Biological Properties

Biological Properties
Immunoglobulins Biological Properties

Introduction Many important biological properties are attributed to antibodies that differ depending on isotype These include; - Neutralization of toxins - Immobilization of microorganisms - Neutralization of viral infectivity - Agglutination of microorganisms or antigenic particles - Binding with soluble antigens - Activation of complement - Protection of fetus Immunoglobulins have protective functions which enable the living organism to fight multiple different infections. Neutralization of toxins : whether bacterial toxins, or toxins of reptiles and insects such as venoms, snake and scorpion toxins. Immobilization of organisms: antibodies can fix organisms by interacting with the organism as a whole or with flagella (organs that are essential for micro-organism movement) Neutralization of viral infectivity : can be a result of blocking the ability of the virus to attach (blocking the attachment site of the virus for the receptor) or interaction with virus; blocking a later phase in reproduction of the virus like; penetration, transcription and coating of the virus. Agglutination of microorganism = tamping of micro-organism Binding with soluble antigens: antigens can be soluble like: bacterial products, microbial products and foreign proteins. Those antigens make the body produce antibodies that combine with the antigens to produce immune complexes, and those complexes bind to phagocytic cells that causes the clearness of this antigens (if the antigens here not cleared it will remain in the circulation and continue to stimulate the immune response which may be harmful to the body). Activation of complement is the function of antibodies either in the classical pathway (activated by antigen antibody complexes) or alternative pathway (activated by aggregated immunoglobulins). Complement system is essential for phagocytosis and to mount an inflammatory response that are essential for the completion of the function of immune response. Protection of fetus: is achieved by antibodies -coming from the maternal circulation- that cross the placenta and provide protection from the organisms that the mother is immune. If the mother is not immune to a certain organism then those organisms maybe harmful to the fetus.

Immunoglobulin Structure-Function Relationship
Cell surface antigen receptor on B cells - Allows B cells to sense their antigenic environment - Connects extracellular space with intracellular signalling machinery Secreted antibody - Neutralization - Arming/recruiting effector cells - Complement fixation Immunoglobulins can be either attached to B-cells and work as a receptor for antigen or it can be secreted.

Immunoglobulins must interact with a
Immunoglobulins are Bifunctional Proteins Immunoglobulins must interact with a small number of specialized molecules : - Fc receptors on cells - Complement proteins - Intracellular cell signalling molecules Whilst simultaneously recognising an infinite array of antigenic determinants. Immunoglobulins can interact with a small number of specialized molecule (above mentioned) through its Fc fragment (that has very small variation), on the other hand they can interact with infinite numbers of antigens ( as antigens interact with Fab which carries a huge variability).

Why do antibodies need an Fc region?
The (Fab)2 fragment can – Detect antigen Precipitate antigen Block the active sites of toxins or pathogen- associated molecules Block interactions between host and pathogen- Precipitate the antigen through binding to it. Block the active sites of toxins or pathogen associated molecules that neutralize infectivity or the effect of the toxin.

but the (Fab)2 can not activate
Inflammatory and effector functions associated with cells Inflammatory and effector functions of complement The trafficking of antigens into the antigen processing pathways Such functions require the Fc fragment as those functions are complementary to antigen binding (e.g phagocytic function and all the above mentioned function).

