IMMUNOLOGY

Monoclonal antibodies

Monoclonal antibodies (mAb or moAb) are monospecific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell, in contrast to polyclonal antibodies which are made from several different immune cells. Monoclonal antibodies have monovalent affinity, in that they bind to the same epitope.

Given almost any substance, it is possible to produce monoclonal antibodies that specifically bind to that substance; they can then serve to detect or purify that substance. This has become an important tool in biochemistry, molecular biology and medicine. When used as medications, the non-proprietary drug name ends in -mab (see "Nomenclature of monoclonal antibodies").

Production

Hybridoma cell production

Monoclonal antibodies are typically made by fusing myeloma cells with the spleen cells from a mouse that has been immunized with the desired antigen. However, recent advances have allowed the use of rabbit B-cells to form a Rabbit Hybridoma. Polyethylene glycol is used to fuse adjacent plasma membranes, but the success rate is low so a selective medium in which only fused cells can grow is used.

Purification of monoclonal antibodies

After obtaining either a media sample of cultured hybridomas or a sample of ascites fluid, the desired antibodies must be extracted. The contaminants in the cell culture sample would consist primarily of media components such as growth factors, hormones, and transferrins. In contrast, the in vivo sample is likely to have host antibodies, proteases, nucleases, nucleic acids, and viruses.

Antibody heterogeneity

Product heterogeneity is common to monoclonal antibody and other recombinant biological production and is typically introduced either upstream during expression or downstream during manufacturing.

Recombinant

The production of recombinant monoclonal antibodies involves technologies, referred to as repertoire cloning or phage display/yeast display. Recombinant antibody engineering involves the use of viruses or yeast to create antibodies, rather than mice. These techniques rely on rapid cloning of immunoglobulin gene segments to create libraries of antibodies with slightly different amino acid sequences from which antibodies with desired specificities can be selected.

Chimeric antibodies

Early on, a major problem for the therapeutic use of monoclonal antibodies in medicine was that initial methods used to produce them yielded mouse, not human antibodies. While structurally similar, differences between the two are sufficient to invoke an immune response occurred when murine monoclonal antibodies were injected into humans and resulted in their rapid removal from the blood, systemic inflammatory effects, and the production of human anti-mouse antibodies (HAMA).[citation needed]

Applications

Diagnostic tests

Once monoclonal antibodies for a given substance have been produced, they can be used to detect the presence of this substance. The Western blot test and immuno dot blot tests detect the protein on a membrane. They are also very useful in immunohistochemistry, which detect antigen in fixed tissue sections and immunofluorescence test, which detect the substance in a frozen tissue section or in live cells.

Therapeutic treatment

Cancer treatment

One possible treatment for cancer involves monoclonal antibodies that bind only to cancer cell-specific antigens and induce an immunological response against the target cancer cell. Such mAb could also be modified for delivery of a toxin, radioisotope, cytokine or other active conjugate; it is also possible to design bispecific antibodies that can bind with their Fab regions both to target antigen and to a conjugate or effector cell. In fact, every intact antibody can bind to cell receptors or other proteins with its Fc region.

Autoimmune diseases

Monoclonal antibodies used for autoimmune diseases include infliximab and adalimumab, which are effective in rheumatoid arthritis, Crohn's disease and ulcerative Colitis by their ability to bind to and inhibit TNF-α.[30] Basiliximab and daclizumab inhibit IL-2 on activated T cells and thereby help prevent acute rejection of kidney transplants.[30] Omalizumab inhibits human immunoglobulin E (IgE) and is useful in moderate-to-severe allergic asthma.

HYPERSENSITIVITY REACTIONS  

Hypersensitivity (also called hypersensitivity reaction) refers to undesirable reactions produced by the normal immune system, including allergies and autoimmunity. These reactions may be damaging, uncomfortable, or occasionally fatal.Hypersensitivity refers to excessive, undesirable (damaging, discomfort-producing and sometimes fatal) reactions produced by the normal immune system. Hypersensitivity reactions require a pre-sensitized (immune) state of the host. Hypersensitivity reactions can be divided into four types: type I, type II, type III and type IV, based on the mechanisms involved and time taken for the reaction. Frequently, a particular clinical condition (disease) may involve more than one type of reaction.

 TYPE I HYPERSENSITIVITY

Type I hypersensitivity is also known as immediate or anaphylactic hypersensitivity. The reaction may involve skin (urticaria and eczema), eyes (conjunctivitis), nasopharynx (rhinorrhea, rhinitis), bronchopulmonary tissues (asthma) and gastrointestinal tract (gastroenteritis). The reaction may cause a range of symptoms from minor inconvenience to death. The reaction usually takes 15 - 30 minutes from the time of exposure to the antigen, although sometimes it may have a delayed onset (10 - 12 hours).

Immediate hypersensitivity is mediated by IgE. The primary cellular component in this hypersensitivity is the mast cell or basophil. The reaction is amplified and/or modified by platelets, neutrophils and eosinophils. A biopsy of the reaction site demonstrates mainly mast cells and eosinophils.

The mechanism of reaction involves preferential production of IgE, in response to certain antigens (often called allergens). The precise mechanism as to why some individuals are more prone to type-I hypersensitivity is not clear. However, it has been shown that such individuals preferentially produce more of TH2 cells that secrete IL-4, IL-5 and IL-13 which in turn favor IgE class switch. IgE has very high affinity for its receptor (Fcε; CD23) on mast cells and basophils.

A subsequent exposure to the same allergen cross links the cell-bound IgE and triggers the release of various pharmacologically active substances (figure 1). Cross-linking of IgE Fc-receptor is important in mast cell triggering. Mast cell degranulation is preceded by increased Ca++ influx, which is a crucial process; ionophores which increase cytoplasmic Ca++ also promote degranulation, whereas, agents which deplete cytoplasmic Ca++ suppress degranulation.

TYPE II HYPERSENSITIVITY

Type II hypersensitivity is also known as cytotoxic hypersensitivity and may affect a variety of organs and tissues. The antigens are normally endogenous, although exogenous chemicals (haptens) which can attach to cell membranes can also lead to type II hypersensitivity. Drug-induced hemolytic anemia, granulocytopenia and thrombocytopenia are such examples. The reaction time is minutes to hours. Type II hypersensitivity is primarily mediated by antibodies of the IgM or IgG classes and complement (Figure 2). Phagocytes and K cells may also play a role.

The lesion contains antibody, complement and neutrophils. Diagnostic tests include detection of circulating antibody against the tissues involved and the presence of antibody and complement in the lesion (biopsy) by immunofluorescence. The staining pattern is normally smooth and linear, such as that seen in Goodpasture's nephritis (renal and lung basement membrane) (figure 3A) and pemphigus (skin intercellular protein, desmosome) (figure 3B).

Treatment involves anti-inflammatory and immunosuppressive agents.

 TYPE III HYPERSENSITIVITY

Type III hypersensitivity is also known as immune complex hypersensitivity. The reaction may be general (e.g., serum sickness) or may involve individual organs including skin (e.g., systemic lupus erythematosus, Arthus reaction), kidneys (e.g., lupus nephritis), lungs (e.g., aspergillosis), blood vessels (e.g., polyarteritis), joints (e.g., rheumatoid arthritis) or other organs. This reaction may be the pathogenic mechanism of diseases caused by many microorganisms.

The reaction may take 3 - 10 hours after exposure to the antigen (as in Arthus reaction). It is mediated by soluble immune complexes. They are mostly of the IgG class, although IgM may also be involved. The antigen may be exogenous (chronic bacterial, viral or parasitic infections), or endogenous (non-organ specific autoimmunity: e.g., systemic lupus erythematosus, SLE). The antigen is soluble and not attached to the organ involved. Primary components are soluble immune complexes and complement (C3a, 4a and 5a). The damage is caused by platelets and neutrophils (Figure 4). The lesion contains primarily neutrophils and deposits of immune complexes and complement. Macrophages infiltrating in later stages may be involved in the healing process.

TYPE IV HYPERSENSITIVITY

Type IV hypersensitivity is also known as cell mediated or delayed type hypersensitivity. The classical example of this hypersensitivity is tuberculin (Montoux) reaction (figure 5) which peaks 48 hours after the injection of antigen (PPD or old tuberculin). The lesion is characterized by induration and erythema.

Type IV hypersensitivity is involved in the pathogenesis of many autoimmune and infectious diseases (tuberculosis, leprosy, blastomycosis, histoplasmosis, toxoplasmosis, leishmaniasis, etc.) and granulomas due to infections and foreign antigens. Another form of delayed hypersensitivity is contact dermatitis (poison ivy (figure 6), chemicals, heavy metals, etc.) in which the lesions are more papular. Type IV hypersensitivity can be classified into three categories depending on the time of onset and clinical and histological presentation (Table 3).

Mechanisms of damage in delayed hypersensitivity include T lymphocytes and monocytes and/or macrophages. Cytotoxic T cells (Tc) cause direct damage whereas helper T (TH1) cells secrete cytokines which activate cytotoxic T cells and recruit and activate monocytes and macrophages, which cause the bulk of the damage (figure 4). The delayed hypersensitivity lesions mainly contain monocytes and a few T cells.

Erythroblastosis foetalis

Hemolytic disease of the newborn, also known as hemolytic disease of the fetus and newborn, HDN, HDFN, or erythroblastosis fetalis,[1] is an alloimmune condition that develops in a fetus, when the IgG molecules (one of the five main types of antibodies) produced by the mother pass through the placenta. Among these antibodies are some which attack the red blood cells in the fetal circulation; the red cells are broken down and the fetus can develop reticulocytosis and anemia. This fetal disease ranges from mild to very severe, and fetal death from heart failure (hydrops fetalis) can occur. When the disease is moderate or severe, many erythroblasts are present in the fetal blood and so these forms of the disease can be called erythroblastosis fetalis (or erythroblastosis foetalis).

