Immunochemical methods use antigens and antibodies as tools to detect microorganisms. Antigens are sub stances recognized as “foreign” in the human body. Anti gens are usually high-molecular-weight proteins or carbohydrates that elicit the production of other proteins, called antibodies, in a human or animal host. Antibodies attach to the antigens and aid the host in removing the infectious agent. 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 epi topes. Figure 1 shows the multiple molecules within group A Streptococcus (Streptococcus pyogenes) that are recognized by the immune system as antigenic.

Fig1. Group A Streptococcus (Streptococcus pyogenes) contains many antigenic structural components and produces various  antigenic enzymes, each of which may elicit a specific antibody  response from the infected host. 

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 com parisons 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 2.

Fig2.  Production of a monoclonal antibody. 

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 suit able 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.

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مواضيع ذات صلة

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