Four distinct roles of Fc binding proteins
They are essential for many of the biological functions of antibodies: 1- The movement of Ab across cell membranes : poly IgR for dimeric IgA & to some extent, pentameric IgM 2- The transfer of lgG from mother to fetus across the placenta : FcRN 3- Trigger effector functions : Opsonization or ADCC 4- Cross-linking of FcR which generates immunoregulatory signals that affect cell activation, differentiation, etc. which are similar to signal transduction from BcR 1- it happens through IgA that is synthesized by intraepithelial lymphocytes then it is bound to poly IgR that allow dimeric IgA to cross epithelium into the luminal side of the mucus membrane, also pentameric IgM cross in a similar process. 2- FcRN: is a receptor on placenta that binds to IgG and allow it to cross the placenta, without such binding IgG can’t cross the placenta. ** it was thought that IgG can cross the placenta due its molecular weight but IgE, Igd and IgA have similar molecular weight but don’t cross the placenta ,so it was discovered that the ability of IgG to cross the placenta is due to the structure of Fc fragment of IgG. 3- Fc is required to trigger effector functions; as IgG binds to its Fc receptor on macrophages and so it is an opsonin , it also has a receptor on natural killer cells (NK) and that is how NK kill by ADCC ( antibody dependant cell-mediated cytotoxicity). 4-Cross-linking of FcR which generates immunoregulatory signals that affect cell activation, differentiation: IgE is present on basophiles and mast cells and its binding to those cells is the first step in initiating an allergic reaction as allergen binds to IgE which is present on those cells. Similarly the Fc fragment of immunoglobulin on B-lymphoctes is essintial to create the signal that will initiate a response to cascade the events leading to the activation of B-lymphocytes; cross-linking of B-cell receptor by an antigen is essential to create a signal on B-lymphocytes.

Accessory signal- transducing polypeptide
The structure of a number of human Fc -receptors Accessory signal- transducing polypeptide Fc -binding polypeptide Fc fragment has an important role and it does its role via a receptor; that is usually high affinity receptor. Those different receptors are found on the surface of different cells each for a different kind of immunoglobulin (know the different kinds of receptor that are in the figure).

Biological Properties of IgG
Distributed equally between the intravascular and extravascular spaces Except for IgG3 which has a rapid turnover (half life=7days), the half life of IgG is approximately 23 days IgG has the longest half life of all immunoglobulin isotypes making it the most suitable for passive immunization Interestingly, as the concentration of IgG in the serum increases, the rate of IgG catabolism increases (half life days) It is the most important immunoglobulin. Most abundant immunoglobulin in serum up to (70%-80%) of immunoglobulin. Distributed equally between the intravascular and extravascular spaces so it is present in the CSF, serum, peritoneal fluid, GI fat, GIT fluid. You can find IgG in all fluids. As IgG has the longest half-life, it is the most abundant. When IgG is produced in high concentration it remain for a long time in the serum as in the cases of measles, yellow fever and hepatitis A the concentration of IgG produced is very huge that they remain in the serum for life-time. IgG production is associated with generation of memory B-lymphocytes that can produce IgG in secondary immune response which maintains IgG in the serum of normal individual. About the 4th point : it is a mechanism that the body uses in order to avoid high concentration of immunoglobulin. There is always a ratio between albumins and globulins and this ratio is imbalanced in the case of high production of immunoglobulin causing hypergammaglobulinemia favoring one reactant over the other causing the increase of the rate of catabolism of immunoglobulins.