Causes

Antibodies are produced when the body is exposed to an antigen foreign to the make-up of the body. If a mother is exposed to a foreign antigen and produces IgG (as opposed to IgM which does not cross the placenta), the IgG will target the antigen, if present in the fetus, and may affect it in utero and persist after delivery. The three most common models in which a woman becomes sensitized toward (i.e., produces IgG antibodies against) a particular antigen are:

• Fetal-maternal hemorrhage can occur due to trauma, abortion, childbirth, ruptures in the placenta during pregnancy, or medical procedures carried out during pregnancy that breach the uterine wall. In subsequent pregnancies, if there is a similar incompatibility in the fetus, these antibodies are then able to cross the placenta into the fetal bloodstream to attach to the red blood cells and cause hemolysis. In other words, if a mother has anti-RhD (D being the major Rhesus antigen) IgG antibodies as a result of previously carrying a RhD-positive fetus, this antibody will only affect a fetus with RhD-positive blood.

• The woman may have received a therapeutic blood transfusion. ABO blood group system and the D antigen of the Rhesus blood group system typing are routine prior to transfusion. Suggestions have been made that women of child bearing age or young girls should not be given a transfusion with Rhc-positive blood or Kell1-positive blood to avoid possible sensitization, but this would strain the resources of blood transfusion services, and it is currently considered uneconomical to screen for these blood groups. HDFN can also be caused by antibodies to a variety of other blood group system antigens, but Kell and Rh are the most frequently encountered.

The third sensitization model can occur in women of blood type O. The immune response to A and B antigens, that are widespread in the environment, usually leads to the production of IgM anti-A and IgM anti-B antibodies early in life.

Recombinant Vaccines

A biological preparation, which evokes an immune response when administered into the body, is termed as vaccines. This usually consists of parts of pathogen in its weakened state or its products. This triggers an immune response from the body to the particular disease without actually causing the disease.

Catering to the needs of large number of diseases, numerous vaccines for a variety of diseases has been developed and still continues to do so.

Recombinant vaccines:
Biotechnology sector has also played its part in developing vaccines against certain diseases. Such vaccine which makes use of recombinant DNA technology is known as recombinant vaccines. It is also known as subunit vaccines.

Recombinant vaccines can be broadly grouped into two kinds:

(i) Recombinant protein vaccines: This is based on production of recombinant DNA which is expressed to release the specific protein used in vaccine preparation

(ii) DNA vaccines: Here the gene encoding for immunogenic protein is isolated and used to produce recombinant DNA which acts as vaccine to be injected into the individual.

Steps involved:
Production of recombinant vaccines involves the following steps:

(i) First and foremost, it is important that the protein which is crucial to the growth and development of the causative organism be identified.

(ii) The corresponding gene is then isolated applying various techniques. Further to this, an extensive study of the gene explains the gene expression pattern involved in the production of corresponding protein.

(iii) This gene is then integrated into a suitable expression vector to produce a recombinant DNA.

(iv) This rDNA is used as vaccines or is introduce into another host organism to produce immunogenic proteins which acts as vaccines.

Recombinant protein vaccines:
A pathogen upon infection produces proteins, vital for its functions, which elicit an immune response from the infected body. The gene encoding such a protein is isolated from the causative organism and used to develop a recombinant DNA. This DNA is expressed in another host organism, like genetically engineered microbes; animal cells; plant cells; insect larvae etc, resulting in the release of the appropriate proteins which are then isolated and purified. These when injected into the body, causes immunogenic response to be active against the corresponding disease providing immunity against future attack of the pathogen.
Based on the proteins involved in evoking immune response recombinant protein vaccines are of two types:

Whole protein vaccines: The whole immunogenic protein is produced in another host organism which is isolated and purified to act as vaccines.

Polypeptide vaccines: It is known that in the immunogenic protein produced, the actual immunogenic property is limited to one or two polypeptides forming the protein. The other parts of the protein may be successful in evoking an immune response but do not actually cause the disease. For eg: in the case of cholera caused by Vibrio cholerae, consists of three polypeptide chains like A1, A2, and B. The A polypeptides are toxic while B is non-toxic. Thus while producing vaccines, the polypeptide B is produced by rDNA technology and used for vaccination.

DNA vaccines:
It refers to the recombinant vaccines in which the DNA is used as a vaccine. The gene responsible for the immunogenic protein is identified, isolated and cloned with corresponding expression vector. Upon introduction into the individuals to be immunized, it produces a recombinant DNA. This DNA when expressed triggers an immune response and the person becomes successfully vaccinated. The mode of delivery of DNA vaccines include: direct injection into muscle; use of vectors like adenovirus, retrovirus etc; invitro transfer of the gene into autologous cells and reimplantation of the same and particle gun delivery of the DNA.
In certain cases, the responsible gene is integrated into live vectors which are introduced into individuals as vaccines. This is known as live recombinant vaccines. Eg: vaccinia virus. Live vaccinia virus vaccine (VV vaccine) with genes corresponding to several diseases, when introduced into the body elicit an immune response but does not actually cause the diseases.

Advantages:
(i) Since it does not involve actual pathogen, recombinant vaccines is considered to be safe than the conventional vaccines.
(ii) It induces both humoral and cellular immune response resulting in effective vaccination.

Major histocompatibility complex

Major histocompatibility complex (MHC) is a cell surface molecule encoded by a large gene family in all vertebrates. MHC molecules mediate interactions of leukocytes, also called white blood cells (WBCs), which are immune cells, with other leukocytes or body cells. MHC determines compatibility of donors for organ transplant as well as one's susceptibility to an autoimmune disease via crossreacting immunization. In humans, MHC is also called human leukocyte antigen (HLA).

Protein molecules—either of the host's own phenotype or of other biologic entities—are continually synthesized and degraded in a cell. Occurring on the cell surface, each MHC molecule displays a molecular fraction, called epitope, of a protein, somewhat like a hot dog (epitope) within a bun (MHC).[1] The presented antigen can be either self or nonself. On the cell membrane, the MHC population in its entirety is like a meter indicating the balance of proteins within the cell.

The MHC gene family is divided into three subgroups—class I, class II, and class III. Diversity of antigen presentation, mediated by MHC classes I and II, is attained in multiple ways: (1) the MHC's genetic encoding is polygenic, (2) MHC genes are highly polymorphic and have many variants, (3) several MHC genes are expressed from both inherited alleles.

MHC proteins

MHC proteins have immunoglobulin-like structure.

Class I

MHC I occurs as an α chain composed of three domains—α1, α2, α3. The α1 rests upon a unit of the non-MHC molecule β2 microglobulin (encoded on human chromosome 15). The α3 subunit is transmembrane, anchoring the MHC class I molecule to the cell membrane. The peptide being presented is held by the floor of the peptide-binding groove, in the central region of the α1/α2 heterodimer (a molecule composed of two nonidentical subunits). The genetically encoded and expressed sequence of amino acids, the sequence of residues, of the peptide-binding groove's floor determines which particular peptide residues it binds.[4]

Class II

MHC class two is formed of two chains, α and β, each having two domains—α1 and α2 and β1 and β2—each chain having a transmembrane domain, α2 and β2, respectively, anchoring the MHC class II molecule to the cell membrane.[5] The peptide-binding groove is formed of the heterodimer of α1 and β1.

MHC class II molecules in humans have five to six isotypes. Classic molecules present peptides to CD4+ lymphocytes. Nonclassic molecules, accessories, with intracellular functions, are not exposed on cell membranes, but in internal membranes in lysosomes, normally loading the antigenic peptides onto classic MHC class II molecules.

[edit] Class III

Class III molecules have physiologic roles unlike classes I and class II, but are encoded between them in the short arm of human chromosome 6. Class III molecules include several secreted proteins with immune functions: components of the complement system (such as C2, C4, and B factor), cytokines (such as TNF-α, LTA, LTB), and heat shock proteins (hsp).

[edit] Antigen processing and presentation

Peptides are processed and presented by two classical pathways:

• In MHC class II phagocytes such as macrophages and immature dendritic cells uptake entities by phagocytosis into phagosomes—though B cells exhibit the more general endocytosis into endosomes—which fuse with lysosomes whose acidic enzymes cleave the uptaken protein into many different peptides. Via physicochemical dynamics in molecular interaction with the particular MHC class II variants borne by the host, encoded in the host's genome, a particular peptide exhibits immunodominance and loads onto MHC class II molecules. These are trafficked to and externalized on the cell surface.[6]

• In MHC class I any nucleated cell normally presents cytosolic peptides, mostly self peptides derived from protein turnover and defective ribosomal products. During viral infection, intracellular microorganism infection, or cancerous transformation, such proteins degraded in the proteosome are as well loaded onto MHC class I molecules and displayed on the cell surface. T lymphocytes can detect a peptide displayed at 0.1%-1% of the MHC molecules.

IMPORTANT ASPECTS OF MHC 

• Although there is a high degree of polymorphism for a species, an individual has maximum of six different class I MHC products and only slightly more class II MHC products (considering only the major loci).  
• Each MHC molecule has only one binding site.  The different peptides a given MHC molecule can bind all bind to the same site, but only one at a time. 
• Because each MHC molecule can bind many different peptides, binding is termed degenerate. 
• MHC polymorphism is determined only in the germline.  There are no recombinational mechanisms for generating diversity. 
• MHC molecules are membrane-bound; recognition by T cells requires cell-cell contact. 
• Alleles for MHC genes are co-dominant.  Each MHC gene product is expressed on the cell surface of an individual nucleated cell. 
• A peptide must associate with a given MHC of that individual, otherwise no immune response can occur.  That is one level of control.
• Mature T cells must have a T cell receptor that recognizes the peptide associated with MHC.  This is the second level of control. 
• Cytokines (especially interferon-γ) increase level of expression of MHC. 
• Peptides from the cytosol associate with class I MHC and are recognized by Tc cells.  Peptides from within vesicles associate with class II MHC and are recognized by Th cells. 
• Polymorphism in MHC is important for survival of the species.

ORGANS OF THE IMMUNE SYSTEM

 

Organs of the Immune System:

• The immune system is made up of many different organs and tissues dispersed throughout the body.
• PRIMARY lymphoid organs are the sites of lymphocyte birth &/ or maturation, in an Ag independent fashion.
• SECONDARY lymphoid organs are the sites of mature lymphocyte selection and expansion in an Ag dependent fashion.

Primary Lymphoid Tissues:

• generate and/or mature and educate cells of the immune system
• maturation and central selection
• diversity created in an Ag independent fashion

Bone Marrow------------------ B cells

Thymus------------------------- T cells

Secondary Lymphoid Tissues:

• site where mature, immunocompetent lymphocytes are exposed to Ag.
• Ag is collected and brought to tissue and lymphocytes are exposed to Ag.
• lymph nodes-- Ag from intracellular tissue fluids
• spleen-------- blood-borne Ag
• MALT------- Ag from mucosal surfaces. Includes Peyer's patches, tonsils, and adenoids

PRIMARY LYMPHOID ORGANS:

Organization is simple as they are sites of growth and maturation and not sites where cells and Ag need to mix.

