Antibodies are large Y-shaped proteins. They are recruited by the immune system to identify and neutralize foreign objects like bacteria and viruses.
Each antibody has a unique target known as the antigen present on the invading organism. This antigen is like a key that helps the antibody in identifying the organism. This is because both the antibody and the antigen have similar structure at the tips of their “Y” structures.
Just like every lock has a single key, an antibody has a single antigen key. When the key is inserted into the lock, the antibody activates, tagging or neutralizing its target. The production of antibodies is the main function of the humoral immune system.
Immunoglobulins are basically proteins that function as antibodies. The terms antibody and immunoglobulin are often used interchangeably.
Immunoglobulins are found in blood and other tissues and fluids. They are made by the plasma cells that are derived from the B cells of the immune system. B cells of the immune system become plasma cells when activated by the binding of a specific antigen on its antibody surfaces. In some cases, the interaction of the B cell with a T helper cell is also necessary.
Antigens are classically defined as any foreign substance that elicits an immune response. They are also called immunogens. The specific region on an antigen that an antibody recognizes and binds to is called the epitope, or antigenic determinant.
An epitope is usually made up of a 5-8 amino acid long chain on the surface of the protein. The chain of amino acids does not exist in a 2 dimensional structure but appears as a 3 dimensional structure. An epitope may only be recognized in its form as it exists in solution, or its native 3D form. If the epitope exists on a single polypeptide chain, it is a continuous, or linear epitope. The antibody may bind to only fragments or denatured segments of a protein or to the native basic protein.
Serum containing antigen-specific antibodies is called antiserum. There are five classes of immunoglobulins including IgM, IgG, IgA, IgD, and IgE.
The basic structure of all antibodies are same. There are four polypeptide chains held together by disulfide bonds. These four polypeptide chains form a symmetrical molecular structure.
There are two identical halves with the antigen binding sites between the ends of the heavy and light chains on both sides. There is a hinge in the center between heavy chains to allow flexibility to the protein. The two light chains are identical to each other. They contain around 220 amino acids while the heave chains have 440 amino acids.
There are two types of light chain among all classes of immunoglobulin, a lambda chain and a kappa chain. Both are similar in function. Each type of immunoglobulin has a different type of heavy chain.
The antibody binds to specific antigens. This signals the other cells of the immune system to get rid of the invading microbes. The strength of binding between the antibody and an antigen at a single binding site is known as the antibody’s affinity for the antigen. The affinity between the antibody and the antigen binding site is determined by the type of bond formed.
Since an antigen can have multiple different epitopes, a number of antibodies can bind to the protein. When two or more antigen binding sites are identical, an antibody can form a stronger bond with the antigen.
Antibodies or immunoglobulins come in a variety of forms. Based on differences in the amino acid sequences at the constant region of the heavy chains they are further classified into five classes. These are:
Each of the forms has a small difference in the constant region of the heavy chain. Based on the differences the Igs are classified into subclasses. These are detected by serological means.
The subclasses include:
The Immunoglobulins are further classified by the type of light chain that they have. Light chain types are based on differences in the amino acid sequence in the constant region of the light chain. There are two types of light chains – Kappa and Lambda side chains.
Based on the light chains there are further subtypes. For example the Lambda subtypes include:
a) Lambda 1
b) Lambda 2
c) Lambda 3
d) Lambda 4
These are monomeric structures that exist as single molecules. These are the most versatile immunoglobulins and can carry out all functions of Ig molecules. This forms the largest portion in the serum and is also found in extravascular spaces. This is the only immunoglobulin that crosses the placenta. It also fixes molecules called complements. It binds to cells and enhances phagocytosis.
These are also monomeric structures. They are found in secretions as a dimer having a J chain. IgA can move across mucosa without degradation. It is the second most abundant Ig in serum. It is the major class of Ig in secretions i.e. in tears, saliva, colostrum (initial breast milk), mucus etc. and is important in mucosal immunity. It binds to PMN cells and lymphocytes. It does not normally fix complement.
These have an extra domain on the mu chain (CH4) and another protein covalently bound via S-S. These exist is J shapes as polymers. Usually they form pentamers or clusters of 5. It is the first Ig to be made by fetus. It is the third most abundant Ig in serum. It fixes with complements and is a good agglutinating Ig that leads to elimination of microbes. It is also able to bind some cells via Fc receptors.
