Molecular Biotechnology. Principles and Applications - Glick B., Pasternak J. 2002

Molecular Biotechnology of Microbial Systems
Molecular Diagnostics
Immunodiagnostic Methods

Many immunological detection systems offer high sensitivity and Specificity while remaining relatively simple. They are widely used for drug testing, evaluating and monitoring various oncological diseases, determining specific metabolites, and identifying and controlling pathogenic microorganisms, but they also have their limitations. If the target molecule is a protein, it is necessary to ensure the expression of its encoding genes and to create conditions that prevent the masking or blocking of the antibody-binding site.

Traditional diagnostic Procedures for infectious agents rely either on a set of CHARACTERISTICS OF THE pathogen or, preferably, on a single, unique, easily distinguishable feature. Clinical microbiologists seek to identify the minimum set of biological characteristics that can reliably detect and identify pathogens. For example, some pathogens produce specific biochemical compounds that must be detected in a biological sample. Often, such a marker molecule can be detected directly through a highly specific biochemical assay. However, this approach inevitably leads to a proliferation of individualized detection systems for pathogens. A universal method capable of detecting any marker molecule regardless of its chemical nature would be highly preferable. The method based on the identification of antigen-antibody complexes is precisely such an approach.

Enzyme-Linked Immunosorbent Assay

A variety of approaches can determine whether an antibody has bound to its target antigen. One of these is the enzyme-linked immunosorbent assay (ELISA), which is frequently used for Diagnostics. The Procedure involves the following steps (Fig. 9.1).

1. The sample in which the specific molecule or microorganism is to be detected is immobilized on a solid support, such as a plastic microtiter plate, typically containing 96 wells (Fig. 9.1, A).

2. An antibody specific to the marker molecule (the primary antibody) is added to the immobilized sample, and the well is then washed to remove unbound primary Antibodies (Fig. 9.1, B).

3. A secondary antibody is added, which specifically binds to the primary antibody and does not interact with the marker molecule (Fig. 9.1, C). This antibody is conjugated to an enzyme (such as alkaline phosphatase, peroxidase, or urease) that catalyzes The conversion of a colorless substrate into a colored product. The well is washed to remove unbound secondary antibody-enzyme conjugate molecules.

4. A colorless substrate is added (Fig. 9.1, D).

5. Qualitative or Quantitative determination of the colored product is performed.

If the primary antibody does not bind to the target in the sample, it is removed During the first wash. Since the secondary antibody-enzyme conjugate then has nothing to bind to, it is removed during the second wash, and the sample remains colorless. If binding to the target occurs, the secondary antibody attaches to the primary one, and the conjugated enzyme catalyzes The formation of an easily detectable colored product.

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Fig. 9.1. Detection of a target antigen using ELISA. E—enzyme conjugated to the secondary antibody

The fundamental principle of ELISA is the specific binding of the primary antibody to the target. If the target molecule is a protein, its purified preparation is typically used to generate antibodies, which are then used to detect the target. The antibodies produced in the serum (antiserum) of an immunized animal (usually a rabbit) bind to different antigenic determinants (epitopes) of the target molecule. Such a mixture of antibodies is called a polyclonal preparation. The Use of polyclonal antibodies has two drawbacks that are significant for some diagnostic Methods: 1) the content of individual antibodies in a polyclonal preparation can vary from batch to batch; 2) polyclonal antibodies cannot be used when it is necessary to distinguish between two similar targets, i.e., when the pathogenic (target) and non-pathogenic (non-target) forms differ by a single determinant. However, these problems are entirely resolvable, as methods have been developed to produce antibody preparations directed against a single antigenic determinant, namely, monoclonal antibody preparations.

Monoclonal Antibodies

In mammals, evolution has produced a complex array of cellular systems that protect the body against toxic substances and infectious agents. An integral part of this protective response is the induced production of specific Proteins (antibodies) by Cells of The Lymphatic system. These antibodies bind to foreign substances (Antigens) and, with the help of other immune system proteins, including The Complement System, neutralize their effects. In response to an immunological stimulus, each antibody-producing Cell synthesizes and secretes a single type of antibody that recognizes a specific site (an epitope or antigenic determinant) on the antigen molecule with high affinity. Since an antigen molecule typically possesses several different epitopes, antibodies against each of them are produced by distinct cells of The Immune System. Such antibodies, which collectively interact with the given antigen, are called polyclonal.

As early as the beginning of this century, when nothing was known about the polyclonality of antibodies, it was clear that their specificity could be used to suppress infections. Later, antibodies began to be used as diagnostic tools to detect toxic compounds in clinical samples. Unfortunately, the efficacy of polyclonal antibody preparations varies from batch to batch because, during immunization, antibody-producing cells may be more strongly stimulated by certain determinants of a given antigen in some cases, while in others, the immune system responds more actively to different epitopes of the same antigen. This can affect the ability of different preparations to neutralize antigens, as individual epitopes have varying immunogenicity (stimulating capacity). Consequently, a given batch of polyclonal antibodies may contain few molecules directed against the primary epitope, making it less effective than the previous one.

