Antibodies | Wikipedia audio article

Antibodies | Wikipedia audio article


An antibody (Ab), also known as an immunoglobulin
(Ig), is a large, Y-shaped protein produced mainly by plasma cells that is used by the
immune system to neutralize pathogens such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule
of the pathogen, called an antigen, via the fragment antigen-binding (Fab) variable region. Each tip of the “Y” of an antibody contains
a paratope (analogous to a lock) that is specific for one particular epitope (similarly, analogous
to a key) on an antigen, allowing these two structures to bind together with precision. Using this binding mechanism, an antibody
can tag a microbe or an infected cell for attack by other parts of the immune system,
or can neutralize its target directly (for example, by inhibiting a part of a microbe
that is essential for its invasion and survival). Depending on the antigen, the binding may
impede the biological process causing the disease or may activate macrophages to destroy
the foreign substance. The ability of an antibody to communicate
with the other components of the immune system is mediated via its Fc region (located at
the base of the “Y”), which contains a conserved glycosylation site involved in these interactions. The production of antibodies is the main function
of the humoral immune system.Antibodies are secreted by B cells of the adaptive immune
system, mostly by differentiated B cells called plasma cells. Antibodies can occur in two physical forms,
a soluble form that is secreted from the cell to be free in the blood plasma, and a membrane-bound
form that is attached to the surface of a B cell and is referred to as the B-cell receptor
(BCR). The BCR is found only on the surface of B
cells and facilitates the activation of these cells and their subsequent differentiation
into either antibody factories called plasma cells or memory B cells that will survive
in the body and remember that same antigen so the B cells can respond faster upon future
exposure. In most cases, interaction of the B cell with
a T helper cell is necessary to produce full activation of the B cell and, therefore, antibody
generation following antigen binding. Soluble antibodies are released into the blood
and tissue fluids, as well as many secretions to continue to survey for invading microorganisms. Antibodies are glycoproteins belonging to
the immunoglobulin superfamily. They constitute most of the gamma globulin
fraction of the blood proteins. They are typically made of basic structural
units—each with two large heavy chains and two small light chains. There are several different types of antibody
heavy chains that define the five different types of crystallisable fragments (Fc) that
may be attached to the antigen-binding fragments. The five different types of Fc regions allow
antibodies to be grouped into five isotypes. Each Fc region of a particular antibody isotype
is able to bind to its specific Fc Receptor (except for IgD, which is essentially the
BCR), thus allowing the antigen-antibody complex to mediate different roles depending on which
FcR it binds. The ability of an antibody to bind to its
corresponding FcR is further modulated by the structure of the glycan(s) present at
conserved sites within its Fc region. The ability of antibodies to bind to FcRs
helps to direct the appropriate immune response for each different type of foreign object
they encounter. For example, IgE is responsible for an allergic
response consisting of mast cell degranulation and histamine release. IgE’s Fab paratope binds to allergic antigen,
for example house dust mite particles, while its Fc region binds to Fc receptor ε. The allergen-IgE-FcRε interaction mediates
allergic signal transduction to induce conditions such as asthma.Though the general structure
of all antibodies is very similar, a small region at the tip of the protein is extremely
variable, allowing millions of antibodies with slightly different tip structures, or
antigen-binding sites, to exist. This region is known as the hypervariable
region. Each of these variants can bind to a different
antigen. This enormous diversity of antibody paratopes
on the antigen-binding fragments allows the immune system to recognize an equally wide
variety of antigens. The large and diverse population of antibody
paratope is generated by random recombination events of a set of gene segments that encode
different antigen-binding sites (or paratopes), followed by random mutations in this area
of the antibody gene, which create further diversity. This recombinational process that produces
clonal antibody paratope diversity is called V(D)J or VJ recombination. Basically, the antibody paratope is polygenic,
made up of three genes, V, D, and J. Each paratope locus is also polymorphic, such that
during antibody production, one allele of V, one of D, and one of J is chosen. These gene segments are then joined together
using random genetic recombination to produce the paratope. The regions where the genes are randomly recombined
together is the hyper variable region used to recognise different antigens on a clonal
basis. Antibody genes also re-organize in a process
called class switching that changes the one type of heavy chain Fc fragment to another,
creating a different isotype of the antibody that retains the antigen-specific variable
region. This allows a single antibody to be used by
different types of Fc receptors, expressed on different parts of the immune system.==History==The first use of the term “antibody” occurred
in a text by Paul Ehrlich. The term Antikörper (the German word for
antibody) appears in the conclusion of his article “Experimental Studies on Immunity”,
published in October 1891, which states that, “if two substances give rise to two different