These exist as monomers. They have low serum levels. It is found primarily on B cells surface and serves as a receptor for antigens. It does not fix complement.
Antibodies are used extensively as diagnostic tools in many different formats. The term applied for antibody based diagnostic tests is “immunoassay”. Antibody-based immunoassays are the most commonly used confirmatory diagnostic assays and is the fastest growing technologies for the analysis of biomolecules.
Examples of immunoassay include titer of antibodies directed against Epstein-Barr virus or Lyme disease estimated from the blood. In absence of these antibodies it is presumed that 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.
For immunoassays, levels of individual classes of immunoglobulins are measured by nephelometry (or turbidimetry) to characterize the antibody profile of patient. The Coombs test is also used for antibody screening in blood transfusion preparation. This test is used for antibody screening in antenatal women as well. Immunoassays are used in multiple sclerosis, psoriasis, and many forms of cancer including non-Hodgkin's lymphoma, colorectal cancer, head and neck cancer and breast cancer.
Then there is the use of radiolabelled antibodies that can be used in the diagnosis of diseases as well. These radiolabelled antibodies are used for diagnosis or detection of whole cells, receptors and enzymes. There is also enzyme labelled immune assays.
In the past, most immunoassays were based on polyclonal antisera drawn from immunized rabbits, which provided a good immune response despite having limited antigens. This changed with the advent of monoclonal antibodies, as described in 1975 by Köhler and Milstein.
Monoclonal antibodies revolutionized the use of antibodies in therapy called immunotherapeutics. Monoclonal antibodies have become a large part of immunodiagnostics as well.
Trends in antibody based diagnosis show advances in assay specificity, detection technologies and sensitivity. Sensitivity and specificity is ensured depending on whether or not the antigen to be quantified competes with labelled antigen for a limited number of antibody binding sites.
Another important new technology with particular importance in diagnostics is flow cytometric analysis. This uses many new monoclonal antibodies against different cell-surface structures and helps flow cytometry in diagnosis if blood cancers. Flow cytometry has also more recently been used in the monitoring of disease and in the evaluation of tumor response to therapy.
As of 2011, 35 monoclonal antibody preparations have been approved by the U.S. Food and Drug Administration for use in humans. Some of these include:
Rhesus factor, also known as Rhesus D (RhD) antigen, is an antigen found on red blood cells. Presence of the antigen makes a person Rhesus-positive (Rh+) and absence makes a person Rhesus-negative (Rh–). 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, there may be sensitization of an Rh- mother to the Rh antigen on the blood cells of the Rh+ child. This may put the remainder of the pregnancy, and any subsequent pregnancies at risk of fetal death due to hemolysis.
For treatment Rho antibodies (specific for human Rhesus D (RhD) antigen) are used. Anti-RhD antibodies are administered as part of a prenatal treatment regimen to prevent sensitization that may occur when a Rhesus-negative mother has a Rhesus-positive fetus.
Antibodies are also used in structure prediction. 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. This, however, is a difficult process. Computational approaches provide a cheaper and faster alternative to crystallography but results are more equivocal.
Antibodies are thus methods that can be used to predict structures of biomolecules. Online web servers such as ''Web Antibody Modeling'' (WAM) and ''Prediction of Immunoglobulin Structure'' (PIGS) enables computational modeling of antibody variable regions.
Antibodies are proteins with around 150 kDa molecular weight. They have a similar basic structure comprising of four polypeptide chains held together by disulfide bonds. These four polypeptide chains form a symmetrical molecular structure. There is a hinge in the center between heavy chains to allow flexibility to the protein. There are:
There are two types of light chain among all classes of immunoglobulin, a lambda chain and a kappa chain. Both are similar in function. Each type of immunoglobulin has a different type of heavy chain. Depending on the heavy chains they are classified into five classes.
Apart from amino acids there are sugar molecules as well. Thus antibodies are glycoproteins rather than proteins alone. The immunoglobulins exists as monomers (e.g. only one Ig unit) or as dimmers (two molecules. E.g. IgA) or as tetramers (four molecules e.g. teleost fish IgM) or exists as pentamers (five molecules e.g. in mammalian IgM)
Immunoglobulin fragments produced by proteolytic digestion by enzymes are the basis of study of structure/function relationships.