Functions of IgG Agglutination and precipitation
Passage through placenta - The IgG isotype, except for IgG2, is the only isotype that can pass through the placenta as of the 3rd to 4th month of gestation - Passage is mediated by the FC portion - Role in health and disease Opsonization - Bridges microorganisms or particulate antigens to phagocytic cells There are two major types of antigen antibody reaction: agglutination, and precipitation. The reaction that results depends on the solubility of the reactant and both reactant solubility can vary as the antigen can be insoluble (e.g. bacteria) and the antibody can be also insoluble ( all immunoglobulins are soluble in serum but IgM and IgD can be insoluble if they attached to B-cells) so we can have three cases ( the doctor just mentioned two of them): 1- both reactants are soluble --- the reaction is precipitation Precipitation is the formation of precipitate of large complexes as the result of interacting soluble antibodies with soluble antigen; IgG is very efficient as antigen-antibody reactant making precipitate when the antigen is soluble actually. Actually precipitate can be in vitro as in the body the complexes that are formed are removed by phagocytic cells. 2- if the antigen is insoluble (e.g. bacteria, erythrocytes) and the antibody is soluble the result reaction is agglutination ( as IgG clamps the antigens; bring them together) As we know that IgG has just two Fab ( 2 antigen binding sites) and it is relatively short so it may not be able to bind large number of cells so the agglutination maybe invisible because of size limit ( although agglutination is a visible reaction) and we can make it visible by adding anti-IgG so the reaction will become larger. IgG2 has limited ability or doesn’t go through the placenta. The passage of IgG through the placenta can beneficial for the baby as to immunize it against the diseases that the mother is immune to, on the other hand it can cause a disease called hemolytic disease of newborn or Erythroblastosis fetalis; as the mother can have a different blood groups than the fetus the most important blood group that can be different is : the Rh; is composed of six antigens –in wiki 5 antigens- they are C , c , E , e , D (d indicates negative for D antigen and it is used to indicate that the individual has negative Rh ). If the mother is negative for Rh and the fetus is positive, transplacental hemorrhage may result from the exposure of fetus blood to the mother’s early in pregnancy or the mother could have synthesized anti-Rh from a previous pregnancy, previous abortion or even blood transfusion. So as a consequence of this sensitization, in the next pregnancy, transplacental hemorrhage will result in the production of large amounts of IgGs, and this is a secondary immune response. Those IgGs will pass through the placenta and reach the blood of the fetus and cause “hemolytic disease of the newborn” and this will result in what is called hydrops fetalis in which the baby will die in the uterus because of severe hemolysis. Sometimes we do intrauterine blood exchange in order to save the fetus. About opsonizations: there are two opsonins; IgG and C3b.

Activation of Complement - Classical or alternative pathway
ADCC - NK cells Activation of Complement - Classical or alternative pathway Neutralization of toxins - Excellent function against toxins such as tetanus and botulinum toxins - Inactivation of snake or scorpion venoms by blocking the active site -ADCC : Antibody dependent cell-mediated cytotoxicity. It is a killing mechanism mediated by antibodies by natural killer cells. -Activation of the complement is achieved by IgG as being part of an immune complex (Ag-Ab complex) and this is the classical pathway, or aggregated IgGs and this is the alternative pathway. -Neutralization of the toxins: IgG is the BEST to neutralize the active site of the toxin. The snake venom can cause severe hemolysis, and thus severe hemolytic anemia.

Immobilization of Bacteria
- IgG molecules are efficient in immobilizing bacteria - Reaction of IgG specific to flagella cause organisms to clump arresting their movement Neutralization of Viruses - IgG is an efficient virus neutralizing antibody - Act by inhibiting attachment, penetration, uncoating, or later steps Immobilization of Bacteria either as a whole or by the reaction with the flagella, so the bacteria will be agglutinated by IgGs.

Important Differences Between IgG Subclasses
% of total IgG 70 20 7 3 Half -life 23 Complement binding + +++ - Placental passage ++ Binding of Monocytes IgG is the most abundant immunoglobulin The most important and the most abundant type of IgGs is IgG1, and it performs all functions with a half-life of 23 days. Other subtypes are less important and differ from each other. IgG2 differ that it is unable to cross the placenta. IgG3 differ that it has a short half-life, it doesn’t really affect the total IgG half-life as it has a low concentration. IgG4 does not bind to the complement or to the monocytes.

IgA dimerisation and secretion
IgA is the major isotype of antibody secreted at mucosal surfaces Exists in serum as a monomer, or as a J chain-linked dimer, that is formed in a similar manner to IgM pentamers. S C S C J C s IIgA as a serum immunoglobulin has no important function. Actually the function of serum immunoglobulins are limited to IgM and IgG (IgM is the first to be produced in an immune response then comes the IgG then the IgA that is produced at a later phase). But IgG is very important in secretions it is the only arm of humeral-mucosal immunity (humeral immune response at the mucosal surface) but there are also short comings for mucosal immunity mediated by IgA because of the transient nature of its protection, as protection provided by IgA antibodies lasts for about 6 months to 2 years only, and it acts on the respiratory tract, GIT as well as the genitourinary tract. Whereas serum protection can last for decades, so the protection provided by IgG can last for several years or decades. *IgG is responsible for serum protection IgA1 is the most abundant both in serum and secretions. IgA exists in two subclasses IgA1 is mostly found in serum and made by bone marrow B cells IgA2 is mostly found in mucosal secretions, colostrum and milk and is made by B cells located in the mucosa

