ANTIBODIES

Glycoprotein substances synthesized by plasma cells in response to antigenic stimulation. Structurally all antibodies are immunoglobulin but functionally, not all immunoglobulin are antibodies. Immunoglubulins are considered to be the humoral branch of the immune response

Complement fixing antibodies Blocking or Inhibitory antibodies

ACCORDING TO THEIR IN-VITRO BEHAVIOR

PROPERTIES OF ANTIBODIES Protein in nature With high molecular weight Present in serum/plasma, saliva, semen, CSF and other body fluids CLASSIFICATION OF ANTIBODIES IgG IgA IgM IgE IgD ELECTROPHORESED AT PH=8.6

STRUCTURE OF IMMUNOGLOBULIN Four (4) polypeptide chains: 2 identical LIGHT chains 2 identical HEAVY chains. Both light and heavy chains are held together by COVALENT DISULFIDE BONDS. Heavy chains are interconnected by DISULFIDE LINKAGES in the HINGE region. 2 terminal regions: Carboxyterminal - with constant amino acid sequence (constant region). Aminoterminal - with varying antibody specificity (variable region).

ACCORDING TO ITS SEDIMENTATION CONSTANT

TREATMENT WITH PAPAIN WILL PRODUCE 3 FRAGMENTS ACCORDING TO TEMPERATURE AT WHICH THEY REACT Cold antibodies Warm antibodies ACCORDING TO OCCURRENCE Natural antibodies Immune antibodies ACCORDING TO THE SPECIES WHICH PRODUCE THEM Isoantibodies Heterophile antibodies ACCORDING TO ITS REACTION WITH AN ANTIGEN Agglutinins Precipitins Agglutinoids Hemagglutinins Lysins Opsonins Neutralizing antibodies Allergic antibodies Antitoxins One Fc fragment - fragment crystalline; involve in the Ig biologic function involved in complement fixation, placental transfer of Ig and serve as the binding site for cell Two Fab fragments - capable of antigen binding even without the Fc but cannot agglutinate of precipitate

Treatment with pepsin results in digestion of Fc fragment, leaving 2 Fab fragments. The two Fab fragments has two antigen combining sites (Bivalent), therefore, it is capable of antigen binding, precipitation and agglutination.

DOMAINS OF IMMUNOGLOBULIN Variable region - responsible for specificity; the antigen binding site CH2 - binds with complement, specifically C1q, and initiates complement pathway CH3 - responsible for cytotropic reactions involving macrophages and monocytes, mast cells, cytotoxic killer cells and B cells CL - responsible for the light chain type: either kappa or lambda

IgG

DOMAINS Globular regions on polypeptide chain stabilized by intrachain disulfide bonds. Domains on the heavy chain VH, CH1, CH2, CH3, (CH4) Domains in the light chain VL, CL DISULFIDE BONDS chemical bonds essential for the normal 3 dimensional structure of Ig. 2 types of disulfide bonds: Interchain Intrachain REGIONS ON POLYPEPTIDE CHAIN

Predominant Ig among humans comprising 75-80% of the total Ig pool. Has 4 major sublasses: IgG1, IgG2, IgG3, IgG4 Equally distributed in the different fluid compartments with detectable amounts in CSF and urine Readily diffusible IgG antibody response appears later than IgM in primary response but they form the major antibody of the secondary immune response. Maternal IgG is actively and selectively transferred across the placenta to the fetus and imparts passive protection to the newborn for 6-9 months

Functions of IgG: IgM Provides immunity for the newborn Complement fixation Opsonization Neutralization of toxins and viruses Participation in agglutination and precipitation reactions The largest of the Ig accounting for 5-10% of the total immunoglobulin pool. Star-shaped in the free state; crab-like in Ag-Ab reaction. The earliest to appear in the 1o immune response but it does not persist for long. Maternal IgM does not cross the placenta. IgM detection in newborn is a useful indicator of intrauterine infection A powerful agglutinin of a particulate antigen

IMMUNOGLOBULIN VARIABILITY Isotypic Variation- refers to the different heavy and light chain classes and subclasses Allotypic variation- refers to the genetic variation within a species involving different alleles at a given locus Idiotypic variation- refers to the diversity at the binding site and in particular relates to the hypervariable segments of the antibody combining site (paratope).