THYMUS:

T cell progenitor cells enter the Thymus from Bone Marrow to mature and to be "educated". The thymus is a flat, bilobed organ situated over the heart. Lobes are covered by a CAPSULE and are divided into LOBULES by connective tissue strands called TRABECULAE . The lobules are divided into 2 regions;

Cortex:

• outer compartment
• high thymocyte concentration
• site of progenitor T cell growth
• Nurse cells in outer area of cortex nourish thymocytes

Medulla:

• cell density much less
• cells are mature and are SINGLE POSITIVE either CD 4+8- or CD 4-8+

Both the cortex and medulla are composed of a mesh of cells forming the STROMA or framework of the tissue. These cells include epithelial cells, Interdigitating cells, and macrophage. These cells make contact with thymocytes to stimulate growth, carry out +ve and -ve selection, and remove dead cells (90-95% of cells die).

BONE MARROW

The Bursa of Fabricius is the site of B cell development in birds. The bone marrow and possibly other sites (Peyer's patches in sheep and cattle) play this role in mammals, but the lack of information on the organ and how it works limits our knowledge of B cell development. Details of B cell development and selection will be discussed later (chapter 11).

SECONDARY LYMPHOID ORGANS:

• site of stimulation of mature lymphocytes
• create sites for Ag-lymphocyte interaction
• Lymph, produced by the leakage of fluid into tissue spaces (interstitial fluids) is returned to the blood via lymphatic vessels of increasing size via the thoracic duct that empties into the left subclavian vein.
• foreign Ag collects in lymph and is moved by the lymphatic system along with lymphocytes to the lymphoid tissues to induce an immune response
• lymphoid tissues can include simple lymphoid follicles or more organized collections of follicles (Peyer's patches, tonsils, appendix) or complex organizations of B and T cell rich organs (lymph nodes, spleen).

Primary follicle-- network of follicular dendritic cells and small resting B cells not yet stimulated by Ag.

Secondary follicle-- Ag stimulated ring of B cells around a Germinal Center of proliferating B cells, B memory cells, and plasma cells, mixed with macrophage and follicular dendritic cells.

LYMPH NODE

Architecture provides microenvironment for lymphocytes to effectively encounter and respond to Ag.

• Encapsulated bean shaped organ where reticular network holds lymphocytes, macrophage, and dendritic cells
• Lymph enters the node via Afferent Lymphatic Vessels and lymph born Ags entering node are trapped by phagocytic cells and reticular dendritic cells
• In the Paracortex (T-cell rich area) Ag is displayed by APC to Th cells
• Th cells and Ag activated B cells move to a primary follicle in the cortex and give rise to a secondary follicle

SPLEEN

• Large ovoid secondary lymphoid organ
• Covered by a capsule it too is divided into compartments by trabeculae
• Gains Ag from blood not lymph

MUCOSAL-ASSOCIATED LYMPHOID TISSUE (MALT)

• Mucosal membranes of the digestive, respiratory, and urogenital systems are the major sites of Ag entry into the body
• MALT ranges from loose clusters of lymphoid cells in the intestinal lamina propria, to more complex organizations as in the Peyer's Patches, tonsils, and appendix.
• Very active areas of the immune system. The numbers of plasma cells in the MALT >> then the number of plasma cells in the spleen + lymph node + bone marrow.

Radioimmunoassay

Radioimmunoassay (RIA) is a very sensitive in vitro assay technique used to measure concentrations of antigens (for example, hormone levels in the blood) by use of antibodies. As such, it can be seen as the inverse of a radiobinding assay, which quantifies an antibody by use of corresponding antigens.

Although the RIA technique is extremely sensitive and extremely specific, requiring specialized equipment, it remains the least expensive method to perform such tests. It requires special precautions and licensing, since radioactive substances are used. Today it has been supplanted by the ELISA method, where the antigen-antibody reaction is measured using colorimetric signals instead of a radioactive signal. However, because of its robustness, consistent results and low price per test, RIA methods are again becoming popular. It is generally simpler to perform than a bioassay.

The RAST test (radioallergosorbent test) is an example of radioimmunoassay. It is used to detect the causative allergen for an allergy.

Method

To perform a radioimmunoassay, a known quantity of an antigen is made radioactive, frequently by labeling it with gamma-radioactive isotopes of iodine attached to tyrosine. This radiolabeled antigen is then mixed with a known amount of antibody for that antigen, and as a result, the two specifically bind to one another. Then, a sample of serum from a patient containing an unknown quantity of that same antigen is added. This causes the unlabeled (or "cold") antigen from the serum to compete with the radiolabeled antigen ("hot") for antibody binding sites. As the concentration of "cold" antigen is increased, more of it binds to the antibody, displacing the radiolabeled variant, and reducing the ratio of antibody-bound radiolabeled antigen to free radiolabeled antigen. The bound antigens are then separated from the unbound ones, and the radioactivity of the free antigen remaining in the supernatant is measured using a gamma counter. Using known standards, a binding curve can then be generated which allows the amount of antigen in the patient's serum to be derived.

The technique of radioimmunoassay has revolutionized research and clinical practice in many areas, e.g.,

• blood banking
• diagnosis of allergies
• endocrinology

Agglutination

Agglutination is the clumping of particles. The word agglutination comes from the Latin agglutinare, meaning "to glue."[1]

This occurs in biology in three main examples:

1. The clumping of cells such as bacteria or red blood cells in the presence of an antibody. The antibody or other molecule binds multiple particles and joins them, creating a large complex. An example occurs when people are given blood transfusions of the wrong blood group.
2. The coalescing of small particles that are suspended in a solution; these larger masses are then (usually) precipitated.
3. An allergic reaction type occurrence where cells become more compacted together to prevent foreign materials entering them. This is usually the result of an antigen in the vicinity of the cells.

Agglutination Tests

Agglutination/Hemagglutination
When the antigen is particulate, the reaction of an antibody with the antigen can be detected by agglutination (clumping) of the antigen. The general term agglutinin is used to describe antibodies that agglutinate particulate antigens. When the antigen is an erythrocyte the term hemagglutination is used. All antibodies can theoretically agglutinate particulate antigens but IgM, due to its high valence, is particularly good agglutinin and one sometimes infers that an antibody may be of the IgM class if it is a good agglutinating antibody.

Qualitative agglutination test
Agglutination tests can be used in a qualitative manner to assay for the presence of an antigen or an antibody. The antibody is mixed with the particulate antigen and a positive test is indicated by the agglutination of the particulate antigen. (Figure 7).

For example, a patient's red blood cells can be mixed with antibody to a blood group antigen to determine a person's blood type. In a second example, a patient's serum is mixed with red blood cells of a known blood type to assay for the presence of antibodies to that blood type in the patient's serum.

Quantitative agglutination test
Agglutination tests can also be used to measure the level of antibodies to particulate antigens. In this test, serial dilutions are made of a sample to be tested for antibody and then a fixed number of red blood cells or bacteria or other such particulate antigen is added. Then the maximum dilution that gives agglutination is determined. The maximum dilution that gives visible agglutination is called the titer. The results are reported as the reciprocal of the maximal dilution that gives visible agglutination. Figure 8 illustrates a quantitative hemagglutination test.

Prozone effect - Occasionally, it is observed that when the concentration of antibody is high (i.e. lower dilutions), there is no agglutination and then, as the sample is diluted, agglutination occurs (See Patient 6 in Figure 8). The lack of agglutination at high concentrations of antibodies is called the prozone effect. Lack of agglutination in the prozone is due to antibody excess resulting in very small complexes that do not clump to form visible agglutination.

 

Applications of agglutination tests

i. Determination of blood types or antibodies to blood group antigens.

ii. To assess bacterial infections

e.g. A rise in titer of an antibody to a particular bacterium indicates an infection with that bacterial type. N.B. a fourfold rise in titer is generally taken as a significant rise in antibody titer.

Animal Cell Culture Media : Natural and Artificial Media

Animal cell culture can be described as in vitro maintenance and propagation of animal cells using a suitable nutrient media. Culturing is a process of growing animal cells artificially. The most important and essential step in animal cell culture is selecting appropriate growth medium for invitro cultivation. The selection of the medium depends on the type of cells to be cultured and also the purpose of the culture. Purpose of animal cell culture can be growth, differentiation, or even production of desired products like pharmaceutical compounds.

Animal cells are cultured using a completely natural media, or an artificial media along with some of the natural products.

Natural Media:

In the early years of this in vitro cultivation of animal cell culture technique natural media are obtained from biological sources were used. For eample
1. Body fluid such as plasma, serum, lymph, amniotic fluid and much more are used. These fluids used as animal cell culture media after testing for toxicity and sterility.

2. Tissue extract such as extract of liver, spleen, bone marrow and leucocyes also used as animal cell culture media. But most commonly used tissue extract is from chick embryo.

3. Plasma clots are also used as media for animal cell culture and now they are commercially produced as culture media.

4. Bovine embryo extract are also prepared using bovine embryos of up to 10days age, and are used as animal cell culture media.

Artificial Media:

1. The artificial media contains partly or fully defined components.

2. The basic criteria for choosing a artificial media for animal cell culture are
The culture media should provide all the required nutrients to the cell.

3. Media should maintain the physiological pH at around 7 with the help of buffering system.

4. The animal cell culture media should be sterile, and isotonic to the culturing cells. The basis for the animal cell culture media is the balanced salt solution, which are used to create a physiological pH and osmolarity required to maintain the animal cells in vitro or in laboratory conditions.

5. For promoting cell growth and proliferation, many types of animal cell culture media are designed by adding or varying different constituents. For example serum containing media and serum-free media.

What is a bioreactor?

• An apparatus for growing organisms (yeast, bacteria, or animal cells) under

controlled conditions.

• Used in industrial processes to produce pharmaceuticals, vaccines, or

antibodies

• Also used to convert raw materials into useful byproducts such as in the

bioconversion of corn into ethanol.

• Bioreactors supply a homogeneous (same throughout) environment by

constantly stirring the contents.