Antikörper, then they themselves must be different”. However, the term was not accepted immediately
and several other terms for antibody were proposed; these included Immunkörper, Amboceptor,
Zwischenkörper, substance sensibilisatrice, copula, Desmon, philocytase, fixateur, and
Immunisin. The word antibody has formal analogy to the
word antitoxin and a similar concept to Immunkörper (immune body in English). As such, the original construction of the
word contains a logical flaw; the antitoxin is something directed against a toxin, while
the antibody is a body directed against something. The study of antibodies began in 1890 when
Kitasato Shibasaburō described antibody activity against diphtheria and tetanus toxins. Kitasato put forward the theory of humoral
immunity, proposing that a mediator in serum could react with a foreign antigen. His idea prompted Paul Ehrlich to propose
the side-chain theory for antibody and antigen interaction in 1897, when he hypothesized
that receptors (described as “side-chains”) on the surface of cells could bind specifically
to toxins – in a “lock-and-key” interaction – and that this binding reaction is the
trigger for the production of antibodies. Other researchers believed that antibodies
existed freely in the blood and, in 1904, Almroth Wright suggested that soluble antibodies
coated bacteria to label them for phagocytosis and killing; a process that he named opsoninization. In the 1920s, Michael Heidelberger and Oswald
Avery observed that antigens could be precipitated by antibodies and went on to show that antibodies
are made of protein. The biochemical properties of antigen-antibody-binding
interactions were examined in more detail in the late 1930s by John Marrack. The next major advance was in the 1940s, when
Linus Pauling confirmed the lock-and-key theory proposed by Ehrlich by showing that the interactions
between antibodies and antigens depend more on their shape than their chemical composition. In 1948, Astrid Fagreaus discovered that B
cells, in the form of plasma cells, were responsible for generating antibodies.Further work concentrated
on characterizing the structures of the antibody proteins. A major advance in these structural studies
was the discovery in the early 1960s by Gerald Edelman and Joseph Gally of the antibody light
chain, and their realization that this protein is the same as the Bence-Jones protein described
in 1845 by Henry Bence Jones. Edelman went on to discover that antibodies
are composed of disulfide bond-linked heavy and light chains. Around the same time, antibody-binding (Fab)
and antibody tail (Fc) regions of IgG were characterized by Rodney Porter. Together, these scientists deduced the structure
and complete amino acid sequence of IgG, a feat for which they were jointly awarded the
1972 Nobel Prize in Physiology or Medicine. The Fv fragment was prepared and characterized
by David Givol. While most of these early studies focused
on IgM and IgG, other immunoglobulin isotypes were identified in the 1960s: Thomas Tomasi
discovered secretory antibody (IgA); David S. Rowe and John L. Fahey discovered IgD;
and Kimishige Ishizaka and Teruko Ishizaka discovered IgE and showed it was a class of
antibodies involved in allergic reactions. In a landmark series of experiments beginning
in 1976, Susumu Tonegawa showed that genetic material can rearrange itself to form the
vast array of available antibodies.==Forms==
The membrane-bound form of an antibody may be called a surface immunoglobulin (sIg) or
a membrane immunoglobulin (mIg). It is part of the B cell receptor (BCR), which
allows a B cell to detect when a specific antigen is present in the body and triggers
B cell activation. The BCR is composed of surface-bound IgD or
IgM antibodies and associated Ig-α and Ig-β heterodimers, which are capable of signal
transduction. A typical human B cell will have 50,000 to
100,000 antibodies bound to its surface. Upon antigen binding, they cluster in large
patches, which can exceed 1 micrometer in diameter, on lipid rafts that isolate the
BCRs from most other cell signaling receptors. These patches may improve the efficiency of
the cellular immune response. In humans, the cell surface is bare around
the B cell receptors for several hundred nanometers, which further isolates the BCRs from competing
influences.==Antibody–antigen interactions==
The antibody’s paratope interacts with the antigen’s epitope. An antigen usually contains different epitopes
along its surface arranged discontinuously, and dominant epitopes on a given antigen are
called determinants. Antibody and antigen interact by spatial complementarity
(lock and key). The molecular forces involved in the Fab-epitope
interaction are weak and non-specific – for example electrostatic forces, hydrogen bonds,
hydrophobic interactions, and van der Waals forces. This means binding between antibody and antigen
is reversible, and the antibody’s affinity towards an antigen is relative rather than
absolute. Relatively weak binding also means it is possible
for an antibody to cross-react with different antigens of different relative affinities. Often, once an antibody and antigen bind,
they become an immune complex, which functions as a unitary object and can act as an antigen
in its own right, being countered by other antibodies. Similarly, haptens are small molecules that
provoke no immune response by themselves, but once they bind to proteins, the resulting
complex or hapten-carrier adduct is antigenic.==Isotypes==
Antibodies can come in different varieties known as isotypes or classes. In placental mammals there are five antibody
isotypes known as IgA, IgD, IgE, IgG, and IgM. They are each named with an “Ig” prefix that
stands for immunoglobulin (a name sometimes used interchangeably with antibody) and differ
