Immunoglobulin (Ig)

Immunoglobulins are glycoprotein molecules that are produced by plasma cells in response to an immunogen and which function as antibodies. The immunoglobulins derive their name from the finding that they migrate with globular proteins when antibody-containing serum is placed in an electrical field

FUNCTION
1. Immunoglobulins bind specifically to one or a few closely related antigens. Each immunoglobulin actually binds to a specific antigenic determinant. Antigen binding by antibodies is the primary function of antibodies and can result in protection of the host.

2. The significant biological effects are a consequence of secondary "effector functions" of antibodies.Phagocytic cells, lymphocytes, platelets, mast cells, and basophils have receptors that bind immunoglobulins. This binding can activate the cells to perform some function. Some immunoglobulins also bind to receptors on placental trophoblasts, which results in transfer of the immunoglobulin across the placenta. As a result, the transferred maternal antibodies provide immunity to the fetus and newborn.

STRUCTURE OF IMMUNOGLOBULINS

The basic structure of the immunoglobulins is illustrated in figure 2. Although different immunoglobulins can differ structurally, they all are built from the same basic units.

A. Heavy and Light Chains

All immunoglobulins have a four chain structure as their basic unit. They are composed of two identical light chains (23kD) and two identical heavy chains (50-70kD)

B. Disulfide bonds

1. Inter-chain disulfide bonds - The heavy and light chains and the two heavy chains are held together by inter-chain disulfide bonds and by non-covalent interactions The number of inter-chain disulfide bonds varies among different immunoglobulin molecules.

2. Intra-chain disulfide binds - Within each of the polypeptide chains there are also intra-chain disulfide bonds.

C. Variable (V) and Constant (C) Regions

When the amino acid sequences of many different heavy chains and light chains were compared, it became clear that both the heavy and light chain could be divided into two regions based on variability in the amino acid sequences. These are the:

1. Light Chain - VL (110 amino acids) and CL (110 amino acids)

2. Heavy Chain - VH (110 amino acids) and CH (330-440 amino acids)\(x = {-b \pm \sqrt{b^2-4ac} \over 2a}\)h the arms of the antibody molecule forms a Y. It is called the hinge region because there is some flexibility in the molecule at this point.

E. Domains

Three dimensional images of the immunoglobulin molecule show that it is not straight as depicted in figure 2A. Rather, it is folded into globular regions each of which contains an intra-chain disulfide bond (figure 2B-D). These regions are called domains.

1. Light Chain Domains - VL and CL

2. Heavy Chain Domains - VH, CH1 - CH3 (or CH4)

F. Oligosaccharides

Carbohydrates are attached to the CH2 domain in most immunoglobulins. However, in some cases carbohydrates may also be attached at other locations. 

IMMUNOGLOBULIN FRAGMENTS: STRUCTURE/FUNCTION RELATIONSHIPS

Immunoglobulin fragments produced by proteolytic digestion –

A.  Fab 
Digestion with papain breaks the immunoglobulin molecule in the hinge region before the H-H inter-chain disulfide bond Figure 6. This results in the formation of two identical fragments that contain the light chain and the VH and CH1 domains of the heavy chain.

Antigen binding – These fragments are  called the Fab fragments because they contained the antigen binding sites of the antibody. Each Fab fragment is monovalent whereas the original molecule was divalent. The combining site of the antibody is created by both VH and VL. 

B. Fc 
Digestion with papain also produces a fragment that contains the remainder of the two heavy chains each containing a CH2 and CH3 domain. This fragment was called Fc because it was easily crystallized.

Effector functions – The effector functions of immunoglobulins are mediated by this part of the molecule. Different functions are mediated by the different domains in this fragment . 

Treatment of immunoglobulins with pepsin results in cleavage of the heavy chain after the H-H inter-chain disulfide bonds resulting in a fragment that contains both antigen binding sites . This fragment is called F(ab’)2because it is divalent. The Fc region of the molecule is digested into small peptides by pepsin. The F(ab’)2binds antigen but it does not mediate the effector functions of antibodies.
 

ANTIGEN-ANTIBODY REACTIONS

Affinity of the antigen-antibody reaction refers to the intensity of the attraction between antigen and antibody molecule.
Antigen-antibody reactions

Reaction test            Modified test

Precipitation  -> Immunoelectrophoresis, Immunoprecipitation
Agglutination -> Latex agglutination, Indirect, Haemagglutination , Coagglutination ,Coombs test

Neutralization  -> Measurement of LD, Plaque assays

Complement fixation  -> Conglutination

Immunofluorescence ->  Indirect immunofiuorescence, Immunoofluoremetric Assay

Enzyme immunoassay -> Enzyme linked, Immunosorbent assay

Radioimmunoassay -> Immunoradiometric assay

Avidity is the strength of the bond after the formation of antigen-antibody complex.