• Bioreactors give the cells a controlled environment by ensuring the same

temperature, pH, and oxygen levels.

Required properties of bioreactors

• Simplicity of design

• Large number of organisms per unit volume

• Uniform distribution of micro-organisms

• Simple and effective oxygen supply

• Low energy requirement

• Uniform distribution of energy

• providing information about the formation of 3D tissue

Types of bioreactors

You can classify bioreactors based on three different parameters.

1. Sterility of the container :

Sterile : used for the production of antibiotics or vitamins.

Not sterile : used for example in conventional fermentations

such as in the production of beer, or more modern

as the treatment of water

2. Conditions imposed by the bioprocess

Organisms growing in bioreactors may be:

• Suspended-

• Immobilized-A simple method, where cells are immobilized, is a Petri dish with agar gel.

Large scale immobilized cell bioreactors are:

• moving media, also known as Moving Bed Biofilm Reactor (MBBR);

• Packed bed;

• Fibrous bed;

• Membrane.

3.Methods of cultivation of micro-organisms

BATCH culture

A typical batch reactor consists of a tank with

an agitator and integral heating/cooling system. These

vessels may vary in size from less than 1 liter to more than

15,000 liters. The advantages of the batch reactor lie with

its versatility. A single vessel can carry out a sequence of

different operations without the need to break containment.

FED-BATCH culture

In a fed-batch reactor, fresh media is continuous or

sometimes periodically added but there is no continuous

removal. The fermenter is emptied or partially emptied

when reactor is full or fermentation is finished. It is

possible to achieve high productivities due to the fact

that controlling the flow rate of the feed entering the

reactor can optimize the growth rate of the cells.

Continuous PERFUSION culture

Perfusion bioreactors involve continuous culture,

feeding, and withdrawal (harvesting) of spent media

for long periods. Perfusion systems accumulate no

waste products. Once established, bioprocessing with

perfusion bioreactors can in many cases be simpler

and experience fewer failures.

CONTINUOUS-FLOW (chemostat) culture

A chemostat (from Chemical environment is static) is

a bioreactor in which fresh medium is continuously

added, while culture liquid is continuously removed to

keep the culture volume constant. By changing the rate

with which medium is added to the bioreactor

the growth rate of the microorganism can be easily

controlled.

Membrane attack complex

The membrane attack complex (MAC) is typically formed on the surface of pathogenic bacterial cells as a result of the activation of the alternative pathway and the classical pathway of the complement system, and it is one of the effector proteins of the immune system. The membrane-attack complex (MAC) forms transmembrane channels. These channels disrupt the phospholipid bilayer of target cells, leading to cell lysis and death.[1][2]

A number of proteins participate in the assembly of the MAC. Freshly activated C5b binds to C6 to form a C5b-6 complex, then to C7 forming the C5b-7 complex. The C5b-7 complex binds to C8, which is composed of three chains (alpha, beta, and gamma), thus forming the C5b-8 complex. C5b-8 subsequently binds to C9[3][4][5] and acts as a catalyst in the polymerization of C9. Active MAC has a subunit composition of C5b-C6-C7-C8-C9.

Structure and function

It is composed of a complex of four complement proteins (C5b, C6, C7, and C8) that bind to the outer surface of the plasma membrane, and many copies of a fifth protein (C9) that hook up to one another, forming a ring in the membrane. C6-C9 all contain a common MACPF domain.[6] This region is homologous to cholesterol-dependent cytolysins from Gram-positive bacteria.[7]

The ring structure formed by C9 is a pore in the membrane that allows free diffusion of molecules in and out of the cell. If enough pores form, the cell is no longer able to survive.

Initiation: C5-C7

The membrane attack complex is initiated when the complement protein C5 convertase cleaves C5 into C5a and C5b.

Another complement protein, C6, binds to C5b.

The C5bC6 complex is bound by C7.

This junction alters the configuration of the protein molecules exposing a hydrophobic site on C7 that allows the C7 to insert into the phospholipid bilayer of the pathogen.

Polymerization: C8-C9

Similar hydrophobic sites on C8 and C9 molecules are exposed when they bind to the complex, so they can also insert into the bilayer.

C8 is a complex made of the two proteins C8-beta and C8 alpha-gamma.

C8 alpha-gamma has the hydrophobic area that inserts into the bilayer. C8 alpha-gamma induces the polymerization of 10-16 molecules of C9 into a pore-forming structure known as the membrane attack complex.

• It has a hydrophobic external face allowing it to associate with the lipid bilayer.

• It has a hydrophilic internal face to allow the passage of water.

[edit] Inhibition

CD59 acts to inhibit the complex. This exists on body cells to protect them from MAC. A rare condition, paroxysmal nocturnal haemoglobinuria, results in red cells which lack CD59. These red cells can therefore be lysed by MAC.

Hybridoma technology

Hybridoma technology is a technology of forming hybrid cell lines (called hybridomas) by fusing a specific antibody-producing B cell with a myeloma (B cell cancer) cell that is selected for its ability to grow in tissue culture and for an absence of antibody chain synthesis. The antibodies produced by the hybridoma are all of a single specificity and are therefore monoclonal antibodies.

Hybridoma technology is used to produce a hybrid cell. These hybrid cells are produced by fusing B-lymphocyte with tumour cell and they are called as myeloma cells. Thus these hybrid cells have got the ability to produce antibodies due to the B-lymphocyte genetic material and also capacity to divide indefinitely in the culture due to the presence of tumour cell or myeloma cells involved in the production of hybrid cells. Therefore, these hybrid cells produced from hybridoma technology are cultured in laboratory or passaged or subcultured using mouse peritoneal cavity and these cells produces monoclonal antibodies, and this technology is called as hybridoma technology.

When an antigen reacts with B-lymphocyte receptors, lymphocytes divide rapidly and produce a clone of B cells, all these B cells produce antibodies against that specific antigen and this is called as clonal selection. That is B-lymphocytes produce only one type of antibodies which are specific to only one type of antigen or antigenic determinant. But fully differentiated antibody producing B-lymphocyte cells known as plasma cells does not divide when cultured in a laboratory.

Procedure of Hybridoma Technology:

1. B-lymphocytes are extracted from the spleen of an animal, but usually it is extracted from the mouse, which has been immunized with the required antigen against which monoclonal antibodies are produced. Mouse is immunized by giving antigen injection along with an adjuvant via subcutaneously or by peritoneal cavity; this is followed by booster doses of the antigen. Adjuvant is nonantigenic in nature but they stimulate the immune system.

2. This immunization with specific antigen increases the specific antibody producing B-lymphocytes; this considerably increases the chances of obtaining the required hybridoma cells or clones.

3. This specific antibody producing B-lymphocytes are then mixed with the selected myeloma cells and are induced to fuse to form hybrid cells. The myeloma cells are selected based on some criteria like these cells themselves should not produce antibodies and also they should contain a genetic markers such as HGPRT. This genetic marker helps in easy selection of the resulting hybrid cells.

4. When HGPRT myeloma cells are fused with specific antibody producing B-lymphocytes, the resulting cell population will have the mixture of cell population such as hybrid cells, myeloma cells, B-lymphocytes.

5. This mixture of cell population is then cultured in selective media known as HAT medium along with the drug aminopterin. The HGPRT myeloma cells cannot divide in the HAT medium due to the presence of aminopterin. The Specific antibody producing B-lymphocytes are unable to divide continuously in the culture medium, therefore eventually they die.

6. Only the hybridoma cells have got the ability to divide and proliferate on the HAT medium because genome from the B-lymphocyte makes them HGPRT positive and genome from the myeloma cells they can divide indefinitely. Thus only the hybridoma cells or fused cells are selected using selective media called as HAT medium.

Identification and Isolation of the Hybridoma Cells:

Hybridoma cells producing specific antibodies for the antigen used to immunize the animal (mouse) are identified and isolated by following method,

1. The specific antibodies present in the each microwell are identified using one of the methods such as precipitation method or agglutination method. Most commonly used and most sensitive and rapid method is ELISA (Enzyme Linked Immunosorbant Assay)

2. Wells which contain the antibodies specific to the antigens are identified and hybridoma cells are isolated from these wells and cultured (cloned). This ensures that these hybridoma cells have the capacity to produce same single type of antibodies specific to the antigen used.

3. After these hybridoma cells are multiplied using in vitro or in vivo method.

Mass Production of Antibodies:

4. The in vivo production involves injecting hybridoma cells into the peritoneal cavity of the animal (mouse), then ascetic fluid is isolated and then antibodies are isolated from it.

5. In vitro method hybridoma cells are cultured in suitable culture media and then antibodies are isolated and purified.

Uses:

This hybridoma technology is used to produce monoclonal antibodies.

Innate immune system

The innate immune system, also known as non-specific immune system and first line of defense,[1] comprises the cells and mechanisms that defend the host from infection by other organisms in a non-specific manner. This means that the cells of the innate system recognize and respond to pathogens in a generic way, but unlike the adaptive immune system, it does not confer long-lasting or protective immunity to the host.[2] Innate immune systems provide immediate defense against infection, and are found in all classes of plant and animal life.

The innate immune system is thought to constitute an evolutionarily older defense strategy, and is the dominant immune system found in plants, fungi, insects, and in primitive multicellular organisms.[3]

The major functions of the vertebrate innate immune system include:

• Recruiting immune cells to sites of infection, through the production of chemical factors, including specialized chemical mediators, called cytokines.
• Activation of the complement cascade to identify bacteria, activate cells and to promote clearance of dead cells or antibody complexes.
• The identification and removal of foreign substances present in organs, tissues, the blood and lymph, by specialised white blood cells.
• Activation of the adaptive immune system through a process known as antigen presentation.
• Acting as a physical and chemical barrier to infectious agents.

Antigen processing

Antigen processing is a biological process that prepares antigens for presentation to special cells of the immune system called T lymphocytes. This process involves two distinct pathways for processing of antigens from an organism's own (self) proteins or intracellular pathogens (e.g. viruses), or from phagocytosed pathogens (e.g. bacteria); subsequent presentation of these antigens on class I or class II MHC molecules is dependent on which pathway is used. Both MHC class I and II are required to bind antigen before they are stably expressed on a cell surface.

While the conventional distinction between the two pathways is useful, there are instances where extracellular-derived peptides are presented in the context of MHC class I and cytosolic peptides are presented in the context of MHC class II.