B Secretory IgA and transcytosis Epithelial cell J
‘Stalk’ of the pIgR is degraded to release IgA containing part of the pIgR - the secretory component J C S s J C S s J C S s IgA and pIgR are transported to the apical surface in vesicles Epithelial cell J C S s The secretory IgA is produced from intraepithelial B cells -that are located in the submucosa- they produces dimers of IgA, then this dimer binds to poly IgR and both; the receptor and IgA are internalized by the epithelial cells, which adds the secretary piece to the immunoglobulin and secrete it to the lumen of the mucosa where it provides protection. pIgR & IgA are internalised Polymeric Ig receptors are expressed on the basolateral surface of epithelial cells to capture IgA produced in the mucosa J C S s B cells located in the submucosa produce dimeric IgA B

Properties of IgA Serum IgA: Half life of 5.5 days, has no important biologic functions Secretory IgA: - Important primary immunologic defense against local infections on mucosal surfaces - No complement activity, therefore, no bacterial lysis - Bactericidal for Gram negative bacteria in the presence of lysozyme - Antiviral activity - Agglutinating activity It is bactericidal for Gram negative bacteria in the presence of lysozyme and not alone. Agglutinating activity of insoluble agents.

IgA facts and figures Heavy chains: a1 or a2 - Alpha 1 or 2 Half-life: IgA days IgA days Serum levels (mgml-1): IgA IgA % of Ig in serum: IgA IgA Complement activation: IgA1 - by alternative and lectin pathway IgA2 - No Interactions with cells: Epithelial cells by pIgR Phagocytes by IgA receptor Transplacental transfer: No There is no complement activation by classical pathway but by alternative pathway (only IgA1). IgA interacts with the epithelial cells by the Polymeric immunoglobulin receptor [pIgR], and interacts with the phagocytic cells by the IgA receptor. IgA is inefficient at causing inflammation because it doesn’t activate the complement or other cells. Vulnerability :susceptibility IgA1 can’t be truncated because the hinge is heavily glycosylated. To reduce vulnerability to microbial proteases the hinge region of IgA2 is truncated. In IgA1 the hinge is heavily glycosylated. IgA is inefficient at causing inflammation and elicits protection by excluding, binding, cross-linking microorganisms and facilitating phagocytosis

Biologic Properties of IgM
Predominantly found in the intravascular space Half life is about 5 days It is the only immunoglobulin class synthesized by the fetus beginning at approximately 5 months of gestation It is the first antibody to be produced and its presence indicates a recent infection -IgM is the first to be produced in the immune response. -IgM is NOT present extravascularly. It is the only immunoglobulin class synthesized by the fetus beginning at approximately 5 months of gestation >> this fact is very important because it enables the diagnosis of the intrauterine infections like rubella … etc. If the fetus has immunoglobulins of IgG class we cannot distinguish if it is from a maternal or a fetal source unless we wait for a while and measure the concentration again (if it is maternal then the concentration will decline, but if it is fetal then the concentration will rise). But if the baby has IgM it is for sure an intrauterine infection. - The only bacterial infection that crosses the placenta is syphillis, and the only parasitic infection is toxoplasmosis, and the remaining are viruses (many viruses can cause intrauterine infection).

