Immunoglobulin and Monoclonal antibodies Reported by Ganesh M

Immunoglobulin and Monoclonal antibodies Reported by Ganesh M. Sc. D endo student

What is immunoglobulin ? Immunoglobulin's 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

Immunoglobulin Fragments Structure/Function Relationships Fab Ag binding Valence = 1 Specificity determined by VH and VL Fc ( crystallizable) Effector functions Papain Fc Fab

Immunoglobulin Fragments Structure/Function Relationships Ag Binding Complement Binding Site Binding to Fc Receptors Placental Transfer

Ig. M Structure Ig. M normally exists as a pentamer (19 S immunoglobulin) but it can also exist as a monomer. In the pentameric form all heavy chains are identical and all light chains are identical. Thus, the valence is theoretically 10. Ig. M has an extra domain on the mu chain (CH 4) and it has another protein covalently bound via a S-S bond called the J chain. This chain functions in polymerization of the molecule into a pentamer.

Ig. M Properties a) Ig. M is the third most common serum Ig. b) Ig. M is the first Ig to be made by the fetus and the first Ig to be made by a virgin B cells when it is stimulated by antigen. c) As a consequence of its pentameric structure, Ig. M is a good complement fixing Ig. Thus, Ig. M antibodies are very efficient in leading to the lysis of microorganisms.

Ig. M Properties d) As a consequence of its structure, Ig. M is also a good agglutinating Ig. Thus, Ig. M antibodies are very good in clumping microorganisms for eventual elimination from the body. e) Ig. M binds to some cells via Fc receptors. f) B cell surface Ig

Surface Ig. M exists as a monomer and lacks J chain but it has an extra 20 amino acids at the C-terminus to anchor it into the membrane. Cell surface Ig. M functions as a receptor for antigen on B cells.

Surface Ig. M is non covalently associated with two additional proteins in the membrane of the B cell called Ig-alpha and Ig-beta. These additional proteins act as signal transducing molecules since the cytoplasmic tail of the Ig molecule itself is too short to transduce a signal.

Ig. D

Ig. D Properties a) Ig. D is found in low levels in serum; its role in serum uncertain. b) Ig. D is primarily found on B cell surfaces where it functions as a receptor for antigen. Ig. D on the surface of B cells has extra amino acids at C-terminal end for anchoring to the membrane. It also associates with the Ig-alpha and Ig-beta chains. c) Ig. D does not bind complement.

Ig. E structure

Ig. E Properties a) Ig. E is the least common serum Ig since it binds very tightly to Fc receptors on basophils and mast cells even before interacting with antigen. b) Involved in allergic reactions - As a consequence of its binding to basophils an mast cells, Ig. E is involved in allergic reactions. Binding of the allergen to the Ig. E on the cells results in the release of various pharmacological mediators that result in allergic symptoms.

Ig. E Properties c) Ig. E also plays a role in parasitic helminth diseases. Since serum Ig. E levels rise in parasitic diseases, measuring Ig. E levels is helpful in diagnosing parasitic infections. Eosinophils have Fc receptors for Ig. E and binding of eosinophils to Ig. Ecoated helminths results in killing of the parasite. d) Ig. E does not fix complement.

clinical implications of human immunoglobulin classes Ig. M Increases (in adults) in: a) Waldenström's macroglobulinemia b) Trypanosomiasis c) Actinomycosis d) Carrión's disease (bartonellosis) e) Malaria f) Infectious mononucleosis g) Lupus erythematosus h) Rheumatoid arthritis I) Dysgammaglobulinemia (certain cases) Note: In the newborn, a level of Ig. M above 20 ng. /dl is an indication of in utero stimulation of the immune system and stimulation by the rubella virus, the cytomegalovirus, syphilis, or toxoplasmosis.

clinical implications of human immunoglobulin classes Ig. M Decreases in a) Agammaglobulinemia b) Lymphoproliferative disorders (certain cases) c) Lymphoid aplasia d) Ig. G and Ig. A myeloma e) Dysgammaglobulinemia f) Chronic lymphoblastic leukemia

clinical implications of human immunoglobulin classes Ig. D Increases in a) Chronic infections b) Ig. D myelomas

clinical implications of human immunoglobulin classes Ig. E Increases in a) Atopic skin diseases such as eczema b) Hay fever c) Asthma d) Anaphylactic shock e) Ig. E-myeloma

clinical implications of human immunoglobulin classes Ig. E Decreases in a) Congenital agammaglobulinemia b) Hypogammaglobulinemia due to faulty metabolism or synthesis of immunoglobulins

Monoclonal antibodies are antibodies that are identical because they were produced by one type of immune cell, all clones of a single parent cell Polyclonal antibodies are antibodies that are derived from different cell lines 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.