The Endogenous Pathway

The endogenous pathway is used to present cellular peptide fragments on the cell surface on MHC class I molecules. If a virus had infected the cell, viral peptides would also be presented, allowing the immune system to recognize and kill the infected cell. Worn out proteins within the cell become ubiquitinated, marking them for proteasome degradation. Proteasomes break the protein up into peptides that include some around nine amino acids long (suitable for fitting within the peptide binding cleft of MHC class I molecules). Transporter associated with antigen presenting (TAP), a protein that spans the membrane of the rough endoplasmic reticulum, transports the peptides into the lumen of the rough endoplasmic reticulum (ER). Also within the rough ER, a series of chaperone proteins, including calnexin, calreticulin, ERp57, and Binding immunoglobulin protein (BiP) facilitates the proper folding of class I MHC and its association with β2 microglobulin. The partially folded MHC class I molecule then interacts with TAP via tapasin (the complete complex also contains calreticulin and Erp57 and, in mice, calnexin). Once the peptide is transported into the ER lumen it binds to the cleft of the awaiting MHC class I molecule, stabilizing the MHC and allowing it to be transported to the cell surface by the golgi apparatus.

[edit] The Exogenous Pathway

The exogenous pathway is utilized by specialized antigen presenting cells to present peptides derived from proteins that the cell has endocytosed. The peptides are presented on MHC class II molecules. Proteins are endocytosed and degraded by acid-dependent proteases in endosomes; this process takes about an hour.[1]

The nascent MHC class II protein in the rough ER has its peptide-binding cleft blocked by Ii (the invariant chain; a trimer) to prevent it from binding cellular peptides or peptides from the endogenous pathway. The invariant chain also facilitates MHC class II's export from the ER in a vesicle. This fuses with a late endosome containing the endocytosed, degraded proteins. The invariant chain is then broken down in stages, leaving only a small fragment called CLIP which still blocks the peptide binding cleft. An MHC class II-like structure, HLA-DM, removes CLIP and replaces it with a peptide from the endosome. The stable MHC class-II is then presented on the cell surface.

In Cross-presentation, peptides derived from extracellular proteins are presented in the context of MHC class I.

Phagocytosis

Phagocytosis in three steps:
1. Unbound phagocyte surface receptors do not trigger phagocytosis.
2. Binding of receptors causes them to cluster.
3. Phagocytosis is triggered and the particle is taken up by the phagocyte.

Phagocytosis (from Ancient Greek φαγεῖν (phagein) , meaning "to devour", κύτος, (kytos) , meaning "cell", and -osis, meaning "process") is the cellular process of engulfing solid particles by the cell membrane to form an internal phagosome by phagocytes and protists. Phagocytosis was revealed by Ilya Mechnikov in 1882. Phagocytosis is a specific form of endocytosis involving the vesicular internalization of solids such as bacteria, and is, therefore, distinct from other forms of endocytosis such as the vesicular internalization of various liquids. Phagocytosis is involved in the acquisition of nutrients for some cells, and, in the immune system, it is a major mechanism used to remove pathogens and cell debris. Bacteria, dead tissue cells, and small mineral particles are all examples of objects that may be phagocytosed.

Engulfment of material is facilitated by the actin-myosin contractile system. The phagosome of ingested material is then fused with the lysosome, leading to degradation.

Degradation can be oxygen-dependent or oxygen-independent.

• Oxygen-dependent degradation depends on NADPH and the production of reactive oxygen species. Hydrogen peroxide and myeloperoxidase activate a halogenating system, which leads to the creation of hypochlorite and the destruction of bacteria.[2]
• Oxygen-independent degradation depends on the release of granules, containing proteolytic enzymes such as defensins, lysozyme, and cationic proteins. Other antimicrobial peptides are present in these granules, including lactoferrin, which sequesters iron to provide unfavourable growth conditions for bacteria.

It is possible for cells other than dedicated phagocytes (such as dendritic cells) to engage in phagocytosis.[3]

[edit] In apoptosis

Following apoptosis, the dying cells need to be taken up into the surrounding tissues by macrophages in a process called efferocytosis. One of the features of an apoptotic cell is the presentation of a variety of intracellular molecules on the cell surface, such as calreticulin, phosphatidylserine (From the inner layer of the plasma membrane), annexin A1, and oxidised LDL. These molecules are recognised by receptors on the cell surface of the macrophage such as the phosphatidylserine receptor or by soluble (free floating) receptors such as thrombospondin 1, Gas-6, and MFG-E8, which themselves then bind to other receptors on the macrophage such as CD36 and alpha-v beta-3 integrin. Defects in apoptotic cell clearance is usually associated with impaired phagocytosis of macropghages. Accumulation of apoptotic cell remnants often causes autoimmune disorders, thus pharmacological potentiation of phagocytosis has a medical potential in treatment of certain forms of autoimmune disorders.[4][5][6][7]

Additional information on phagocytosis of apoptotic cells could be found in the book: “Phagocytosis of dying cells: from molecular mechanisms to human diseases” (Eds DV Krysko and P Vandenabeele, 2009, Springer).

[edit] In protists

In many protists, phagocytosis is used as a means of feeding, providing part or all of their nourishment. This is called phagotrophic nutrition, as distinguished from osmotrophic nutrition, which takes place by absorption.

• In some, such as amoeba, phagocytosis takes place by surrounding the target object with pseudopods, as in animal phagocytes. In humans, Entamoeba histolytica can phagocytose red blood cells.[8] This process is known as "erythrophagocystosis", and is considered the only reliable way to distinguish Entamoeba histolytica from noninvasive species such as Entamoeba dispar.[9]

• Ciliates also engage in phagocytosis.[10] In ciliates there is a specialized groove or chamber in the cell where phagocytosis takes place, called the cytostome or mouth.

The resulting phagosome may be merged with lysosomes containing digestive enzymes, forming a phagolysosome. The food particles will then be digested, and the released nutrients are diffused or transported into the cytosol for use in other metabolic processes.

Mixotrophy can involve phagotrophic nutrition and phototrophic nutrition.[11]

Antigen-presenting cell

An antigen-presenting cell (APC) or accessory cell is a cell that displays foreign antigen complexes with major histocompatibility complex (MHC) on their surfaces. T-cells may recognize these complexes using their T-cell receptors (TCRs). These cells process antigens and present them to T-cells.

Types

APCs fall into two categories: professional or non-professional.

T cells cannot recognise, and therefore cannot respond to, 'free' antigen. T cells can only 'see' an antigen that has been processed and presented by cells via carrier molecules like MHC and CD1 molecules. Most cells in the body can present antigen to CD8+ T cells via MHC class I molecules and, thus, act as "APCs"; however, the term is often limited to specialized cells that can prime T cells (i.e., activate a T cell that has not been exposed to antigen, termed a naive T cell). These cells, in general, express MHC class II as well as MHC class I molecules, and can stimulate CD4+ ("helper") cells as well as CD8+ ("cytotoxic") T cells, respectively. (Almost all nucleated cells express MHC class I receptors, including professional APCs. If a virus infects a macrophage or dendritic cell, it will try to promote its own destruction through cytotoxic T cells. However, dendritic cells can ingest viruses through pinocytosis and therefore activate the adaptive immune response to create antibodies for the virus through class II MHC receptors.)

To help distinguish between the two types of APCs, those that express MHC class II molecules are often called professional antigen-presenting cells.

[edit] Professional APCs

Professional APCs are very efficient at internalizing antigen, either by phagocytosis or by receptor-mediated endocytosis, and then displaying a fragment of the antigen, bound to a class II MHC molecule, on their membrane. The T cell recognizes and interacts with the antigen-class II MHC molecule complex on the membrane of the antigen-presenting cell. An additional co-stimulatory signal is then produced by the antigen-presenting cell, leading to activation of the T cell. The expression of co-stimulatory molecules is a defining feature of professional APCs.

There are three main types of professional antigen-presenting cell:

• Dendritic cells (DCs), which have the broadest range of antigen presentation, and are probably the most important APC. Activated DCs are especially potent TH cell activators because, as part of their composition, they express co-stimulatory molecules such as B7.
• Macrophages, which are also CD4+ and are therefore also susceptible to infection by HIV.
• Certain B-cells, which express (as B cell receptor) and secrete a specific antibody, can internalize the antigen, which bind to its BCR and present it incorporated to MHC II molecule, but are inefficient APC for most other antigens.
• Certain activated epithelial cells

[edit] Non-professional

A non-professional APC does not constitutively express the Major Histocompatibility Complex class II (MHC class II) proteins required for interaction with naive T cells; these are expressed only upon stimulation of the non-professional APC by certain cytokines such as IFN-γ. Non-professional APCs include:

• Fibroblasts (skin)
• Thymic epithelial cells
• Thyroid epithelial cells
• Glial cells (brain)
• Pancreatic beta cells
• Vascular endothelial cells

ELISA

Enzyme-linked immunosorbent assay (ELISA) is a test that uses antibodies and color change to identify a substance.

ELISA is a popular format of a "wet-lab" type analytic biochemistry assay that uses a solid-phase enzyme immunoassay (EIA) to detect the presence of a substance, usually an antigen, in a liquid sample or wet sample.

The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality-control check in various industries.

Antigens from the sample are attached to a surface. Then, a further specific antibody is applied over the surface so it can bind to the antigen. This antibody is linked to an enzyme, and, in the final step, a substance containing the enzyme's substrate is added. The subsequent reaction produces a detectable signal, most commonly a color change in the substrate.

Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). After the antigen is immobilized, the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody that is linked to an enzyme through bioconjugation. Between each step, the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step, the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample.

Of note, ELISA can perform other forms of ligand binding assays instead of strictly "immuno" assays, though the name carried the original "immuno" because of the common use and history of development of this method. The technique essentially requires any ligating reagent that can be immobilized on the solid phase along with a detection reagent that will bind specifically and use an enzyme to generate a signal that can be properly quantified. In between the washes, only the ligand and its specific binding counterparts remain specifically bound or "immunosorbed" by antigen-antibody interactions to the solid phase, while the nonspecific or unbound components are washed away. Unlike other spectrophotometric wet lab assay formats where the same reaction well (e.g. a cuvette) can be reused after washing, the ELISA plates have the reaction products immunosorbed on the solid phase which is part of the plate, so are not easily reusable.