Sensitivity refers to the ability of the test to detect even very minute quantities of antigen or antibody. A test shall be called as highly sensitive if false negative results are absent or minimal.

Specificity refers to the ability of the test to detect reactions between homologous antigens and antibodies only, and with no other. In a highly specific test, false positive reactions will be minimal or absent.

CHEMICAL AGENTS

Chlorine and iodine are most useful disinfectant Iodine as a skin disinfectant and chlorine as a water disinfectant have given consistently magnificent results. Their activity is almost exclusively bactericidal and they are effective against sporulating organisms also. 
Mixtures of various surface acting agents with iodine are known as iodophores and these are used for the sterilization of dairy products.

Apart from chlorine, hypochlorite, inorganic chioramines are all good disinfectants but they act by liberating chlorine. 

Hydrogen peroxide in a 3% solution is a harmless but very weak disinfectant whose primary use is in the cleansing of the wound.
 
Potassium permanganate is another oxidising agent which is used in the treatment of urethntzs. 

Formaldehyde — is one of the least selective agent acting on proteins. It is a gas that is usually employed as its 37% solution, formalin. 

When used in sufficiently high concentration it destroys the bacteria and their spores.

Classification of chemical sterilizing agents

Chemical disinfectant

Interfere with membrane functions

•    Surface acting agents : Quaternary ammonium, Compounds, Soaps and fatty acids

•    Phenols : Phenol, cresol, Hexylresorcinol

•    Organic solvent : Chloroform, Alcohol

Denatures proteins

•    Acids and alkalies : Organic acids, Hydrochloric acid , Sulphuric acid

Destroy functional groups of proteins

•    Heavy metals :  Copper, silver , Mercury

•    Oxidizing agents: Iodine, chlorine, Hydrogen peroxide

•    Dyes : Acridine orange, Acriflavine

•    Alkylating agents : Formaldehyde, Ethylene oxide

Applications and in-use dilution of chemical disinfectants

Alcohols : Skin antiseptic Surface disinfectant, Dilution used 70%

Mercurials : Skin antiseptic Surface disinfectant Dilution Used 0.1 %

Silver nitrate : Antiseptic (eyes and burns)  Dilution Used 1 %

Phenolic compound : Antiseptic skin washes  Dilution Used .5 -5 %

Iodine : Disinfects inanimate object, Skin antiseptic Dilution used  2%

Chlorine compounds  : Water treatment Disinfect inanimate objects , Dillution used 5 %

Quaternary ammonium Compounds : Skin antiseptic , Disinfects inanimate object, Dilution Used < 1 %

Glutaraldehyde: Heat sensitve instruments, Dilution used 1-2 %

Cold sterilization can be achieved by dipping the precleaned instrument in 2% solution of gluteraldehyde for 15-20 minutes. This time is sufficient to kill the vegetative form as well as spores ofthe organisms that are commonly encountered in the dentistry.

Ethylene oxide is an a agent extensively used in gaseous sterilization. It is active against all kinds of bacteria and their spores. but its greatest utility is in sterilizing those objects which are damaged by heat (e.g. heart lung machine). It is also used to sterlise fragile, heat sensitive equipment, powders as well as components of space crafts.

Evaluation of Disinfectants

Two methods which are widely employed are:

 Phenol coefficient test, Kelsey -Sykes test
 
These tests determine the capacity of disinfectant as well as their ability to retain their activity.
 

Cell Functions:
-> Autolysis

- degradative reactions in cells caused by indigenous intracellular enzymes – usually occurs after cell death
- Irreversible (along with Coagulative necrosis or infarcts) – reversible: fatty degeneration, & hydropic degeneration

-> Autolysin:
•    Ab causing cellular lysis in the presence of complement
•    Autolytic enzymes produced by the organism degrade the cell’s own cell wall structures

-> In the presence of cephalosporins & penicillins, growing bacterial cells lyse
•    W/o functional cell wall structures, the bacterial cell bursts

-> Heterolysis: cellular degradation by enzymes derived from sources extrinsic to the cell (e.g., bacteria)

-> Necrosis: sum of intracellular degradative reactions occurring after individual cell death w/in a living organism

ANTIGENS

Immunogen
A substance that induces a specific immune response.

Antigen (Ag)
A substance that reacts with the products of a specific immune response.

Hapten
A substance that is non-immunogenic but which can react with the products of a specific immune response. Haptens are small molecules which could never induce an immune response when administered by themselves but which can when coupled to a carrier molecule. Free haptens, however, can react with products of the immune response after such products have been elicited. Haptens have the property of antigenicity but not immunogenicity.