in their biological properties, functional locations and ability to deal with different
antigens, as depicted in the table. The different suffixes of the antibody isotypes
denote the different types of heavy chains the antibody contains, with each heavy chain
class named alphabetically: α (alpha), γ (gamma), δ (delta), ε (epsilon), and μ
(mu). This gives rise to IgA, IgG, IgD, IgE, and
IgM, respectively. The antibody isotype of a B cell changes during
cell development and activation. Immature B cells, which have never been exposed
to an antigen, express only the IgM isotype in a cell surface bound form. The B lymphocyte, in this ready-to-respond
form, is known as a “naive B lymphocyte.” The naive B lymphocyte expresses both surface
IgM and IgD. The co-expression of both of these immunoglobulin
isotypes renders the B cell ready to respond to antigen. B cell activation follows engagement of the
cell-bound antibody molecule with an antigen, causing the cell to divide and differentiate
into an antibody-producing cell called a plasma cell. In this activated form, the B cell starts
to produce antibody in a secreted form rather than a membrane-bound form. Some daughter cells of the activated B cells
undergo isotype switching, a mechanism that causes the production of antibodies to change
from IgM or IgD to the other antibody isotypes, IgE, IgA, or IgG, that have defined roles
in the immune system.==Structure==
Antibodies are heavy (~150 kDa) globular plasma proteins. The size of an antibody molecule is about
10 nm. They have sugar chains (glycans) added to
conserved amino acid residues. In other words, antibodies are glycoproteins. The attached glycans are critically important
to the structure and function of the antibody. Among other things the expressed glycans can
modulate an antibody’s affinity for its corresponding FcR(s).The basic functional unit of each antibody
is an immunoglobulin (Ig) monomer (containing only one Ig unit); secreted antibodies can
also be dimeric with two Ig units as with IgA, tetrameric with four Ig units like teleost
fish IgM, or pentameric with five Ig units, like mammalian IgM. The variable parts of an antibody are its
V regions, and the constant part is its C region.===Immunoglobulin domains===
The Ig monomer is a “Y”-shaped molecule that consists of four polypeptide chains; two identical
heavy chains and two identical light chains connected by disulfide bonds. Each chain is composed of structural domains
called immunoglobulin domains. These domains contain about 70–110 amino
acids and are classified into different categories (for example, variable or IgV, and constant
or IgC) according to their size and function. They have a characteristic immunoglobulin
fold in which two beta sheets create a “sandwich” shape, held together by interactions between
conserved cysteines and other charged amino acids.===Heavy chain===There are five types of mammalian Ig heavy
chain denoted by the Greek letters: α, δ, ε, γ, and μ. The type of heavy chain present defines the
class of antibody; these chains are found in IgA, IgD, IgE, IgG, and IgM antibodies,
respectively. Distinct heavy chains differ in size and composition;
α and γ contain approximately 450 amino acids, whereas μ and ε have approximately
550 amino acids. Each heavy chain has two regions, the constant
region and the variable region. The constant region is identical in all antibodies
of the same isotype, but differs in antibodies of different isotypes. Heavy chains γ, α and δ have a constant
region composed of three tandem (in a line) Ig domains, and a hinge region for added flexibility;
heavy chains μ and ε have a constant region composed of four immunoglobulin domains. The variable region of the heavy chain differs
in antibodies produced by different B cells, but is the same for all antibodies produced
by a single B cell or B cell clone. The variable region of each heavy chain is
approximately 110 amino acids long and is composed of a single Ig domain.===Light chain===In mammals there are two types of immunoglobulin
light chain, which are called lambda (λ) and kappa (κ). A light chain has two successive domains:
one constant domain and one variable domain. The approximate length of a light chain is
211 to 217 amino acids. Each antibody contains two light chains that
are always identical; only one type of light chain, κ or λ, is present per antibody in
mammals. Other types of light chains, such as the iota
(ι) chain, are found in other vertebrates like sharks (Chondrichthyes) and bony fishes
(Teleostei). It has been shown that the rearrangement of
lambda light chains of Ig in humans can lead to a deletion of some protein-coding genes
located between variable subgenes of lambda, but this is likely not pathogenic in any way,
since these genes are not expressed in B cells.===CDRs, Fv, Fab and Fc regions===
Different parts of an antibody have different functions. Specifically, the “arms” (which are generally
identical) contain sites that can bind to specific molecules, enabling recognition of
specific antigens. This region of the antibody is called the
Fab (fragment, antigen-binding) region. It is composed of one constant and one variable
domain from each heavy and light chain of the antibody. The paratope at the amino terminal end of
the antibody monomer is shaped by the variable domains from the heavy and light chains. The variable domain is also referred to as
the FV region and is the most important region for binding to antigens. To be specific, variable loops of β-strands,
three each on the light (VL) and heavy (VH) chains are responsible for binding to the
antigen. These loops are referred to as the complementarity
determining regions (CDRs). The structures of these CDRs have been clustered
and classified by Chothia et al. and more recently by North et al.