Functions of IgM: Complement fixation Agglutination Opsonization Neutralization of toxins Surface receptor for antigens (on B-cells)

IgA

15-20% of human serum Ig pool. Found in serum in small amounts but predominant in sero-mucous secretions of he respiratory tract, genito-urinary tract and GI tracts. It is also found in tears, sweat, saliva, colostrum and breastmilk.

provided by genetic elements in the nucleus of the cell rather from the antigen CLONAL SELECTION THEORY Individual lymphocytes are genetically pre-programmed to produce one type of immunoglobulin, and that specific antigen finds or selects those particular cells capable of responding to it, causing these to proliferate. Repeated contact with the antigen would continually increase a lymphocyte pool MONOCLONAL ANTIBODIES Heterogeneity of the antiserum- phenomenon where in several B cell clones are involved, each producing a slightly different Ab molecule to the myriad antigenic determinants, encompassing the whole antigen Polyclonal antiserum antiserum coming from many antibody producing B cell clones Monoclonal antibody product of one to one cell hybridization; antibody produced to a single determinant by a single hybridized B cell Cell lines grew continuously in culture because of the cancerous myeloma hybrid, forming a little tumor

FORMS OF IgA Serum IgA- can agglutinate motile infectious agents thus promoting their phagocytosis but they cannot activate the complement system Secretory IgA- a polymeric form stabilized a short polypeptide chain. It is known as the antiseptic paint of mucous membranes. It can activate the bacteriolytic activity through the alternate pathway of complement system and only in the presence of lysozyme Heat labile immunoglobulin, accounts for less than 1% of the total serum Ig but is known to be present in large quantitites on the membrane of many circulating immunocompetent B lymphocytes. Detectable by highly sensitive assay requiring radiolabelled antisera Precise biological action is not known but it may play a role in antigen-triggered lymphocyte differentiation. Heat labile immunoglobulin. Least abundant Ig in the serum accounting for only 0.004% of the total serum Ig. Synthesized locally by plasma cell present in the mucous membrane of the GI and respiratory tracts. It is unable to fix the complement via the classical pathway. It is homocytotropic due to its affinity for cells of the host species, particularly for tissue mast cells and blood basophils. Because of its ability to attach to the human skin, it is associated with immediate hypersensitivity reactions but also, apparently , with immunity to certain helminthic parasites. Also known as reaginic antibody/nuissance antibody

IgD

ADVANTAGES OF MONOCLONAL ANTIBODIES Exact same antibody can be produced by different groups of workers Large amounts of antibody can be produced thus providing a steady supply of reagents

IgE

DRAWBACKS OF MONOCLONAL ANTIBODIES Do not form lattice necessary for precipitation, so cannot be used in precipitation assays, radial immunoassay, immunoelectrophoresis or agar gel diffusion May not fix complement, hence not useful in complement fixation tests May recognize Ag of low reactivity as in recipients of multiple transfusions

PRODUCTION OF HYBRID ANTIBODY MOLECULES DNA transfection immunoglobulin gene DNA can be introduced into myeloma cells; DNA can be custom-altered to yield modified monoclonal antibodies Recombinant antibodies can be produced with the desired antigen binding capacity and fused to a portion of a molecule with enzymatic function

THEORIES OF ANTIBODY PROUCTION EHRLICHS SIDE CHAIN THEORY Certain cells has had specific surface receptors for antigen that were present before contact with antigen occurred. Once antigen was introduced, it would select the cell with proper receptors , combination would take place and then receptors will break off and enter the circulation as antibody molecules. New receptors will be formed in place of those broken off and this process could be repeated. THE TEMPLATE THEORY Antibody producing cells are capable of synthesizing a generalized type of antibody, and when contact with an antigen occurs, the antigen serve as a mold or template and alters protein synthesis so that antibody with a specific fit is made. The molded antibody then enters the circulation, while the antigen remains behind to direct further synthesis SELECTIVE THEORY Assumes that antibodies are synthesized in a manner similar to that of other proteins. Instructions for their synthesis are

SOME APPLICATIONS OF MONOCLONAL ANTIBODIES Enumeration of human lymphocyte sub populations Analysis of viral antigens Analysis of putative protective antigens Analysis of immunologically competent cell surface molecules Blood grouping HLA typing Diagnosis of cancer In autoimmunity and immune deficiency In the control of fertility Monoclonal mutants