Antibodies are proteins that are present on the B-cell membrane and are secreted by plasma cells. Serum antibodies are also sometimes referred to as “immunoglobulins.” Antibody functions as the effector of the humoral response by binding to antigen and neutralizing it or facilitating its elimination by 1) cross-linking several antigens, forming clusters that are more readily ingested by phagocytic cells or 2) by binding to antigen on a microorganisms thereby activating the complement system, resulting in lysis of the organism. Antibody can also neutralize toxins or viral particles by coating them, which prevents them form binding to host cells

The interaction between an antibody and an antigen depends on 4 types of noncovalent forces: (1) ionic bonds between oppositely charged residues; (2) hydrogen bonds in which a hydrogen atom is shared between 2 electronegative atoms; (3) hydrophobic interactions in which water forces hydrophobic groups together to maximize hydrogen bonding of water molecules, and (4) van der Waals interactions between the outer electron clouds of 2 atoms.

Definitions:

Affinity: See outline

Crossblocking: refers to an antibody that lowers the amount of binding of a reference antibody to an epitope on an antigen. Suitable methods for determining whther a first antibody crossblocks binding of a second antibody to an eitope are known in the art. For example, crosslbocking antibodies can be identified by comparing the binding of a reference antibody in the presence and absence of a test antibody (US6,355,245). 

Cross-reactivity:
Although Ag-Ab reactions are highly specific, in some cases antibody elicited by one antigen can cross-react with an unrelated antigen where the two antigens share identical or very similar epitopes. This happens, for example, between microbial antigens present on common intestinal bacteria in the gut and red blood cells. Such microbial antigens induce the formation of antibodies in individuals lacking similar blood group antigens (ABO blood group antigens which are glycoproteins expressed on red blood cells) on their red blood cells (In individuals possessing these antigens, complementary antibodies would be eliminated during time that antibodies that recognize self epitopes are negatively selected). The blood group antibodies, although elicited by microbial antigens, will cross-react with similar oligosaccharides on foreign red blood cells. This provides the basis for blood testing. A type A individual has anti-B antibodies; a type B individual has anti-A and a type O individual has anti-A and anti-B. Some vacines also exhibit cross-reactivity. For example, vaccinia virus which causes cowpox expresses cross-reacting epitopes with variola virus which causes smallpox. This fact serves as the basis for using vaccinia virus to induce immunity to smallpox.
Antigens are foreign substances that induce an immune response when injected into an animal. Most large molecules, including virtually all proteins and many polysaccharides can serve as antigens. Those parts of an antigen that combine with the antigen-binding site on either an antibody molecule or a lymphocyte receptor are called antigenic determinants or epitopes. Most antigens have a variety of epitopes that can stimulate the production of antibodies, specific T cell responses, or both. Some epitopes of an antigen produce a greater response than others and are said to be immunodominant.

Dissociation constant KD: See outline

Isoelectric point or pI: of a protein-antibody is the pH at which it has not net electrical charge. Biological molecules such as proteins are comprised of amino acids which may be positive, negative, neutral or polar in nature and together give a protein its overall charge. At a pH below its pI, a protein carries a net positive charge while at a pH about its pI it carries a net negative charge. Lack of charge may have certain consequences on a protein. For example, protiens are often minimally soluble in water or buffers near their pI, which can lead to difficulties in purificaiton or formulation of therapeutics and often precipitate out of solution. The pI of a protein may be determined mathematically be several methods including the Henderson Hasselbalch equation. The pI may be computed by this equation by taking into account eh acid’dissociate constant or pKa of nine different chemical groups, including the side chains of seven amino acids, aspartic acid, glutamin acid, lysine, histidine, arginine, tyrosine and cysteine as well as the amino and carboxy terminal amino acid residues of the protein. Alternatively, the pI of a protein may be determined experimentally using isolectic focusing. For example, when a protien is in a pH region below its pI, it will be positively charged and so will migrate toward a cathode. As it migrates, however, the charge will decrease until the protein reaches the pH region that corresponds to its pI. At this point it has no net charge and so migration ceases. As a result, the protiens become focused into sharp stationary bands with each protein positioned at a point in the pH gradient corresponding to its pI.

Evans, US Patent Application 13140554, discloses a method for determining isolectric points of proteins by identifying surface exposed amino acid resiudes in a sequence of amino acid residues of the protein, assigning a pKa value to the surface exposed amino acid residues and calculating the pI of the protein fomr the pKa values assigned to the surface exposed amino acid residues. 