In 1975, Georges Kohler and Cesar Milstein first fused lymphocytes to produce a cell line which was both immortal and a producer of specific antibodies. The two scientists were awarded the Nobel Prize for Medicine in 1984 for the development of this "hybridoma. " The value of hybridomas to the field was not truly appreciated until about 1987, when MAbs were regularly produced in rodents for diagnostics.

Monoclonal antibodies Antibodies produced from a single clone of B cells. Produced by fusing a B cell secreting the desired antibody with a myeloma cell capable of growing indefinitely in tissue culture. Monoclonal antibodies all have identical antigen-binding sites. Thus they all bind to the same epitope with the same affinity. They are all of the same antibody class (isotype).

Hybridomas Technique B lymphocytes can mutate into tumor cells that result in a type of cancer termed myeloma. - Myeloma cells become “immortal” and will grow indefinitely in culture. - Fusion of a single activated B cell and a myeloma cell will create a hybridoma that can grow indefinitely in culture

Hybridoma Selection The “HAT Trick” Myeloma cells have been genetically engineered such that they can not use hypoxanthine, aminopterin, and thymidine (HAT medium) as a source for nucleic acid biosynthesis and will die in culture. Only B cells that have fused with the engineered myeloma cells will survive in culture when grown in HAT medium.

Polyclonal antibodies Produced by: Many B cell clones Monoclonal Antibodies A single B cell clone Bind to: Multiple epitopes of all antigens used in the immunization A single epitope of a single antigen Antibody class: A mixture of different Ab classes (isotypes) All of a single Ab class Ag-binding sites: A mixture of Abs with different antigen-binding sites All Abs have the same antigen binding site Potential for crossreactivity: High Low

Monoclonal Antibodies Purified antigen molecule Inject antigen into mouse Kill the mouse and remove the spleen (containing antibody generating cells) spleen

Each cell may have produced a different antibody spleen fuse dissociate + A Hybrid cell that can be cultured “immortalized” cell

Select one of these "hybrid" cells to subculture…. … and produce a mono clonal population

USES Measuring protein and drug levels in serum Typing tissue and blood Identifying infectious agents Identifying clusters of differentiation for the classification and follow-up therapy of leukemias and lymphomas

USES Identifying tumor metastasis Identifying and quantifying hormones Immuno affinity Purification Used in several diagnostic tests to detect small amounts of drugs, toxins or hormones, e. g. monoclonal antibodies to HCG used in pregnancy test kits or diagnosis of AIDS by the ELISA test.

USES monoclonal antibody can be coupled to another molecule like a fluorescent molecule to aid in imaging the target Or with a strongly-radioactive atom, such as Iodine-131 to aid in killing the target. Used in the radioimmuno detection and radioimmuno therapy of cancer, and some new methods can even target only cancerous cells.

USES Monoclonal antibodies can be used to treat viral diseases, traditionally considered "untreatable". In fact, there is some evidence to suggest that antibodies may lead to a cure for AIDS. Monoclonal antibodies can be used to classify strains of a single pathogen, e. g. Neisseria gonorrhoeae can be typed using m. AB. To identify and to trace specific cells or molecules in an organism.

USES OKT 3 (immunosuppressant ) an antibody to the T 3 antigen of T cells, is used to alleviate the problem of organ rejection in patients who have had organ transplants.

Affinity chromatography: 1. Bind antibody to a support matrix (e. g. sepharose gel) 2. Add protein mixture - antigen binds to antibody on support 3. Wash to remove unbound material 4. Lower p. H - antibody releases the antigen - which is now free of contaminants

Affinity chromatography - antibody purification. Antigen can be bound to the support matrix in order to purify antigen-specific antibody from a polyclonal antiserum. From http: //users. rcn. com/jkimball. ma. ultranet/Biology. Pages/A/Affi nity. Chrom. html

Tumor diagnosis in vitro in vivo 99 m. Tc-m. Ab

Monoclonal antibodies for cancer treatment Three mechanisms that could be responsible for the cancer treatment. 1. m. Abs act directly when binding to a cancer specific antigens and induce immunological response to cancer cells. Such as inducing cancer cell apoptosis, inhibiting growth, or interfering with a key function

Monoclonal antibodies for cancer treatment 2. m. Abs can be modified for delivery of a toxin, radioisotope, cytokine or other active conjugates. 3. 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

Drawbacks of Monoclonal Antibodies Since monoclonal antibodies produce antibodies to a single determinant, they do not form the lattice necessary for precipitation and so cannot be used in precipitation assays, radial immunodiffusion, immunoelectrophoresis or agar gel diffusion. Very high titer antibodies also pose certain difficulties

ANY QUESTION …… Thank you all