Types

"Indirect" ELISA

The steps of "indirect" ELISA follows the mechanism below:-

• A buffered solution of the antigen to be tested for is added to each well of a microtiter plate, where it is given time to adhere to the plastic through charge interactions.
• A solution of nonreacting protein, such as bovine serum albumin or casein, is added to block any plastic surface in the well that remains uncoated by the antigen.
• The primary antibody is added, which binds specifically to the test antigen coating the well. This primary antibody could also be in the serum of a donor to be tested for reactivity towards the antigen.
• A secondary antibody is added, which will bind the primary antibody. This secondary antibody often has an enzyme attached to it, which has a negligible effect on the binding properties of the antibody. In other cases, as in the diagram to the left, the primary antibody itself is conjugated to the enzyme.
• A substrate for this enzyme is then added. Often, this substrate changes color upon reaction with the enzyme. The color change shows the secondary antibody has bound to primary antibody, which strongly implies the donor has had an immune reaction to the test antigen. This can be helpful in a clinical setting, and in research.
• The higher the concentration of the primary antibody present in the serum, the stronger the color change. Often, a spectrometer is used to give quantitative values for color strength.

Sandwich ELISA

A sandwich ELISA. (1) Plate is coated with a capture antibody; (2) sample is added, and any antigen present binds to capture antibody; (3) detecting antibody is added, and binds to antigen; (4) enzyme-linked secondary antibody is added, and binds to detecting antibody; (5) substrate is added, and is converted by enzyme to detectable form.

1. A less-common variant of this technique, a "sandwich" ELISA, is used to detect sample antigen.

Competitive ELISA

A third use of ELISA is through competitive binding. The steps for this ELISA are somewhat different from the first two examples:

1. Unlabeled antibody is incubated in the presence of its antigen (sample).
2. These bound antibody/antigen complexes are then added to an antigen-coated well.
3. The plate is washed, so unbound antibody is removed. (The more antigen in the sample, the less antibody will be able to bind to the antigen in the well, hence "competition".)
4. The secondary antibody, specific to the primary antibody, is added. This second antibody is coupled to the enzyme.
5. A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.
6. The reaction is stopped to prevent eventual saturation of the signal.

Multiple and portable ELISA

A new technique uses a solid phase made up of an immunosorbent polystyrene rod with eight to 12 protruding ogives. The entire device is immersed in a test tube containing the collected sample and the following steps (washing, incubation in conjugate and incubation in chromogens) are carried out by dipping the ogives in microwells of standard microplates filled with reagents.

The advantages of this technique are:

1. The ogives can each be sensitized to a different reagent, allowing the simultaneous detection of different antibodies and/or different antigens for multiple-target assays.
2. The sample volume can be increased to improve the test sensitivity in clinical (blood, saliva, urine), food (bulk milk, pooled eggs) and environmental (water) samples.
3. One ogive is left unsensitized to measure the nonspecific reactions of the sample.
4. The use of laboratory supplies for dispensing sample aliquots, washing solution and reagents in microwells is not required, facilitating the development of ready-to-use lab kits and on-site testing.

[edit] Applications

Because the ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in a sample, it is a useful tool for determining serum antibody concentrations (such as with the HIV test[9] or West Nile virus). It has also found applications in the food industry in detecting potential food allergens, such as milk, peanuts, walnuts, almonds, and eggs.[10] ELISA can also be used in toxicology as a rapid presumptive screen for certain classes of drugs.

• detection of Mycobacterium antibodies in tuberculosis
• detection of rotavirus in feces
• detection of hepatitis B markers in serum
• detection of enterotoxin of E. coli in feces
• detection of HIV antibodies in blood samples

Immunological memory

The ability of the body to defend itself against specific invading agents such as bacteria, toxins, viruses and foreign tissues is called immunity. Immunity is said to have a memory for most invading agents encountered before, because a second encounter with the same agent prompts a rapid and vigorous response. This is called immunological memory which leads to a perception that an individual is immune to a particular agent.

There are two types of immune response: cell-mediated (CMI) associated with specialised blood cells called T-cells, and antibody mediated associated with specialised blood cells called B-cells. Both immune responses act on substances called antigens.

Antigens are defined by their ability to provoke an immune response (immunogenicity). Immunogenicity causes the proliferation of specific T-cells or the production of particular antibodies by B-cells. Antigens also said to have reactivity: the ability to react specifically with T-cells, or antibodies.

T-cells are produced in the bone marrow and migrate to the thymus to mature. They then reside in lymphatic tissue until threatened by a foreign antigen when they move out on a ‘seek and destroy’ mission.

There are four main types of T-cells:

Helper T-cells – which assist in both CMI and AMI. They secrete lymphokines (cytokines) which are hormones that stimulate other cells in the body to resist invading antibodies. They display the protein CD4 on their surface;

Killer T-cells – which kill antigens directly once stimulated by agents released by the helper T-cells. They display the protein CD8 on their surface;

Suppressor T-cells – are a controversial cell that is believed to dampen or suppress the immune response; and

Memory T-cells - which recognise the original invading antigen. When the antigen returns thousands of memory cells are available to initiate a far swifter reaction than occurred during the first invasion.

B-cells are produced and mature in the bone marrow. When the body is invaded by a foreign antigen they do not migrate like T-cells rather they stay put in lymphatic tissue, the spleen, lymph nodes, and the GIT. When activated they differentiate into plasma cells that secrete antibodies into lymph and blood. Helper T-cells amplify antibody production. The antibodies circulate the cardiovascular and lymphatic systems to reach the site of infection.

B-cells that are activated but do not differentiate into plasma cells remain as memory B-cells, ready to respond more rapidly and forcefully should the same antigen reappear at a future time.

Complement system

The complement system helps or “complements” the ability of antibodies and phagocytic cells to clear pathogens from an organism. It is part of the immune system called the innate immune system[1] that is not adaptable and does not change over the course of an individual's lifetime. However, it can be recruited and brought into action by the adaptive immune system.

The complement system consists of a number of small proteins found in the blood, generally synthesized by the liver, and normally circulating as inactive precursors (pro-proteins). When stimulated by one of several triggers, proteases in the system cleave specific proteins to release cytokines and initiate an amplifying cascade of further cleavages. The end-result of this activation cascade is massive amplification of the response and activation of the cell-killing membrane attack complex. Over 25 proteins and protein fragments make up the complement system, including serum proteins, serosal proteins, and cell membrane receptors. They account for about 5% of the globulin fraction of blood serum.

Three biochemical pathways activate the complement system: the classical complement pathway, the alternative complement pathway, and the lectin pathway.[2]

Functions of the Complement

The following are the basic functions of the complement:

1. Opsonization - enhancing phagocytosis of antigens
2. Chemotaxis - attracting macrophages and neutrophils
3. Cell Lysis - rupturing membranes of foreign cells
4. Clumping of antigen-bearing agents

Regulation of the complement system

The complement system has the potential to be extremely damaging to host tissues, meaning its activation must be tightly regulated. The complement system is regulated by complement control proteins, which are present at a higher concentration in the blood plasma than the complement proteins themselves. Some complement control proteins are present on the membranes of self-cells preventing them from being targeted by complement. One example is CD59, also known as protectin, which inhibits C9 polymerisation during the formation of the membrane attack complex. The classical pathway is inhibited by C1-inhibitor, which binds to C1 to prevent its activation.

Role in disease

It is thought that the complement system might play a role in many diseases with an immune component, such as Barraquer-Simons Syndrome, asthma, lupus erythematosus, glomerulonephritis, various forms of arthritis, autoimmune heart disease, multiple sclerosis, inflammatory bowel disease, and ischemia-reperfusion injuries.[20][21] and rejection of transplanted organs.[22]

The complement system is also becoming increasingly implicated in diseases of the central nervous system such as Alzheimer's disease and other neurodegenerative conditions such as spinal cord injuries.[23][24][25]

Deficiencies of the terminal pathway predispose to both autoimmune disease and infections (particularly Neisseria meningitidis, due to the role that the membrane attack complex plays in attacking Gram-negative bacteria).

Mutations in the complement regulators factor H and membrane cofactor protein have been associated with atypical haemolytic uraemic syndrome.[26][27] Moreover, a common single nucleotide polymorphism in factor H (Y402H) has been associated with the common eye disease age-related macular degeneration.[28] Polymorphisms of complement component 3, complement factor B, and complement factor I, as well as deletion of complement factor H-related 3 and complement factor H-related 1 also affect a person's risk of developing age-related macular degeneration.[29] Both of these disorders are currently thought to be due to aberrant complement activation on the surface of host cells.

Mutations in the C1 inhibitor gene can cause hereditary angioedema, an autoimmune condition resulting from reduced regulation of the complement pathway.

Mutations in the MAC components of complement, especially C8, are often implicated in recurrent Neisserial infection.

Organ Specific Autoimmune diseases

Organ-specific autoimmune diseases, as the name suggests, are defined as disorders in which the body’s immune response attacks healthy cells in a specific organ. Autoimmune hepatitis, Hashimoto’s thyroiditis, Graves’ disease, type 1 diabetes, Addison’s disease, and Sjögren’s syndrome are some of the more common organ-specific autoimmune conditions.

Autoimmune hepatitis. Autoimmune hepatitis is a chronic autoimmune disorder that affects the liver. The liver becomes inflamed when the body’s immune cells wrongly identify liver cells as foreign invaders. Autoimmune hepatitis is sometimes referred to as chronic active hepatitis (CAH), but autoimmunity is not its only cause. Allergic reactions to medications, excessive alcohol use, and viruses also can cause it. Autoimmune hepatitis is differentiated from these other forms of chronic hepatitis by the presence of auto-antibody markers.

Patients with autoimmune hepatitis may notice the following symptoms: abdominal distention, dark urine, fatigue, itching, loss of appetite, nausea and vomiting, and pale stools. Women may also experience amenorrhea, the absence of menstruation.

Hashimoto’s thyroiditis. Hashimoto’s thyroiditis, also referred to as chronic lymphocytic thyroiditis or autoimmune thyroiditis, is caused by an autoimmune reaction to proteins in the thyroid gland. This inappropriate reaction causes the thyroid gland to swell, which reduces its function. A hypofunctioning thyroid can lead to a variety of symptoms, including constipation, difficulty concentrating, dry skin, enlarged neck or face swelling, fatigue, hair loss, heavy and irregular periods, intolerance to cold, weight gain, and joint stiffness.

