Investigation and Exploitation of Antibody Combining Sites


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The binding event between antibody and antigen marks the foreign invader for destruction via phagocytosis or activation of the complement system.

They differ not only in their physiological roles but also in their structures. From a structural point of view, IgG antibodies are a particular class of immunoglobulins that have been extensively studied, perhaps because of the dominant role they play in a mature immune response. A considerable amount of what we know about the structure of antibodies comes from X-ray crystallographic [2] and immunoglobulin gene sequencing studies. IgG antibodies are Y-shaped proteins composed of two heavy chains and two light chains that are joined by disulfide linkages. The IgG molecule can be broken down into two regions, the Fc and Fab.

The Fc region, so called because it is the fragment of the IgG molecule that most readily crystallizes, is involved in effecting the physiological roles the antibody must play. Two identical Fab fragment are present at the ends of the "Y" in every IgG structure.


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The Fab region is named as such because it is the IgG fragment that contains the antibody binding site. The Fab region contains a region of highly conserved amino acids as well as a region of highly variable amino acids Fv. These variable sequences are confined to 6 protein loops or complementarity determining regions that cluster together at the end of the Fab fragments to form a continuous hypervariable surface. It is this region that is responsible for the binding of foreign antigens.

Click for a labeled picture of an intact antibody. Figure 1. Schematic drawing of an IgG antibody. In order to perform their crucial role in the line of defense, antibodies must be extremely versatile. Indeed, on even a daily basis the immune system encounters a great variety of foreign substances e. As a result, antibodies must be extremely diverse to counter a large number of unexpected and unknown possibilities. Through a complex process of gene splicing, B-cells have been estimated to produce between 1 x 10 8 to 1 x 10 10 IgG antibodies that differ in the composition of their binding sites.

Another needed attribute of antibodies is specificity. In order to distinguish between both self and a multitude of foreign species, antibodies need to have a highly discriminating method of recognition on the molecular level. This specificity is the result of the complementary nature of antibody binding. By taking advantage of the varied chemical properties of the 20 amino acids, the immune system is able to generate an array of antibody binding pockets that can accommodate the shape, charge, and hydrophobicity of seemingly any given antigen.

The complementary nature of antibody binding has been confirmed with the aid of X-ray crystallography. To date, a variety of crystal structures between Fab fragments and small molecules have been solved. In all cases, extensive use of intermolecular attractive forces are employed. For example, in the crystal structure between phosphocholine 1 and the antibody McPC [6] several well-placed amino acid residues can be seen to hold phosphocholine in the antibody binding pocket. In general, McPC, makes critical use of coulombic interactions to recognize this antigen in direct response to the charged nature of the phosphocholine.

For example, the negatively charged carboxylate group of an aspartic acid residue forms a salt bridge with the quarternized amine of the choline molecule. In addition, a guanidinium moiety from an arginine amino acid residue is observed to take advantage of both H-bonding and charge-charge interactions with the negatively-charged phosphate group present at the other end of the phosphocholine molecule. Van der Waals contacts are used extensively by antibodies to conform to the shape of an antigen.

Fab complexes with macromolecules, such as proteins, show that considerably more surface area is accessible to an antibody binding pocket to conform to the shape of an antigen. Interfacial contact between antibody and antigen have been shown to vary between and square angstroms for several complexes of antibody Fabs with lysozyme and with neuraminidase. This exclusion of interfacial water, as evidenced in many complexes between Fabs and antigens, seems to be a common feature of antibody binding.

Immunohistochemistry - Wikipedia

The high degree of complementarity exhibited by antibody binding also endows antibodies with high affinities for their antigens. For a mature immune response, antibody affinities typically fall in the range of 10 5 to 10 12 M The first was the experimentally verified upper limit for on rates 10 5 6 M Two types of antibody samples can be used in the study of antibody-related phenomenon.

The first type, polyclonal antibodies, [13] can be obtained by immunizing a mammal, such as a goat, sheep, mouse, or, most conveniently, a rabbit. After immunization, blood is removed periodically, if desired and the antibodies can be purified directly from the serum.

The name polyclonal is derived from the Greek word for many polys and sprout klon. As implied by the name, polyclonal antibodies originate or "sprout" from a variety of B-cells that differ in the genetic material that encodes for antibody production. In a polyclonal sample, some of the antibodies will be specific for the antigen with which the animal was immunized.

Antibody Domain Exchange Is an Immunological Solution to Carbohydrate Cluster Recognition

The remaining antibodies have been elicited from encounters with other foreign antigens that the animal has been exposed to throughout its lifetime. The second type of antibody sample, the monoclonal antibody, [14] is derived from a more complex process.

B-Cells 1 - What are Antibodies?