When Ig are broken down with papain it breaks at the hinge region before the H-H inter-chain disulfide bond; this results in the formation of two identical fragments that contain the light chain and the VH (Variable Heavy chain) and CH1 (Constant heavy chain) domains of the heavy chain. These fragments are then called Fab since they contained the antigen binding sites of the antibody. Each Fab fragment is monovalent.
Digestion with papain also leads to formation of a fragment that contains rest of the two heavy chains each containing a CH2 and CH3 domain. This fragment is easily crystallized.
When digested with pepsin, the Igs are cleaved at the heavy chain after the H-H inter-chain disulfide bonds. The resulting fragments contain both antigen binding sites. This fragment was called F(ab')2 because it is divalent. This can bind to the antigens but does not lead to effector functions.
The Ig monomer is a "Y"-shaped molecule. It has four polypeptide chains - two identical ''heavy chains'' and two identical ''light chains''. There are five types of mammalian Ig heavy chain denoted by the Greek letters: α, δ, ε, γ, and μ. These form respectively IgA, IgD, IgE, IgG and IgM.
The Ig has a paratope at the amino terminal end of the antibody monomer. This exists at the variable domains from the heavy and light chains. The variable domain is the FV region and is the most important region for binding to antigens. At this region are variable loops of β-strands. On the light chain are three loops - VL and on the heavy chain are three loops VH. These are responsible for binding to the antigen. These loops are referred to as the complementarity determining regions (CDRs). These CDRs are also called idiotypes.
The base of the Y modulates the immune cell activity. This region is called the ''Fc (Fragment, crystallizable) region''. In this area are two heavy chains that contribute two or three constant domains depending on the class of the antibody.
Antibodies play an important role in the immune system. The immunoglobulins present on the B-lymphocyte surface send in signals to the cytoplasmic and nuclear electors. These also deliver the antigen to the cell where it can be destroyed, processed and returned to the cell surface to be presented by MHC class II molecules to antigen-speciﬁc T helper cells.
The T lymphocytes in turn send signals to the B cells for them to mature and recognize the antigens and create antibodies targeted specifically against it.
Antibodies secreted by B lymphocytes are responsible for the humoral immune response. The humoral immune system helps in destroying external pathogens and prevents spread of intracellular infections. This immune system also protects against toxins.
The two structural portions of the antibody, i.e. the variable (Fab) and the constant (Fc) fragments, impart distinct biological functions.
These functions are outlined as follows:
Fc-mediated effector functions:
Antibodies function in the body as a double-edged sword. With one edge they protect the body from microbes and with the other they can cause severe allergic reactions to relatively harmless proteins and other molecules present in food, environment, medicines etc.
IgE is the most important mediator of hypersensitivity or allergic reactions. When it binds to multivalent antigens there is activation of the mast cell, which releases chemical mediators stored in granules and capable of mediating local inﬂammatory reactions. This is called mast cell degranulation.
All microbes trigger an antibody response. Due to diversity in microbes, the antibody needs to adopt variations to allow their interactions with many different antigens.
Human, for example, generate about 10 billion different antibodies. Each of these is specific for distinct epitope of an antigen. Since each of the antibodies are different for each of the antigens, the body needs to be capable for generating these proteins.
The genes coding for these diverse range of immunoglobulins however are limited and do not number similar to the variety of antibodies. To create the variety of antibodies thus the body adopts complex mechanisms from the relatively small number of antibody genes.
Each of the genes for the antibodies are located on specific location (loci) on the chromosomes. The locus for antibodies is a relatively large segment. There are several distinct genes for each domain of the antibody. There is a locus for the heavy chain genes that is found on chromosome 14 and a locus for lambda and kappa light chain genes that is found on chromosomes 22 and 2 respectively in humans.
One of these regions is called the variable domain. This is present in each heavy and light chain of every antibody, but can differ in different antibodies generated from distinct B cells.
The variable domains are located on three loops known as hypervariable regions (HV-1, HV-2 and HV-3). These are also called complementarity determining regions (CDR1, CDR2 and CDR3). The loci for the heavy chains has around 65 different variable domain genes. These differ in their CDRs. When combined, these genes can yield a large variety of combinations for antibodies by permutation and combination.