Functions of IgM Agglutination - Very efficient
- Forms bridges between distant antigenic epitopes Isohemagglutinins - Naturally occurring against RBC antigens - Triggered by exposure to bacteria bearing similar determinants - Transfusion reactions Activation of Complement - Most efficient complement fixing antibody IgM is the most effieint one in agglutination because it has 10 binding sites (they actually can bind just 5 antigens at a time) Isohemagglutinins: are antibodies directed against red blood cells’ antigens, they are usually absent at birth but start to appear by the end of the first year of life (as these antibodies normally reach the titer of 4), they are against the blood group antigen that the individual lack. They are acquired by cross-reactivity with bacterial antigens and an individual will have antibodies against the blood groups that he doesn't have ( even if he was infected with a bacteria that has cross-reactive antigens with his RBCs’ antigens the body will recognize it as self so no AB will be produced) The creation of blood group antigens is a function of transferase enzymes that we inherit; N-acetylgalactosaminyltransferase and galactosyltransferase. This enzyme transfers the sugar and adds it to the red blood cells, so the addition of the sugar will create a protein. The addition of galactose creates the B protein, and the addition of N-acetylgalactosamine creates the A protein. The addition of both creates the AB proteins. If there is no addition, the result will be the O blood group. Activation of complement by IgM by the classical pathway.

Monomeric IgM IgM only exists as a monomer on the surface of B cells
Monomeric IgM has a very low affinity for antigen Cm4 Cm3 Cm2 Cm1 Cm4 contains the transmembrane and cytoplasmic regions. These are removed by RNA splicing to produce secreted IgM

IgM forms pentamers and rarely hexamers
Polymeric IgM IgM forms pentamers and rarely hexamers Cm4 Cm3 Cm2 Cm1 Cm3 binds C1q to initiate activation of the classical complement pathway Cm1 binds C3b to facilitate uptake of opsonised antigens by macrophages Cm4 mediates multimerisation (Cm3 may also be involved) Polymeric are formed in secreted immunoglobulins. Cm4 mediates multimerisation of immunoglobulins.

Multimerisation of IgM
1. Two IgM monomers in the ER (Fc regions only shown) 2. Cysteines in the J chain form disulphide bonds with cysteines from each monomer to form a dimer Cm4 Cm3 Cm2 C Cm4 Cm3 Cm2 C Cm4 Cm3 Cm2 C 3. A J chain detaches leaving the dimer disulphide bonded. s s C C C C s 4. A J chain captures another IgM monomer and joins it to the dimer. It is the secretary piece or a membrane segment that determine if the IgM will be a pentamer or a monomer (this will be discussed later in immunoglobulin diversity ). Cm4 Cm3 Cm2 C s Cm4 Cm3 Cm2 C 5. The cycle is repeated twice more 6. The J chain remains attached to the IgM pentamer.

IgM facts and figures Heavy chain: m - Mu Half-life: 5 to 10 days
% of Ig in serum: 10 Serum level (mgml-1): Complement activation: ++++ by classical pathway Interactions with cells: Phagocytes via C3b receptors Epithelial cells via polymeric Ig receptor Transplacental transfer: No Affinity for antigen: Monomeric IgM - low affinity - valency of Pentameric IgM - high avidity - valency of 10

Biological Properties of IGD &IgE
- No function except B cell maturation - Half life is 2-8 days IgE ( Reaginic antibody) - Half life is 2 days - Binds with high affinity to mast cells and basophils - No agglutination or complement fixing activities - Antiparasitic - Major role in hypersensitivity IgD activate B-cells.

IgD facts and figures Heavy chain: d - Delta Half-life: 2 to 8 days % of Ig in serum: 0.2 Serum level (mgml-1): Complement activation: No Interactions with cells: T cells via lectin like IgD receptor Transplacental transfer: No IgD is co-expressed with IgM on B cells due to differential RNA splicing Level of expression exceeds IgM on naïve B cells IgD plasma cells are found in the nasal mucosa - however the function of IgD in host defence is unknown Ligation of IgD with antigen can activate, delete or anergise B cells IgD may react with T-cells but it is most abundant on naïve B-cells.