The technology associated with the production of monoclonal antibodies [15] is shown schematically in Figure 2. Here, a mammal, almost always an inbred mouse, is immunized with an antigen. After repeated immunizations, the spleen of the animal is removed. Because the spleen is responsible for B-cell production, the spleen cells contain the genetic information that gives rise to antibody production.

Unfortunately, these spleen cells cannot be cultured. As a result, they are fused with "immortal" myeloma cells, so-called because of their ability to proliferate in vitro. The resulting fused cells, called hybridoma cells, are screened with a colorometric enzyme-linked immunoabsorbant assay ELISA. Because a given hybridoma cell is derived from a single B-cell, it produces a monoclonal antibody. Here, the prefix mono-, derived from the Greek word for single monos , is used to indicate that a monoclonal antibody is derived from the genetic code of a unique B-cell.

Once a single hybridoma line is selected, it is injected into a healthy mouse. Hybridoma cells, like myeloma cells, have the ability to produce tumors; consequently, after injection with a hybridoma line, a tumor grows inside the host mouse. When this tumor grows, it produces ascites, a fluid that is rich in monoclonal antibodies.

Figure 2. An outline of the procedure used to produce monoclonal antibodies. In this scheme three different antigen-specific antibodies produced via immunization are shown. Hybridoma cells that produce antibodies that are not specific for the antigen are easily screened out with an ELISA. Once antibodies are produced, considerable care needs to be taken in their purification to avoid deleterious effects that may affect their study. The development of protein A and G-based chromatography has brought about considerable advances in the isolation of antibodies, in particular those of the IgG class.

This chromatography utilizes a bacterial protein that binds specifically to the Fc region of IgG antibodies. At neutral to high pH, IgG antibodies are bound and unwanted biological substances, such as albumins and nucleic acids, can be washed through the column. When the pH is lowered, the bound IgG antibodies are eluted from the column. Depending on the experimental situation, either a polyclonal or monoclonal antibody approach may be warranted. Each approach offers certain advantages. In the case of polyclonal antibodies, there are clear technical advantages.

Polyclonal antibodies are inexpensive to produce relative to the cost of monoclonal antibody technology. Finally, high affinity polyclonal antibodies can be isolated merely months after the initial immunization.

Investigation and Exploitation of Antibody Combining Sites

This expeditious production facilitates their rapid study. There are also advantages to the use of polyclonal antibodies from a scientific perspective. Because polyclonal antibodies contain the entire antigen-specific antibody population, they offer a statistically relevant glimpse into the overall picture of an immune response. A similar viewpoint is considerably more difficult, if not impossible, using a monoclonal antibody approach. On the other hand, monoclonal antibodies have certain advantages over polyclonal antibodies. Because of their immortal nature, hybridoma cells can be frozen, thawed, and recultured in vitro.

As a result, for a given monoclonal line, there exists a constant and renewable source of antibodies for study. In addition, the defined composition of a monoclonal antibody allows for its chemical composition, on a molecular level, to be analyzed in detail. For example, X-ray crystallographic and gene sequencing methods can only be applied to monoclonal antibodies. This level of detail is particularly useful when studying mechanistic issues related to binding.

Many of the basic principles in the field of immunochemistry the study of immunological processes on the molecular level are now unreferenced material in the modern literature. Nevertheless, it is important to remember that these principles were established over half a century ago by chemists interested in unraveling the mysteries of antibodies. While commonplace today, the use of chemical methodologies to understand immunological phenomena early on in the field of immunochemistry was considered a revolutionary approach.

One of the first attempts to study antibodies from the viewpoint of chemistry was taken up by Arrhenius. This text was of significance in that Arrhenius applied a mathematical approach founded in physical chemistry to explain various in vivo and in vitro phenomena related to antibody-antigen complex formation. In the preface of the book he wrote:.

Investigation and Exploitation of Antibody Combining Sites Investigation and Exploitation of Antibody Combining Sites
Investigation and Exploitation of Antibody Combining Sites Investigation and Exploitation of Antibody Combining Sites
Investigation and Exploitation of Antibody Combining Sites Investigation and Exploitation of Antibody Combining Sites
Investigation and Exploitation of Antibody Combining Sites Investigation and Exploitation of Antibody Combining Sites
Investigation and Exploitation of Antibody Combining Sites Investigation and Exploitation of Antibody Combining Sites
Investigation and Exploitation of Antibody Combining Sites Investigation and Exploitation of Antibody Combining Sites
Investigation and Exploitation of Antibody Combining Sites Investigation and Exploitation of Antibody Combining Sites
Investigation and Exploitation of Antibody Combining Sites Investigation and Exploitation of Antibody Combining Sites

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