and Nikoloudis et al. In the framework of the immune network theory,
CDRs are also called idiotypes. According to immune network theory, the adaptive
immune system is regulated by interactions between idiotypes. The base of the Y plays a role in modulating
immune cell activity. This region is called the Fc (Fragment, crystallizable)
region, and is composed of two heavy chains that contribute two or three constant domains
depending on the class of the antibody. Thus, the Fc region ensures that each antibody
generates an appropriate immune response for a given antigen, by binding to a specific
class of Fc receptors, and other immune molecules, such as complement proteins. By doing this, it mediates different physiological
effects, including recognition of opsonized particles (binding to FcγR), lysis of cells
(binding to complement), and degranulation of mast cells, basophils, and eosinophils
(binding to FcεR).In summary, the Fab region of the antibody determines antigen specificity
while the Fc region of the antibody determines the antibody’s class effect. Since only the constant domains of the heavy
chains make up the Fc region of an antibody, the classes of heavy chain in antibodies determine
their class effects. Possible classes of heavy chains in antibodies
include alpha, gamma, delta, epsilon, and mu, and they define the antibody’s isotypes
IgA, G, D, E, and M, respectively. This infers different isotypes of antibodies
have different class effects due to their different Fc regions binding and activating
different types of receptors. Possible class effects of antibodies include:
Opsonisation, agglutination, haemolysis, complement activation, mast cell degranulation, and neutralisation
(though this class effect may be mediated by the Fab region rather than the Fc region). It also implies that Fab-mediated effects
are directed at microbes or toxins, whilst Fc mediated effects are directed at effector
cells or effector molecules (see below).==Function==The main categories of antibody action include
the following: Neutralisation, in which neutralizing antibodies
block parts of the surface of a bacterial cell or virion to render its attack ineffective
Agglutination, in which antibodies “glue together” foreign cells into clumps that are attractive
targets for phagocytosis Precipitation, in which antibodies “glue together”
serum-soluble antigens, forcing them to precipitate out of solution in clumps that are attractive
targets for phagocytosis Complement activation (fixation), in which
antibodies that are latched onto a foreign cell encourage complement to attack it with
a membrane attack complex, which leads to the following:
Lysis of the foreign cell Encouragement of inflammation by chemotactically
attracting inflammatory cellsActivated B cells differentiate into either antibody-producing
cells called plasma cells that secrete soluble antibody or memory cells that survive in the
body for years afterward in order to allow the immune system to remember an antigen and
respond faster upon future exposures.At the prenatal and neonatal stages of life, the
presence of antibodies is provided by passive immunization from the mother. Early endogenous antibody production varies
for different kinds of antibodies, and usually appear within the first years of life. Since antibodies exist freely in the bloodstream,
they are said to be part of the humoral immune system. Circulating antibodies are produced by clonal
B cells that specifically respond to only one antigen (an example is a virus capsid
protein fragment). Antibodies contribute to immunity in three
ways: They prevent pathogens from entering or damaging cells by binding to them; they
stimulate removal of pathogens by macrophages and other cells by coating the pathogen; and
they trigger destruction of pathogens by stimulating other immune responses such as the complement
pathway. Antibodies will also trigger vasoactive amine
degranulation to contribute to immunity against certain types of antigens (helminths, allergens).===Activation of complement===
Antibodies that bind to surface antigens (for example, on bacteria) will attract the first
component of the complement cascade with their Fc region and initiate activation of the “classical”
complement system. This results in the killing of bacteria in
two ways. First, the binding of the antibody and complement
molecules marks the microbe for ingestion by phagocytes in a process called opsonization;
these phagocytes are attracted by certain complement molecules generated in the complement
cascade. Second, some complement system components
form a membrane attack complex to assist antibodies to kill the bacterium directly (bacteriolysis).===Activation of effector cells===
To combat pathogens that replicate outside cells, antibodies bind to pathogens to link
them together, causing them to agglutinate. Since an antibody has at least two paratopes,
it can bind more than one antigen by binding identical epitopes carried on the surfaces
of these antigens. By coating the pathogen, antibodies stimulate
effector functions against the pathogen in cells that recognize their Fc region.Those
cells that recognize coated pathogens have Fc receptors, which, as the name suggests,
interact with the Fc region of IgA, IgG, and IgE antibodies. The engagement of a particular antibody with
the Fc receptor on a particular cell triggers an effector function of that cell; phagocytes
will phagocytose, mast cells and neutrophils will degranulate, natural killer cells will
release cytokines and cytotoxic molecules; that will ultimately result in destruction
of the invading microbe. The activation of natural killer cells by
antibodies initiates a cytotoxic mechanism known as antibody-dependent cell-mediated
cytotoxicity (ADCC) – this process may explain the efficacy of monoclonal antibodies used
in biological therapies against cancer. The Fc receptors are isotype-specific, which