Epitope or Antigenic Determinant
That portion of an antigen that combines with the products of a specific immune response.

Antibody (Ab)
A specific protein which is produced in response to an immunogen and which reacts with an antigen.

FACTORS INFLUENCING IMMUNOGENICITY

- Larger the molecule the more immunogenic it is likely to be.

- More complex the substance is chemically the more immunogenic it will be.

- Particulate antigens are more immunogenic than soluble ones and denatured antigens more immunogenic than the native form.

- Antigens that are easily phagocytosed are generally more immunogenic. This is because for most antigens (T-dependant antigens, see below) the development of an immune response requires that the antigen be phagocytosed, processed and presented to helper T cells by an antigen presenting cell (APC).

- Some substances are immunogenic in one species but not in another. Similarly, some substances are immunogenic in one individual but not in others (i.e. responders and non-responders). The species or individuals may lack or have altered genes that code for the receptors for antigen on B cells and T cells or they may not have the appropriate genes needed for the APC to present antigen to the helper T cells.

Method of Administration

1. Dose
The dose of administration of an immunogen can influence its immunogenicity. There is a dose of antigen above or below which the immune response will not be optimal.

2. Route
Generally the subcutaneous route is better than the intravenous or intragastric routes. The route of antigen administration can also alter the nature of the response

3. Adjuvants
Substances that can enhance the immune response to an immunogen are called adjuvants. The use of adjuvants, however, is often hampered by undesirable side effects such as fever and inflammation.

TYPES OF ANTIGENS

T-independent Antigens
T-independent antigens are antigens which can directly stimulate the B cells to produce antibody without the requirement for T cell help In general, polysaccharides are T-independent antigens. The responses to these antigens differ from the responses to other antigens.
Properties of T-independent antigens

1. Polymeric structure
These antigens are characterized by the same antigenic determinant .

2. Polyclonal activation of B cells
Many of these antigens can activate B cell clones specific for other antigens (polyclonal activation). T-independent antigens can be subdivided into Type 1 and Type 2 based on their ability to polyclonally activate B cells. Type 1 T-independent antigens are polyclonal activators while Type 2 are not.

3. Resistance to degradation
T-independent antigens are generally more resistant to degradation and thus they persist for longer periods of time and continue to stimulate the immune system.

T-dependent Antigens
T-dependent antigens are those that do not directly stimulate the production of antibody without the help of T cells. Proteins are T-dependent antigens. Structurally these antigens are characterized by a few copies of many different antigenic determinants as illustrated in the Figure 2.

HAPTEN-CARRIER CONJUGATES

Hapten-carrier conjugates are immunogenic molecules to which haptens have been covalently attached. The immunogenic molecule is called the carrier.

Structure
Structurally these conjugates are characterized by having native antigenic determinants of the carrier as well as new determinants created by the hapten (haptenic determinants). The actual determinant created by the hapten consists of the hapten and a few of the adjacent residues, although the antibody produced to the determinant will also react with free hapten. In such conjugates the type of carrier determines whether the response will be T-independent or T-dependent.

SUPERANTIGENS

When the immune system encounters a conventional T-dependent antigen, only a small fraction (1 in 104 -105) of the T cell population is able to recognize the antigen and become activated (monoclonal/oligoclonal response). However, there are some antigens which polyclonally activate a large fraction of the T cells (up to 25%). These antigens are called superantigens .

Examples of superantigens include: Staphylococcal enterotoxins (food poisoning), Staphylococcal toxic shock toxin (toxic shock syndrome), Staphylococcal exfoliating toxins (scalded skin syndrome) and Streptococcal pyrogenic exotoxins (shock).

PHAGOCYTOSIS AND INTRACELLULAR KILLING

A. Phagocytic cells

1. Neutrophiles/Polymorphonuclear cells

PMNs are motile phagocytic cells that have lobed nuclei. They can be identified by their characteristic nucleus or by an antigen present on the cell surface called CD66. They contain two kinds of granules the contents of which are involved in the antimicrobial properties of these cells. 

The second type of granule found in more mature PMNs is the secondary or specific granule. These contain lysozyme, NADPH oxidase components, which are involved in the generation of toxic oxygen products, and characteristically lactoferrin, an iron chelating protein and B12-binding protein.

2. Monocytes/Macrophages

 Macrophages are phagocytic cells . They can be identified morphologically or by the presence of the CD14 cell surface marker. 

B. Response of phagocytes to infection 

Circulating PMNs and monocytes respond to danger (SOS) signals generated at the site of an infection. SOS signals include N-formyl-methionine containing peptides released by bacteria, clotting system peptides, complement products and cytokines released from tissue macrophages that have encountered bacteria in tissue.
Some of the SOS signals stimulate endothelial cells near the site of the infection to express cell adhesion molecules such as ICAM-1 and selectins which bind to components on the surface of phagocytic cells and cause the phagocytes to adhere to the endothelium. 
Vasodilators produced at the site of infection cause the junctions between endothelial cells to loosen and the phagocytes then cross the endothelial barrier by “squeezing” between the endothelial cells in a process called diapedesis.