Therefore, for the Structure/182.html">Practical Application of antibodies as diagnostic tools or therapeutic components, it was necessary to establish a cell line that could grow in culture and produce a single type of antibody with high affinity for a specific target antigen—monoclonal antibodies. Such a cell line would provide an inexhaustible source of identical antibody molecules. Unfortunately, antibody-synthesizing B lymphocytes (B cells) cannot replicate in culture. The solution to this problem lay in creating a hybrid cell. By acquiring genetic material from a B cell, it could produce antibodies, and by gaining The ability to divide from a compatible cell type, it could grow in culture. It was known that B lymphocytes sometimes undergo malignant transformation to become Cancer (myeloma) cells, thereby gaining the ability to grow in culture while retaining many properties of B cells. Thus, myeloma cells, particularly those that do not produce antibodies themselves, became candidates for fusion with antibody-producing B cells. In the mid-1970s, these ideas became a reality.

Generation and Selection of Hybrid Cells The first step in obtaining a hybrid cell line that produces a single type of antibody is to immunize mice with the antigen. After a series of immunizations over several weeks, the animals are tested for an Immune Response. If a response has developed, the animals are euthanized, and the Spleen is harvested, washed, minced, and gently agitated to release single cells, which include antibody-producing B cells. The spleen cell suspension is mixed with a suspension of myeloma cells deficient in hypoxanthine-guanine phosphoribosyltransferase (HGPRT-). The combined suspension is incubated in 35% polyethylene glycol for a few minutes and then transferred to a medium containing hypoxanthine, aminopterin, and thymidine (HAT medium).

Treatment with polyethylene glycol facilitates Cell Fusion; however, fusion is a rare and largely random event. The mixture contains myeloma cells, spleen cells, as well as fused myeloma-spleen, myeloma-myeloma, and spleen-spleen cells. However, only hybrid myeloma-spleen cells grow in HAT medium; all other cell types fail to proliferate in it. Spleen cells and fused spleen-spleen cells do not grow in culture at all, while HGPRT- myeloma cells and fused myeloma-myeloma cells cannot use hypoxanthine as a precursor for The Biosynthesis of the purine bases guanine and adenine, which are essential for nucleic acid synthesis. However, they possess another natural pathway for purine synthesis involving Dihydrofolate Reductase, which is why the medium contains aminopterin to inhibit this enzyme's activity. Consequently, HGPRT- myeloma cells and fused myeloma-myeloma cells cannot synthesize Purines in HAT medium and die.

Fused spleen-myeloma cells grow in HAT medium because: 1) spleen cells supply functional HGPRT, which can utilize exogenous hypoxanthine from the medium despite the blockage of purine synthesis by aminopterin via dihydrofolate reductase inhibition; 2) myeloma cells are capable of active division. Thymidine is required to bypass the block in pyrimidine synthesis caused by dihydrofolate reductase inhibition. By days 10–14 after cell fusion, only fused spleen-myeloma cells survive and grow in HAT medium. They are then transferred into the wells of plastic microtiter plates and grown in complete culture medium without HAT.

Identification of Hybrid Cell Lines Secreting Specific Antibodies

Next, it is necessary to identify the hybrid cells that produce antibodies against the immunizing antigen. This is typically achieved by screening the culture media containing the secreted antibodies. Medium from wells with growing cells is harvested and transferred to the wells of another microtiter plate that have been pre-coated with a layer of target antigen molecules. If the culture medium contains an antibody (primary antibody) that recognizes one of the epitopes of this antigen, it will bind to the antigen and remain in the wells after washing. A secondary antibody specific to mouse antibodies is then added to the wells. It will bind to any primary antibody that is complexed with the antigen.

The secondary antibody used in the immunoassay is pre-conjugated to an enzyme that converts a colorless substrate into a colored compound. A color change in a well indicates that the original culture medium contained an antibody specific to the antigen (Fig. 9.2). If no such antibody was present in the medium, the secondary antibody will have nothing to bind to and will be washed away during the second wash. The substrate in these wells will remain colorless.

The wells of the original microtiter plate whose medium yields a positive immune response (color change) may contain a mixture of fused cells. To obtain single-cell-derived lines (clones), The Cell suspension from these wells is diluted with culture medium and seeded into other wells. After culturing the resulting clones, the media are tested again to determine which cell line (hybridoma) produces monoclonal antibodies that recognize the target antigen. If more than one specific hybridoma is obtained, further studies are conducted to determine whether the antibodies produced by different clones are directed against the same antigenic determinant. Each clone producing a monoclonal antibody can be maintained in culture almost indefinitely. Additionally, samples can be frozen in liquid nitrogen and used as a future cell source.

Fig. 9.2. Screening for monoclonal antibody-producing cells. Spleen cells are isolated from a mouse immunized with a specific antigen and fused with non-antibody-producing myeloma cells. Fused cells are selected based on their ability to grow on HAT (hypoxanthine, aminopterin, thymidine) medium. Cells producing specific antibodies against the immunizing antigen (hybridoma cells) are identified by immunological methods and subcultured to obtain individual clones. Monoclonal antibodies are harvested from the hybridoma growing in culture and secreting a single type of antibody molecule.

Fig. 9.3. Use of monoclonal antibodies for the detection of various compounds and Diagnosis of infectious diseases.

The use of monoclonal antibodies significantly increases the Specificity of the ELISA method, as they bind to a single, strictly defined antigenic site. To date, a wide range of monoclonal antibodies has been generated that can be used to detect various compounds and pathogens (Fig. 9.3). An alternative to producing monoclonal antibodies from hybridoma cell culture is the selection and production of MONOCLONAL ANTIBODIES AND their fragments (Fv fragments) targeted against the target antigen using E. coli (see Ch. 10).