gives greater flexibility to the immune system, invoking only the appropriate immune mechanisms
for distinct pathogens.===Natural antibodies===
Humans and higher primates also produce “natural antibodies” that are present in serum before
viral infection. Natural antibodies have been defined as antibodies
that are produced without any previous infection, vaccination, other foreign antigen exposure
or passive immunization. These antibodies can activate the classical
complement pathway leading to lysis of enveloped virus particles long before the adaptive immune
response is activated. Many natural antibodies are directed against
the disaccharide galactose α(1,3)-galactose (α-Gal), which is found as a terminal sugar
on glycosylated cell surface proteins, and generated in response to production of this
sugar by bacteria contained in the human gut. Rejection of xenotransplantated organs is
thought to be, in part, the result of natural antibodies circulating in the serum of the
recipient binding to α-Gal antigens expressed on the donor tissue.==Immunoglobulin diversity==
Virtually all microbes can trigger an antibody response. Successful recognition and eradication of
many different types of microbes requires diversity among antibodies; their amino acid
composition varies allowing them to interact with many different antigens. It has been estimated that humans generate
about 10 billion different antibodies, each capable of binding a distinct epitope of an
antigen. Although a huge repertoire of different antibodies
is generated in a single individual, the number of genes available to make these proteins
is limited by the size of the human genome. Several complex genetic mechanisms have evolved
that allow vertebrate B cells to generate a diverse pool of antibodies from a relatively
small number of antibody genes.===Domain variability===The chromosomal region that encodes an antibody
is large and contains several distinct gene loci for each domain of the antibody—the
chromosome region containing heavy chain genes ([email protected]) is found on chromosome 14, and the
loci containing lambda and kappa light chain genes ([email protected] and [email protected]) are found on chromosomes
22 and 2 in humans. One of these domains is called the variable
domain, which is present in each heavy and light chain of every antibody, but can differ
in different antibodies generated from distinct B cells. Differences, between the variable domains,
are located on three loops known as hypervariable regions (HV-1, HV-2 and HV-3) or complementarity
determining regions (CDR1, CDR2 and CDR3). CDRs are supported within the variable domains
by conserved framework regions. The heavy chain locus contains about 65 different
variable domain genes that all differ in their CDRs. Combining these genes with an array of genes
for other domains of the antibody generates a large cavalry of antibodies with a high
degree of variability. This combination is called V(D)J recombination
discussed below.===V(D)J recombination===Somatic recombination of immunoglobulins,
also known as V(D)J recombination, involves the generation of a unique immunoglobulin
variable region. The variable region of each immunoglobulin
heavy or light chain is encoded in several pieces—known as gene segments (subgenes). These segments are called variable (V), diversity
(D) and joining (J) segments. V, D and J segments are found in Ig heavy
chains, but only V and J segments are found in Ig light chains. Multiple copies of the V, D and J gene segments
exist, and are tandemly arranged in the genomes of mammals. In the bone marrow, each developing B cell
will assemble an immunoglobulin variable region by randomly selecting and combining one V,
one D and one J gene segment (or one V and one J segment in the light chain). As there are multiple copies of each type
of gene segment, and different combinations of gene segments can be used to generate each
immunoglobulin variable region, this process generates a huge number of antibodies, each
with different paratopes, and thus different antigen specificities. The rearrangement of several subgenes (i.e. V2 family) for lambda light chain immunoglobulin
is coupled with the activation of microRNA miR-650, which further influences biology
of B-cells. RAG proteins play an important role with V(D)J
recombination in cutting DNA at a particular region. Without the presence of these proteins, V(D)J
recombination would not occur.After a B cell produces a functional immunoglobulin gene
during V(D)J recombination, it cannot express any other variable region (a process known
as allelic exclusion) thus each B cell can produce antibodies containing only one kind
of variable chain.===Somatic hypermutation and affinity maturation
===Following activation with antigen, B cells
begin to proliferate rapidly. In these rapidly dividing cells, the genes
encoding the variable domains of the heavy and light chains undergo a high rate of point
mutation, by a process called somatic hypermutation (SHM). SHM results in approximately one nucleotide
change per variable gene, per cell division. As a consequence, any daughter B cells will
acquire slight amino acid differences in the variable domains of their antibody chains. This serves to increase the diversity of the
antibody pool and impacts the antibody’s antigen-binding affinity. Some point mutations will result in the production
of antibodies that have a weaker interaction (low affinity) with their antigen than the
original antibody, and some mutations will generate antibodies with a stronger interaction
(high affinity). B cells that express high affinity antibodies
on their surface will receive a strong survival signal during interactions with other cells,
whereas those with low affinity antibodies will not, and will die by apoptosis. Thus, B cells expressing antibodies with a
higher affinity for the antigen will outcompete those with weaker affinities for function
and survival allowing the average affinity of antibodies to increase over time. The process of generating antibodies with