 Once in the tissue spaces some of the SOS signals attract phagocytes to the infection site by chemotaxis (movement toward an increasing chemical gradient). The SOS signals also activate the phagocytes, which results in increased phagocytosis and intracellular killing of the invading organisms.

C. Initiation of Phagocytosis 

Phagocytic cells have a variety of receptors on their cell membranes through which infectious agents bind to the cells. These include:

1. Fc receptors – Bacteria with IgG antibody on their surface have the Fc region exposed and this part of the Ig molecule can bind to the receptor on phagocytes. Binding to the Fc receptor requires prior interaction of the antibody with an antigen. Binding of IgG-coated bacteria to Fc receptors results in enhanced phagocytosis and activation of the metabolic activity of phagocytes (respiratory burst).

2. Complement receptors – Phagocytic cells have a receptor for the 3rd component of complement, C3b. Binding of C3b-coated bacteria to this receptor also results in enhanced phagocytosis and stimulation of the respiratory burst. 

3. Scavenger receptors – Scavenger receptors bind a wide variety of polyanions on bacterial surfaces resulting in phagocytosis of bacteria.

4. Toll-like receptors – Phagocytes have a variety of Toll-like receptors (Pattern Recognition Receptors or PRRs) which recognize broad molecular patterns called PAMPs (pathogen associated molecular patterns) on infectious agents. Binding of infectious agents via Toll-like receptors results in phagocytosis and the release of inflammatory cytokines (IL-1, TNF-alpha and IL-6) by the phagocytes.

D. Phagocytosis 

The pseudopods eventually surround the bacterium and engulf it, and the bacterium is enclosed in a phagosome. During phagocytosis the granules or lysosomes of the phagocyte fuse with the phagosome and empty their contents. The result is a bacterium engulfed in a phagolysosome which contains the contents of the granules or lysosomes.

E. Respiratory burst and intracellular killing

During phagocytosis there is an increase in glucose and oxygen consumption which is referred to as the respiratory burst. The consequence of the respiratory burst is that a number of oxygen-containing compounds are produced which kill the bacteria being phagocytosed. This is referred to as oxygen-dependent intracellular killing. In addition, bacteria can be killed by pre-formed substances released from granules or lysosomes when they fuse with the phagosome. This is referred to as oxygen-independent intracellular killing.

1. Oxygen-dependent myeloperoxidase-independent intracellular killing

During phagocytosis glucose is metabolized via the pentose monophosphate shunt and NADPH is formed. Cytochrome B which was part of the specific granule combines with the plasma membrane NADPH oxidase and activates it. The activated NADPH oxidase uses oxygen to oxidize the NADPH. The result is the production of superoxide anion. Some of the superoxide anion is converted to H2O2 and singlet oxygen by superoxide dismutase. In addition, superoxide anion can react with H2O2 resulting in the formation of hydroxyl radical and more singlet oxygen. The result of all of these reactions is the production of the toxic oxygen compounds superoxide anion (O2-), H2O2, singlet oxygen (1O2) and hydroxyl radical (OH•).

2. Oxygen-dependent myeloperoxidase-dependent intracellular killing 

As the azurophilic granules fuse with the phagosome, myeloperoxidase is released into the phagolysosome. Myeloperoxidase utilizes H2O2 and halide ions (usually Cl-) to produce hypochlorite, a highly toxic substance. Some of the hypochlorite can spontaneously break down to yield singlet oxygen. The result of these reactions is the production of toxic hypochlorite (OCl-) and singlet oxygen (1O2).

3. Detoxification reactions 

PMNs and macrophages have means to protect themselves from the toxic oxygen intermediates. These reactions involve the dismutation of superoxide anion to hydrogen peroxide by superoxide dismutase and the conversion of hydrogen peroxide to water by catalase. 

4. Oxygen-independent intracellular killing 

In addition to the oxygen-dependent mechanisms of killing there are also oxygen–independent killing mechanisms in phagocytes: cationic proteins (cathepsin) released into the phagolysosome can damage bacterial membranes; lysozyme breaks down bacterial cell walls; lactoferrin chelates iron, which deprives bacteria of this required nutrient; hydrolytic enzymes break down bacterial proteins. Thus, even patients who have defects in the oxygen-dependent killing pathways are able to kill bacteria. However, since the oxygen-dependent mechanisms are much more efficient in killing, patients with defects in these pathways are more susceptible and get more serious infections.