IgE facts and figures Heavy chain: e - Epsilon Half-life: days Serum level (mgml-1): % of Ig in serum: Complement activation: No Interactions with cells: Via high affinity IgE receptors expressed by mast cells, eosinophils, basophils and Langerhans cells Via low affinity IgE receptor on B cells and monocytes Transplacental transfer: No Very low concentration in serum IgE appears late in evolution in accordance with its role in protecting against parasitic infections Most IgE is absorbed onto the high affinity IgE receptors of effector cells IgE is also closely linked with allergic diseases

Role for IgE on mast cells and basophils
High affinity receptor for IgE IgE binds to Fc receptor on mast cells, as the allergen binds to IgE attached to FcR, this cross linking of IgE will create a signal that transfer via the cell membrane and work on the expulsion of granules of the mast cells and the release of its contents. antigen antigen Antigen comes to the mast cell which already has IgE attached to its receptor

Immunoglobulins properties
IgG1 IgG2 IgG3 IgG4 IgM IgA IgE Classical pathway of complement activation + +/- - ++ Placental transfer Low affinity binding to phagocytes High affinity binding to macrophages and activated neutrophils -/+ High affinity binding to basophils or mast cells

Passive Sero - & Antibody therapy
① In 1890, injection of 0.2ml serum from tetanus-immunized rabbits into the abdominal cavity of mice protected from challenge of virulent tetanus bacteria (Dr. Von Behring) During the 1930s & 1940s, passive immunotherapy based on the transfer of Ab (measles & Hepatitis A) was used in clinical (medical) practice.

The passive transfer of immunity to tetanus
Sero -therapy Tetanus toxoid Immunized horse This mechanism can be used in patients who are sick of tetanus or diphtheria. Immune horse serum (tetanus antitoxin) Patient at risk of tetanus Patient protected The passive transfer of immunity to tetanus by means of antibody

Passive Immunity Immune protection produced by transfer of antibodies to a recipient from a donor Donor has been actively immunized Occurs naturally from mother to fetus during pregnancy and mother to infant during nursing Short-lived protection Passive immunity can be natural (like mother to fetus) or artificial (like injecting immunoglobulins to the host).

Antibody therapy Pooled plasma from thousands of donors -> treatment with solvents & the use of detergents that was highly effective in inactivating viruses. Intravenous immune globulin (IVIG) contains ~1018 Ab (mostly IgG) which may incorporate > 107 different Ab specificities Action mechanism of passively administered Ab. i) Activation of the complement pathway ii) Promotes opsonization, phagocytosis & killing of bacteria iii) mediate the killing of target cells by NK cells (ADCC) iv) neutralizes toxins & viruses When the host is immunodefient and can't make its own antibodies we inject him with large concentration of immunoglobulins with wide specificities. So that those individuals can live a normal life by injecting the antibodies from others.

The conventional polyclonal antiserum contains
a mixture of monoclonal antibodies Production of antibodies can be polyclonal or monoclonal. Polyclonal can be produced in your body as multiple B-lymphocytes are involved.

Monoclonal antibody and Hybridoma
We can select a single B-lymphocytes by producing single cell suspension, fusing them with tumor cells and produce more and more antibodies. The above figures shows the technique of last century to produce monoclonal antibodies that is by using animals and creating of hybridoma (fusing multiple myeloma cell with normal B-lymphocyte to create immortal cells that can produce infinite number of antibodies)

Uses of Monoclonal Antibodies
Diagnostic agents (histology, immunoassays) Experimental probes for cell biology Therapeutic agents What are the advantages over polyclonal antibodies raised by immunisation of larger animals? In diagnosis we use them to target a single molecule like CD5 , CD3 on T-lymphocytes to identify them Therapeutic agent as in cases of cancer we can direct monoclonal antibody to antigens on cancer cells or we can add to this antibody a toxin any other chemotherapeutic agent so the antibodies will act as a poison arrow that target very specific structure on the cancer cell that causes the killing of the tumor cell.

Therapeutic Monoclonal antibodies for killing lymphocytes

CD52 is strongly expressed on lymphocytes and not on blood stem cells
MYELOID CELLS We can eliminate a certain type of cells from a population like eliminating lymphocytes from blood as CD52 is strongly expressed on lymphocytes and not on other kinds of blood cells so we can use anti-CD25 with complement that will lyse all lymphocytes. PLATELETS RED CELLS

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