Researchers believe some people may be genetically predisposed to Hashimoto’s, as patients frequently have concurrent autoimmune conditions or a family history of thyroid problems. The disease occurs in both men and women but women are more prone to it and are most likely to develop the condition between ages 30 and 50. Current estimates are that, in Western countries, between 0.1 and 5 percent of adults are affected by this condition.

Graves’ disease. Like Hashimoto’s disease, Graves’ is an autoimmune disorder that disturbs the thyroid — but with the opposite effect. Graves’ causes the thyroid to overfunction, leading to a series of symptoms, many of which are in contrast to those caused by Hashimoto’s: anxiety, double vision, eye irritation and tearing, frequent bowel movements, heat intolerance, increased appetite, increased sweating, insomnia, rapid or irregular heartbeat, shortness of breath upon exertion, tremor, and weight loss. These types of symptoms are due to the fact that Graves’ disease causes an increase in production of thyroid hormone, which helps regulate metabolism. The hyperfunctioning thyroid caused by Graves’ leads to an increase in metabolism.

Men and women can experience Graves’ disease, but it’s more commonly seen in women, and most commonly between the ages of 20 and 30. In the United States and Europe, an estimated three million people have the condition, with approximately 37,000 new cases per year in the United States.

Type 1 diabetes. Sometimes referred to as insulin-dependent diabetes or juvenile diabetes, type 1 diabetes occurs when a person’s body produces little or no insulin. Insulin is a necessary hormone because it’s what allows sugar to enter the cells. When sugar can’t enter the cells, the body can’t get energy, so type 1 diabetes patients often experience increased hunger — yet tend not to gain weight. They also may need to frequently use the bathroom because when glucose doesn’t go into the cells, it gets stuck in the blood and can cause excessive urination and increased thirst. Abdominal pain, loss of periods, fatigue, nausea, and weight loss are other symptoms of type 1 diabetes.

Addison’s disease. This condition occurs when the adrenal glands, small organs that sit on top of the kidneys, don’t produce enough hormones. Specifically, Addison’s patients don’t produce enough cortisol and aldosterone, two hormones that are produced in the outer portion (the cortex) of the adrenal glands. This is why the disorder is sometimes referred to as adrenal insufficiency. Common symptoms of Addison’s disease include blood pressure changes, diarrhea, patchy skin darkening, fatigue and weakness, loss of appetite, mouth lesions on the inside of the cheeks, salt cravings, nausea and vomiting, and weight loss.

Not all cases of Addison’s disease are considered autoimmune in nature. The condition can be caused by tuberculosis, HIV or fungal infections, blood loss, tumors, or the use of blood-thinning drugs. However, autoimmune attacks on the adrenal glands account for 70 percent of Addison’s cases.

Addison’s disease affects 1 in 100,000 people of all age groups, but most commonly in people between the ages of 30 and 50. Men and women are equally affected by this condition.

Sjögren’s syndrome. This autoimmune condition attacks the glands that produce tears and saliva. As a result, people who have Sjögren’s syndrome experience dry eyes and mouth. Other symptoms may include itching eyes, difficulty swallowing, loss of taste, dental cavities, hoarseness, fatigue, joint pain, and swollen glands. In most cases of Sjögren’s, symptoms are limited to the tear ducts and salivary glands, but in severe cases, the condition also can affect the kidneys and lungs.

Sjögren’s syndrome occurs most often in middle-aged women, although men also can be affected. The condition is rarely seen in children, unless they have another autoimmune disease. While Sjögren’s can occur alone, it’s common to see it show up in people with other autoimmune conditions such as rheumatoid arthritis, lupus, scleroderma, or polymyositis. It is estimated that between one and four million Americans have Sjögren’s syndrome

B cell

B cells belong to a group of white blood cells known as lymphocytes, making them a vital part of the immune system -- specifically the humoral immunity branch of the adaptive immune system. B cells can be distinguished from other lymphocytes, such as T cells and natural killer cells (NK cells), by the presence of a protein on the B cell's outer surface known as a B cell receptor (BCR). This specialized receptor protein allows a B cell to bind to a specific antigen.

The principal functions of B cells are to make antibodies against antigens, to perform the role of antigen-presenting cells (APCs), and to develop into memory B cells after activation by antigen interaction. Recently, a new, suppressive function of B cells has been discovered.[1]

[edit] Development of B cells

Immature B cells are produced in the bone marrow of most mammals. Rabbits are an exception; their B cells develop in the appendix-sacculus rotundus. After reaching the IgM+ immature stage in the bone marrow, these immature B cells migrate to secondary lymphoid tissues (such as the spleen, lymph nodes, Peyer's patches, etc.) where they are called transitional B cells, and some of these cells differentiate into mature B lymphocytes.[3]

[edit] Functions

The human body makes millions of different types of B cells each day that circulate in the blood and lymphatic system performing the role of immune surveillance. They do not produce antibodies until they become fully activated. Each B cell has a unique receptor protein (referred to as the B cell receptor (BCR)) on its surface that will bind to one particular antigen. The BCR is a membrane-bound immunoglobulin, and it is this molecule that allows the distinction of B cells from other types of lymphocyte, as well as being the main protein involved in B cell activation. Once a B cell encounters its cognate antigen and receives an additional signal from a T helper cell, it can further differentiate into one of the two types of B cells listed below (plasma B cells and memory B cells). The B cell may either become one of these cell types directly or it may undergo an intermediate differentiation step, the germinal center reaction, where the B cell will hypermutate the variable region of its immunoglobulin gene ("somatic hypermutation") and possibly undergo class switching. Other functions for B cells include antigen presentation, cytokine production and lymphoid tissue organization.

[edit] Activation of B cells

B cell recognition of antigen is not the only element necessary for B cell activation (a combination of clonal proliferation and terminal differentiation into plasma cells). B cells that have not been exposed to antigen, also known as naïve B cells, can be activated in a T cell-dependent or -independent manner.

[edit] T cell-dependent activation

Once a pathogen is ingested by an antigen-presenting cell such as a macrophage or dendritic cell, the pathogen's proteins are then digested to peptides and attached to a class II MHC protein. This complex is then moved to the outside of the cell membrane. The macrophage is now activated to deliver multiple signals to a specific T cell that recognizes the peptide presented. The T cell is then stimulated to produce autocrines (Refer to Autocrine signalling), resulting in the proliferation and differentiation to effector and memory T cells. Helper T cells (i.e. CD4+ T cells) then activate specific B cells through a phenomenon known as an Immunological synapse. Activated B cells subsequently produce antibodies which assist in inhibiting pathogens until phagocytes (i.e. macrophages, neutrophils) or the complement system for example clears the host of the pathogen(s).

Most antigens are T-dependent, meaning T cell help is required for maximal antibody production. With a T-dependent antigen, the first signal comes from antigen cross linking the B cell receptor (BCR) and the second signal comes from co-stimulation provided by a T cell. T dependent antigens contain proteins that are presented on B cell Class II MHC to a special subtype of T cell called a Th2 cell. When a B cell processes and presents the same antigen to the primed Th cell, the T cell secretes cytokines that activate the B cell. These cytokines trigger B cell proliferation and differentiation into plasma cells. Isotype switching to IgG, IgA, and IgE and memory cell generation occur in response to T-dependent antigens. This isotype switching is known as Class Switch Recombination (CSR). Once this switch has occurred, that particular B cell will usually no longer make the earlier isotypes, IgM or IgD.

[edit] T cell-independent activation

Many antigens are T cell-independent in that they can deliver both of the signals to the B cell. Mice without a thymus (nude or athymic mice that do not produce any T cells) can respond to T independent antigens. Many bacteria have repeating carbohydrate epitopes that stimulate B cells, by cross-linking the IgM antigen receptors in the B cell, responding with IgM synthesis in the absence of T cell help. Conjugate vaccines are made to provide a stronger immune response against these foreign molecules. There are two types of T cell independent activation; Type 1 T cell-independent (polyclonal) activation, and type 2 T cell-independent activation (in which macrophages present several of the same antigen in a way that causes cross-linking of antibodies on the surface of B cells).

Antigen

In immunology, an antigen is a substance that evokes the production of one or more antibodies. Each antibody binds to a specific antigen by way of an interaction similar to the fit between a lock and a key. The substance may be from the external environment or formed within the body. The immune system will try to destroy or neutralize any antigen that is recognized as a foreign and potentially harmful invader. The term originally came from antibody generator[1][2] and was a molecule that binds specifically to an antibody, but the term now also refers to any molecule or molecular fragment that can be bound by a major histocompatibility complex (MHC) and presented to a T-cell receptor.[3] "Self" antigens are usually tolerated by the immune system, whereas "non-self" antigens can be identified as invaders and can be attacked by the immune system.

Polyclonal antibodies

Polyclonal antibodies (or antisera) are antibodies that are obtained from different B cell resources. They are a combination of immunoglobulin molecules secreted against a specific antigen, each identifying a different epitope.

Production

These antibodies are typically produced by inoculation of a suitable mammal, such as a mouse, rabbit or goat. Larger mammals are often preferred as the amount of serum that can be collected is greater. An antigen is injected into the mammal. This induces the B-lymphocytes to produce IgG immunoglobulins specific for the antigen. This polyclonal IgG is purified from the mammal’s serum.

Many methodologies exist for polyclonal antibody production in laboratory animals. Institutional guidelines governing animal use and procedures relating to these methodologies are generally oriented around humane considerations and appropriate conduct for adjuvant (agents which modify the effect of other agents while having few if any direct effects when given by themselves) use. This includes adjuvant selection, routes and sites of administration, injection volumes per site and number of sites per animal. Institutional policies generally include allowable volumes of blood per collection and safety precautions including appropriate restraint and sedation or anesthesia of animals for injury prevention to animals or personnel.

The primary goal of antibody production in laboratory animals is to obtain high titer, high affinity antisera for use in experimentation or diagnostic tests. Adjuvants are used to improve or enhance an immune response to antigens. Most adjuvants provide for an injection site, antigen depot which allows for a slow release of antigen into draining lymph nodes.

Many adjuvants also contain or act directly as:

1. surfactants which promote concentration of protein antigens molecules over a large surface area, and
2. immunostimulatory molecules or properties. Adjuvants are generally used with soluble protein antigens to increase antibody titers and induce a prolonged response with accompanying memory.