increased binding affinities is called affinity maturation. Affinity maturation occurs in mature B cells
after V(D)J recombination, and is dependent on help from helper T cells.===Class switching===
Isotype or class switching is a biological process occurring after activation of the
B cell, which allows the cell to produce different classes of antibody (IgA, IgE, or IgG). The different classes of antibody, and thus
effector functions, are defined by the constant (C) regions of the immunoglobulin heavy chain. Initially, naive B cells express only cell-surface
IgM and IgD with identical antigen binding regions. Each isotype is adapted for a distinct function;
therefore, after activation, an antibody with an IgG, IgA, or IgE effector function might
be required to effectively eliminate an antigen. Class switching allows different daughter
cells from the same activated B cell to produce antibodies of different isotypes. Only the constant region of the antibody heavy
chain changes during class switching; the variable regions, and therefore antigen specificity,
remain unchanged. Thus the progeny of a single B cell can produce
antibodies, all specific for the same antigen, but with the ability to produce the effector
function appropriate for each antigenic challenge. Class switching is triggered by cytokines;
the isotype generated depends on which cytokines are present in the B cell environment.Class
switching occurs in the heavy chain gene locus by a mechanism called class switch recombination
(CSR). This mechanism relies on conserved nucleotide
motifs, called switch (S) regions, found in DNA upstream of each constant region gene
(except in the δ-chain). The DNA strand is broken by the activity of
a series of enzymes at two selected S-regions. The variable domain exon is rejoined through
a process called non-homologous end joining (NHEJ) to the desired constant region (γ,
α or ε). This process results in an immunoglobulin
gene that encodes an antibody of a different isotype.===Specificity designations===
An antibody can be called monospecific if it has specificity for the same antigen or
epitope, or bispecific if they have affinity for two different antigens or two different
epitopes on the same antigen. A group of antibodies can be called polyvalent
(or unspecific) if they have affinity for various antigens or microorganisms. Intravenous immunoglobulin, if not otherwise
noted, consists of a variety of different IgG (polyclonal IgG). In contrast, monoclonal antibodies are identical
antibodies produced by a single B cell.===Asymmetrical antibodies===
Heterodimeric antibodies, which are also asymmetrical and antibodies, allow for greater flexibility
and new formats for attaching a variety of drugs to the antibody arms. One of the general formats for a heterodimeric
antibody is the “knobs-into-holes” format. This format is specific to the heavy chain
part of the constant region in antibodies. The “knobs” part is engineered by replacing
a small amino acid with a larger one. It fits into the “hole”, which is engineered
by replacing a large amino acid with a smaller one. What connects the “knobs” to the “holes”
are the disulfide bonds between each chain. The “knobs-into-holes” shape facilitates
antibody dependent cell mediated cytotoxicity. Single chain variable fragments (scFv) are
connected to the variable domain of the heavy and light chain via a short linker peptide. The linker is rich in glycine, which gives
it more flexibility, and serine/threonine, which gives it specificity. Two different scFv fragments can be connected
together, via a hinge region, to the constant domain of the heavy chain or the constant
domain of the light chain. This gives the antibody bispecificity, allowing
for the binding specificities of two different antigens. The “knobs-into-holes” format enhances
heterodimer formation but doesn’t suppress homodimer formation. To further improve the function of heterodimeric
antibodies, many scientists are looking towards artificial constructs. Artificial antibodies are largely diverse
protein motifs that use the functional strategy of the antibody molecule, but aren’t limited
by the loop and framework structural constraints of the natural antibody. Being able to control the combinational design
of the sequence and three-dimensional space could transcend the natural design and allow
for the attachment of different combinations of drugs to the arms. Heterodimeric antibodies have a greater range
in shapes they can take and the drugs that are attached to the arms don’t have to be
the same on each arm, allowing for different combinations of drugs to be used in cancer
treatment. Pharmaceuticals are able to produce highly
functional bispecific, and even multispecific, antibodies. The degree to which they can function is impressive
given that such a change of shape from the natural form should lead to decreased functionality.==Medical applications=====
Disease diagnosis===Detection of particular antibodies is a very
common form of medical diagnostics, and applications such as serology depend on these methods. For example, in biochemical assays for disease
diagnosis, a titer of antibodies directed against Epstein-Barr virus or Lyme disease
is estimated from the blood. If those antibodies are not present, either
the person is not infected or the infection occurred a very long time ago, and the B cells
generating these specific antibodies have naturally decayed. In clinical immunology, levels of individual
classes of immunoglobulins are measured by nephelometry (or turbidimetry) to characterize
the antibody profile of patient. Elevations in different classes of immunoglobulins
are sometimes useful in determining the cause of liver damage in patients for whom the diagnosis
is unclear. For example, elevated IgA indicates alcoholic
cirrhosis, elevated IgM indicates viral hepatitis and primary biliary cirrhosis, while IgG is
