess antigen prevents lattice formation, resulting in no band formation, the effect is termed postzone. Immunodiffusion is currently used to detect exoantigens produced by the systemic fungi to confirm their presence in culture (Figure 9-3). However, the technique is extremely time-consuming and is no longer used regularly in the clinical laboratory for antigen detection in patient specimens.

Figure 9-3

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