Paratope: The part of the antibody that recognizes the epitope is called a paratope.

pH dependency of Antibodies: See homeostasis

Specificity (potency): of an antibody refers to the number of different types of antigens (antigenic determinants) to which a particular antigen-binding molecule can bind. Specificity can also be determined based on affinity and/or avidity. The affinity, represented by the equilibrium constant for the dissociation of an antigen with an antigen-binding protein (KD) is a measure for the binding strength between an antigenic determinant and an antigen binding site on the antigen binding protein: the lesser the value of the Kc, the stronger the binding strenght between an antigenic determinant and the antigen binding molecule. (alternatively, the affinity can also be expressed as the affinity constant (KA), which is 1/KC).

Specificity can also be determined using IC50 of the antigen (see also receptor kinetics) . IC50 or “half maximal inhibitory concentration” refers to the concentration of a substance (inhibitor, antagonist) that produces a 50% inhibition of a given biological process (e.g., enzyme, antibody, etc). The IC50 of a substance can be determined by constructing a dose-response curve and examining the effect of different concentrations of the antagonist on reversing agonist activity. IC50 vlaues can be calculated for a given antagonist by determining the concentration needed to inhibit half of the maximum biological response of the agonst.

Sensitivity: refers to the percetnage of actual positives that are correctly identified. For example, sensitivity of an antibody to a molecule refers to the lowest concentration of of the molecule that competes with, and reduces, binding of the antibody to its antigen.

Epitopes and Their Mapping

Epitope (antigenic determinant): refers to a site on an antigen to which B and/or T cells respond. B cell epitopes can be formed both from contigous amino acids or noncontiguous amino acids justaposed by tertiary folding of a protein. An epitope typically includes at least 3, and usually at least 5 or 8-10 amino acids in a unique spatial conformation. Antibodies that recognize the same epitope ca be identified in a simple immunoassay showing the ability of one antiboyd to block the binding of another antibody to a target antigen. T cells recognize continous epitopes of about 9 amino acids for CD8 cell or about 13-15 amino acids for CD4 cells. T cells that recognize the eptiope can be identified by in vitor assays that measure antigne dependent proliferation, as determined by 3H-thymidine incorporation by primed T cells in response to an eptiope.

The epitopes of protein target molecules are divided into two categories; conformational epitopes and linear epitopes, based on their structure and interaction with the paratope. A conformational epitope is composed of discontinous secitons ont he target molecule’s amino acid sequence. These epitopes interact with the paratope based on the 3 D surface features and shape or teriary struture of the target molecule. In contrast, linear epitopes interact with the paratope based on their primary structure, the amino acids that make up a linear epitope are a contineous sequence of amino acids from the target molecule. (Josephus, US13/982970).

Most antigens offer multiple epitopes and thus induce proliferation and differentiation of a variety of B-cell clones, each derived form a B cell that recognizes a particular epitope. The resulting serum antibodies are heterogeneous, comprising a mixture of antibodies, each specific for one epitope and are called polyclonal as opposed to a single B or T cell clone which is said to be monoclonal.

Epitope Mapping: Methods for identifying the epitope to which a particular antibody binds are known in the art. For example, the binding epitope of a particular antibody can be identified by measuring the binding of the antibody to several overlapping peptide dragments of a particular antigen. Each of the different overlapping peptides is bound to a unique address on a solid support. Next the antibody is interrogated by contacting it to each of the peptides in the plate. The present or amount of the detectable signal produced by the detectably labeled secondary antibody in a well is an indication that the antibody binds to the particular ppetide fragment associated with the well. (US 20060153836). 

Epitope mapping is a way to determine which epitopes on a peptide/protein are important for antibody neutralization. One scheme to do this is disclosed by Yang (Biocehm J (1998) 330, 497-503 wehre the protein of interest (in this case snake venom protein beta-bungarotoxin) is adminsitered to mixe, moncolonal antibodies directed to the protein are isolated from the mice and those antibodies are contacted with small/secondary peptides (the peptides are blotted on a membrane, incubated with the antibodies and then secondary antibodies and substrate then added)and those antibodies which bind to the secondary antigens are selected. Peptides containing the neutralizing epitopes of the toxin can then be used for imunization and tested for protection of mice from toxin challenge.