elevated in viral hepatitis, autoimmune hepatitis and cirrhosis. Autoimmune disorders can often be traced to
antibodies that bind the body’s own epitopes; many can be detected through blood tests. Antibodies directed against red blood cell
surface antigens in immune mediated hemolytic anemia are detected with the Coombs test. The Coombs test is also used for antibody
screening in blood transfusion preparation and also for antibody screening in antenatal
women.Practically, several immunodiagnostic methods based on detection of complex antigen-antibody
are used to diagnose infectious diseases, for example ELISA, immunofluorescence, Western
blot, immunodiffusion, immunoelectrophoresis, and magnetic immunoassay. Antibodies raised against human chorionic
gonadotropin are used in over the counter pregnancy tests. New dioxaborolane chemistry enables radioactive
fluoride (18F) labeling of antibodies, which allows for positron emission tomography (PET)
imaging of cancer.===Disease therapy===
Targeted monoclonal antibody therapy is employed to treat diseases such as rheumatoid arthritis,
multiple sclerosis, psoriasis, and many forms of cancer including non-Hodgkin’s lymphoma,
colorectal cancer, head and neck cancer and breast cancer.Some immune deficiencies, such
as X-linked agammaglobulinemia and hypogammaglobulinemia, result in partial or complete lack of antibodies. These diseases are often treated by inducing
a short term form of immunity called passive immunity. Passive immunity is achieved through the transfer
of ready-made antibodies in the form of human or animal serum, pooled immunoglobulin or
monoclonal antibodies, into the affected individual.===Prenatal therapy===
Rh factor, also known as Rh D antigen, is an antigen found on red blood cells; individuals
that are Rh-positive (Rh+) have this antigen on their red blood cells and individuals that
are Rh-negative (Rh–) do not. During normal childbirth, delivery trauma
or complications during pregnancy, blood from a fetus can enter the mother’s system. In the case of an Rh-incompatible mother and
child, consequential blood mixing may sensitize an Rh- mother to the Rh antigen on the blood
cells of the Rh+ child, putting the remainder of the pregnancy, and any subsequent pregnancies,
at risk for hemolytic disease of the newborn.Rho(D) immune globulin antibodies are specific for
human RhD antigen. Anti-RhD antibodies are administered as part
of a prenatal treatment regimen to prevent sensitization that may occur when a Rh-negative
mother has a Rh-positive fetus. Treatment of a mother with Anti-RhD antibodies
prior to and immediately after trauma and delivery destroys Rh antigen in the mother’s
system from the fetus. It is important to note that this occurs before
the antigen can stimulate maternal B cells to “remember” Rh antigen by generating memory
B cells. Therefore, her humoral immune system will
not make anti-Rh antibodies, and will not attack the Rh antigens of the current or subsequent
babies. Rho(D) Immune Globulin treatment prevents
sensitization that can lead to Rh disease, but does not prevent or treat the underlying
disease itself.==Research applications==Specific antibodies are produced by injecting
an antigen into a mammal, such as a mouse, rat, rabbit, goat, sheep, or horse for large
quantities of antibody. Blood isolated from these animals contains
polyclonal antibodies—multiple antibodies that bind to the same antigen—in the serum,
which can now be called antiserum. Antigens are also injected into chickens for
generation of polyclonal antibodies in egg yolk. To obtain antibody that is specific for a
single epitope of an antigen, antibody-secreting lymphocytes are isolated from the animal and
immortalized by fusing them with a cancer cell line. The fused cells are called hybridomas, and
will continually grow and secrete antibody in culture. Single hybridoma cells are isolated by dilution
cloning to generate cell clones that all produce the same antibody; these antibodies are called
monoclonal antibodies. Polyclonal and monoclonal antibodies are often
purified using Protein A/G or antigen-affinity chromatography.In research, purified antibodies
are used in many applications. Antibodies for research applications can be
found directly from antibody suppliers, or through use of a specialist search engine. Research antibodies are most commonly used
to identify and locate intracellular and extracellular proteins. Antibodies are used in flow cytometry to differentiate
cell types by the proteins they express; different types of cell express different combinations
of cluster of differentiation molecules on their surface, and produce different intracellular
and secretable proteins. They are also used in immunoprecipitation
to separate proteins and anything bound to them (co-immunoprecipitation) from other molecules
in a cell lysate, in Western blot analyses to identify proteins separated by electrophoresis,
and in immunohistochemistry or immunofluorescence to examine protein expression in tissue sections
or to locate proteins within cells with the assistance of a microscope. Proteins can also be detected and quantified
with antibodies, using ELISA and ELISpot techniques.Antibodies used in research are some of the most powerful,
yet most problematic reagents with a tremendous number of factors that must be controlled
in any experiment including cross reactivity, or the antibody recognizing multiple epitopes
and affinity, which can vary widely depending on experimental conditions such as pH, solvent,
state of tissue etc. Multiple attempts have been made to improve
both the way that researchers validate antibodies and ways in which they report on antibodies. Researchers using antibodies in their work
need to record them correctly in order to allow their research to be reproducible (and
therefore tested, and qualified by other researchers). Less than half of research antibodies referenced
in academic papers can be easily identified. Papers published in F1000 in 2014 and 2015