The recombination of these genes is called ''V(D)J recombination''. This means generation of distinctly different antibodies due to different variable domains on the antibodies. The variable region of each immunoglobulin heavy or light chain is encoded in several pieces on the genes. These are called variable (V), diversity (D) and joining (J) segments.
Isotypes are called class switching. Once the B cells are activated they produce different classes of antibody (IgA, IgE, or IgG). When the heavy chain gene locus undergoes a phenomenon called class switch recombination (CSR), it leads to formation of isotypes. This process results in an immunoglobulin gene that encodes an antibody of a different isotype.
The plasma cells switch from producing IgM to IgG or to another immunoglobulin class. The switch involves a change in the H chain constant domains (CH). In this there is usually no alteration in the L chain or in the variable portion of the H chain and thus there is no change in antigen-binding specificity.
When the antigen binds to the antibody and activates the B cells, they rapidly proliferate and each of these cells contains DNA for antibody formation. The genes coding for the antibodies in these B cells undergo high rate of point mutation or point changes in genetic codes for antibodies. This is called ''somatic hypermutation'' (SHM).
Each SHM results in one nucleotide change per variable gene with each cell division. This mutation leads to formation of a variety of antibodies specific for the antigen. While some of the generated antibodies are weak, some have a stronger affinity for the antigen. Those B cells that produce these strong antibodies as a result of the mutations are preserved and proliferated while the others die off.
The earliest reference to antibodies was from Emil von Behring along with Kitasato Shibasaburo in 1890 who found the presence of a neutralizing substance in the blood that could counter infections. They developed the serum against diphtheria. This they did by transferring serum produced from animals immunized against diphtheria to animals suffering from it. This serum could cure the infected animals. Behring was awarded the Nobel Prize for this work in 1901.
In 1900 Paul Ehrlich hypothesized that there are side chain receptors on cells that bind to a given pathogen. He speculated that this interaction induces the cell exhibiting the receptor to multiply and produce more copies of the same receptor. This theory, called the selective theory was not proven for next five decades.
Between 1901-1920, Landsteiner demonstrated the ABO blood group system (Rh antibody was found in in 1940). In the 1920s, Michael Heidelberger and Oswald Avery observed that antigens could be precipitated by antibodies and went on to show that antibodies were made of protein.
The chemical nature of antibodies was still not known. The biochemical properties of antigen-antibody binding interactions were examined in more detail in the late 1930s by John Marrack. In the next few decades that followed it could be shown that the protective serum could neutralize and precipitate toxins, and clump bacteria. The biomolecule responsible for these actions was termed antitoxin, precipitin and agglutinin.
It was not known that all the three substances were one entity. This was later shown by Elvin A. Kabat in 1939. Kabat in 1938 had also shown heterogeneity of antibodies through ultracentrifugation studies of horses' sera.
Thereafter cell-mediated immunity was found and recognized as different from humoral immunity in 1942 when Merrill Chase successfully transferred immunity against tuberculosis between pigs by transferring white blood cells.
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 depended more on their shape than their chemical composition.
In 1948 Astrid Fagraeus in her doctoral thesis demonstrated that plasma B cells are specifically involved in antibody production. James Gowans in 1959 showed that lymphocytes had a role in mediating both cell-mediated and humoral responses.
Jerne, Talmage and Burnet in the late 1950’s found the clonal selection theory. This proved all the elements of Ehrlich's hypothesis except that the specific receptors that could neutralize the agent were soluble and free and not membrane bound. This was further proven by Sir Gustav Nossal who showed that one clone of B cell always produces only one antibody.
In the 1960’s Edelman, Porter, and Hilschmann elucidated the primary and secondary structure of antibodies. They also found that Bence-Jones proteins were immunoglobulin L-chains. Thomas Tomasi discovered secretory antibody(IgA) and David Rowe and John Fahey identified IgD, and IgE was identified by Kikishige Ishizaka and Teruki Ishizaka as a class of antibodies involved in allergic reactions. In 1974 the role of MHC in antigen presentation was demonstrated by Rolf Zinkernagel and Peter C. Doherty.
In 1975, Kohler and Milstein found the key to monoclonal antibodies. These were the magic bullets that were derived from the progeny of a single immune cell. These were pure and available in potentially unlimited quantities.
Several have been found which recognise human cancers and some of these have been tested in clinical trials. In 1976 Susumu Tonegawa cloned the first antibody gene.