Such antigens by themselves are generally poor immunogens. Most complex protein antigens induce multiple B-cell clones during the immune response, thus, the response is polyclonal. Immune responses to non-protein antigens are generally poorly or enhanced by adjuvants and there is no system memory.

Antibodies are currently also being produced from isolation of human B-lymphocytes to produce specific recombinant polyclonal antibodies. The biotechnology company, Symphogen, produces this type of antibody for therapeutic applications. They are the first research company to develop recombinant polyclonal antibody drugs to reach phase two trials. This production prevents viral and prion transmission.

Inflammation

Inflammation (Latin, īnflammō, "I ignite, set alight") is part of the complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants.[1] The classical signs of acute inflammation are pain (dolor), heat (calor), redness (rubor), swelling (tumor), and loss of function (functio laesa). Inflammation is a protective attempt by the organism to remove the injurious stimuli and to initiate the healing process. Inflammation is not a synonym for infection, even in cases where inflammation is caused by infection. Although infection is caused by a microorganism, inflammation is one of the responses of the organism to the pathogen. However, inflammation is a stereotyped response, and therefore it is considered as a mechanism of innate immunity, as compared to adaptive immunity, which is specific for each pathogen.[2]

Progressive destruction of the tissue would compromise the survival of the organism. However, chronic inflammation can also lead to a host of diseases, such as hay fever, periodontitis, atherosclerosis, rheumatoid arthritis, and even cancer (e.g., gallbladder carcinoma). It is for that reason that inflammation is normally closely regulated by the body.

Inflammation can be classified as either acute or chronic. Acute inflammation is the initial response of the body to harmful stimuli and is achieved by the increased movement of plasma and leukocytes (especially granulocytes) from the blood into the injured tissues. A cascade of biochemical events propagates and matures the inflammatory response, involving the local vascular system, the immune system, and various cells within the injured tissue. Prolonged inflammation, known as chronic inflammation, leads to a progressive shift in the type of cells present at the site of inflammation and is characterized by simultaneous destruction and healing of the tissue from the inflammatory process.

Causes

• Burns
• Chemical irritants
• Frostbite
• Toxins
• Infection by pathogens
• Physical injury, blunt or penetrating
• Immune reactions due to hypersensitivity
• Ionizing radiation
• Foreign bodies, including splinters, dirt and debris
• Stress
• Trauma
• Alcohol

Antibody

An antibody (Ab), also known as an immunoglobulin (Ig), is a large Y-shaped protein produced by B-cells that is used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, called an antigen.[1][2] Each tip of the "Y" of an antibody contains a paratope (a structure 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 blocking a part of a microbe that is essential for its invasion and survival). The production of antibodies is the main function of the humoral immune system.[3]

Antibodies are secreted by a type of white blood cell called a plasma cell. Antibodies can occur in two physical forms, a soluble form that is secreted from the cell, 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 only found 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.[4] 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.[5] 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; the terms antibody and immunoglobulin are often used interchangeably.[6] Antibodies 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, and several different kinds of antibodies, which are grouped into different isotypes based on which heavy chain they possess. Five different antibody isotypes are known in mammals, which perform different roles, and help direct the appropriate immune response for each different type of foreign object they encounter.[7]

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, another name for antibody, and differ in their biological properties, functional locations and ability to deal with different antigens, as depicted in the table.[12]

Antibody isotypes of mammals
Name	Types	Description	Antibody Complexes
IgA	2	Found in mucosal areas, such as the gut, respiratory tract and urogenital tract, and prevents colonization by pathogens.[13] Also found in saliva, tears, and breast milk.
IgD	1	Functions mainly as an antigen receptor on B cells that have not been exposed to antigens.[14] It has been shown to activate basophils and mast cells to produce antimicrobial factors.[15]
IgE	1	Binds to allergens and triggers histamine release from mast cells and basophils, and is involved in allergy. Also protects against parasitic worms.[3]
IgG	4	In its four forms, provides the majority of antibody-based immunity against invading pathogens.[3] The only antibody capable of crossing the placenta to give passive immunity to the fetus.
IgM	1	Expressed on the surface of B cells (monomer) and in a secreted form (pentamer) with very high avidity. Eliminates pathogens in the early stages of B cell mediated (humoral) immunity before there is sufficient IgG.[3][14]

IgG

Immunoglobulin G, or IgG, antibodies play a major role in protecting against infection and disease. IgG antibodies are found throughout the whole body, and they are prevalent in blood serum, accounting for around 80 percent of the total antibodies found in serum, reports Miami University. IgG antibodies can bind to foreign bacteria and viruses and target the foreign microbes for destruction by the immune system.

IgE

Another common type of antibody is immunoglobin E, or IgE, antibodies. Unlike IgG, which activates the immune system in response to pathogens, IgE activates the immune system in response to allergens. The variable region of IgE antibodies allows for selective binding between specific IgE antibodies and specific allergens.

IgA

Immunoglobulin A, or IgA, antibodies play a role in immune system functioning in mucosal tissue. Mucosal membranes line the nasal and oral cavities, respiratory system and digestive tract along with several other tissues. IgA antibodies are secreted within the mucosal lining and released to protect the mucosal lining by binding to and neutralizing foreign particles before they reach the cells within the mucosal cells.

INTERFERON

Interferons play an important role in the first line of defense against viral infections. They are part of the non-specific immune system and are induced at an early stage in viral infection – before the specific immune system has time to respond.

Interferons are made by cells in response to an appropriate stimulus, and are released into the surrounding medium; they then bind to receptors on target cells and induce transcription of  approximately 20-30 genes in the target cells, and this results in an anti-viral state in the target cells.

TYPES OF INTERFERON:

TYPE I interferon:

Interferon-alpha (leukocyte interferon) is produced by virus-infected leukocytes, etc

Interferon-beta (fibroblast interferon) is produced by virus-infected fibroblasts, or virus-infected epithelial cells, etc

TYPE II inteferon

            Interferon-gamma  (immune interferon) is produced by certain activated T-cells and NK cells.

THERAPEUTIC USES OF INTERFERONS

Interferons-alpha and -beta have been used to treat various viral infections. One currently approved use for various types of interferon-a is in the treatment of certain cases of acute and chronic hepatitis C and chronic hepatitis B.

Interferon-gamma has been used to treat a variety of disease in which macrophage activation might play an important role in recovery, eg. lepromatous leprosy, leishmaniasis, toxoplasmosis.

Since interferons have anti-proliferative effects, they have also been used to treat certain tumors such as melanoma and Kaposi’s sarcoma.

SIDE EFFECTS OF INTERFERONS

Common side effects of interferons:

            fever, malaise, fatigue, muscle pains

High levels of interferons can cause kidney, liver, bone marrow and heart toxicity.

Interleukin

Interleukins are a group of cytokines (secreted proteins/

signaling molecules) that were first seen to be expressed by

white blood cells (leukocytes).[1] The term interleukin derives from (inter-) "as a means of communication", and (-leukin) "deriving from the fact that many of these proteins are produced by leukocytes and act on leukocytes". The name is something of a relic, though (the term was coined by Dr. Vern Paetkau, University of Victoria); it has since been found that interleukins are produced by a wide variety of body cells. The function of the immune system depends in a large part on interleukins, and rare deficiencies of a number of them have been described, all featuring

autoimmune diseases or

immune deficiency. The majority of interleukins are synthesized by helper CD4 T lymphocytes, as well as through monocytes, macrophages, and endothelial cells. They promote the development and differentiation of T, B, and hematopoietic cells.

Adjuvant

An adjuvant (from Latin, adiuvare: to aid) is a pharmacological or immunological agent that modifies the effect of other agents, such as a drug or vaccine. They are often included in vaccines to enhance the recipient's
immune response to a supplied antigen, while keeping the injected foreign material to a minimum.

In immunology, an adjuvant is an agent that may stimulate the immune system and increase the response to a vaccine, without having any specific antigenic effect in itself.[1] The word “adjuvant” comes from the Latin word adiuvare, meaning to help or aid.[2] "An immunologic adjuvant is defined as any substance that acts to accelerate, prolong, or enhance antigen-specific immune responses when used in combination with specific vaccine antigens.

Autoimmunity

Autoimmunity is the failure of an organism in recognizing its own constituent parts as self, which allows an immune response against its own cells and tissues. Any disease that results from such an aberrant immune response is termed an autoimmune disease. Autoimmunity is often caused by a lack of germ development of a target body and as such the immune response acts against its own cells and tissues.

Antibody opsonization

Antibody opsonization is the process by which a pathogen is marked for ingestion and destruction by a phagocyte. Opsonization involves the binding of an opsonin, e.g., antibody, to a receptor on the pathogen's cell membrane.[1] After opsonin binds to the membrane, phagocytes are attracted to the pathogen. The Fab portion of the antibody binds to the antigen, whereas the Fc portion of the antibody binds to an Fc receptor on the phagocyte, facilitating phagocytosis.

Macrophage

Macrophages are
cells produced by the differentiation of monocytes in tissues. Human macrophages are about 21 micrometres (0.00083 in) in diameter.[2] Monocytes and macrophages are phagocytes. Macrophages function in both non-specific defense (
innate immunity) as well as help initiate specific defense mechanisms (
adaptive immunity) of vertebrate animals. Their role is to phagocytose, or engulf and then digest, cellular debris and pathogens, either as stationary or as mobile cells. They also stimulate lymphocytes and other immune cells to respond to pathogens. They are specialized phagocytic cells that attack foreign substances, infectious microbes and cancer cells through destruction and ingestion.

Epitope

An epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells. The part of an antibody that recognizes the epitope is called a paratope. Although epitopes are usually non-self proteins, sequences derived from the host that can be recognized are also epitopes.

The epitopes of protein antigens are divided into two categories, conformational epitopes and
linear epitopes, based on their structure and interaction with the paratope.[1] A conformational epitope is composed of discontinuous sections of the antigen's
amino acid sequence. These epitopes interact with the paratope based on the 3-D surface features and shape or
tertiary structure of the antigen. Most epitopes are conformational.[citation needed]

By contrast, linear epitopes interact with the paratope based on their primary structure. A linear epitope is formed by a continuous sequence of amino acids from the antigen.