provide researchers with a guide for reporting research antibody use. The RRID paper, is co-published in 4 journals
that implemented the RRIDs Standard for research resource citation, which draws data from the
antibodyregistry.org as the source of antibody identifiers (see also group at Force11).==Regulations=====
Production and testing===Traditionally, most antibodies are produced
by hybridoma cell lines through immortalization of antibody-producing cells by chemically-induced
fusion with myeloma cells. In some cases, additional fusions with other
lines have created “triomas” and “quadromas”. The manufacturing process should be appropriately
described and validated. Validation studies should
at least include: The demonstration that the process is able
to produce in good quality (the process should be validated) The efficiency of the antibody purification
(all impurities and virus must be eliminated) The characterization of purified antibody
(physicochemical characterization, immunological properties, biological activities, contaminants,
…) Determination of the virus clearance studies===
Before clinical trials===Product safety testing: Sterility (bacteria
and fungi), In vitro and in vivo testing for adventitious viruses, Murine retrovirus testing… Product safety data needed before the initiation
of feasibility trials in serious or immediately life-threatening conditions, it serves to
evaluate dangerous potential of the product. Feasibility testing: These are pilot studies
whose objectives include, among others, early characterization of safety and initial proof
of concept in a small specific patient population (in vitro or in vivo testing).===Preclinical studies===
Testing cross-reactivity of antibody: to highlight unwanted interactions (toxicity) of antibodies
with previously characterized tissues. This study can be performed in vitro (Reactivity
of the antibody or immunoconjugate should be determined with a quick-frozen adult tissues)
or in vivo (with appropriates animal models). More information about in vitro cross-reactivity
testing. Preclinical pharmacology and toxicity testing:
Preclinical safety testing of antibody is designed to identify possible toxicity in
humans, to estimate the likelihood and severity of potential adverse events in humans, and
to identify a safe starting dose and dose escalation, when possible. Animal toxicity studies: Acute toxicity testing,
Repeat-dose toxicity testing, Long-term toxicity testing Safety & Testing | ari.info
Pharmacokinetics and pharmacodynamics testing: Use for determinate clinical dosages, antibody
activities (AUC, pharmacodynamics, biodistribution, …), evaluation of the potential clinical
effects==
Structure prediction and computational antibody design==
The importance of antibodies in health care and the biotechnology industry demands knowledge
of their structures at high resolution. This information is used for protein engineering,
modifying the antigen binding affinity, and identifying an epitope, of a given antibody. X-ray crystallography is one commonly used
method for determining antibody structures. However, crystallizing an antibody is often
laborious and time-consuming. Computational approaches provide a cheaper
and faster alternative to crystallography, but their results are more equivocal, since
they do not produce empirical structures. Online web servers such as Web Antibody Modeling
(WAM) and Prediction of Immunoglobulin Structure (PIGS) enables computational modeling of antibody
variable regions. Rosetta Antibody is a novel antibody FV region
structure prediction server, which incorporates sophisticated techniques to minimize CDR loops
and optimize the relative orientation of the light and heavy chains, as well as homology
models that predict successful docking of antibodies with their unique antigen.The ability
to describe the antibody through binding affinity to the antigen is supplemented by information
on antibody structure and amino acid sequences for the purpose of patent claims. Several methods have been presented for computational
design of antibodies based on the structural bioinformatics studies of antibody CDRs.There
are a variety of methods used to sequence an antibody including Edman degradation, cDNA,
etc.; albeit one of the most common modern uses for peptide/protein identification is
liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). High volume antibody sequencing methods require
computational approaches for the data analysis, including de novo sequencing directly from
tandem mass spectra and database search methods that use existing protein sequence databases. Many versions of shotgun protein sequencing
are able to increase the coverage by utilizing CID/HCD/ETD fragmentation methods and other
techniques, and they have achieved substantial progress in attempt to fully sequence proteins,
especially antibodies. Other methods have assumed the existence of
similar proteins, a known genome sequence, or combined top-down and bottom up approaches. Current technologies have the ability to assemble
protein sequences with high accuracy by integrating de novo sequencing peptides, intensity, and
positional confidence scores from database and homology searches.==Antibody mimetic==
Antibody mimetics are organic compounds, like antibodies, that can specifically bind antigens. They are usually artificial peptides or proteins
with a molar mass of about 3 to 20 kDa. Nucleic acids and small molecules are sometimes
considered antibody mimetics, but not artificial antibodies, antibody fragments and fusion
proteins are composed from these. Common advantages over antibodies are better
solubility, tissue penetration, stability towards heat and enzymes, and comparatively
low production costs. Antibody mimetics such as the Affimer and
the DARPin have being developed and commercialised as research, diagnostic and therapeutic agents.==See also

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