ANTIGEN * An antigen is defined by its antibody * Can be small molecule or macromolecule * Contact/recognition area on the antigen is called the epitope * Can have more than one epitope on an antigen * epitopes are unique rendering specificity to antibody-antigen reactionANTIBODY * Protein specifically synthesised upon contact between an animal and a foreign molecule (antigen) * Consist of constant region and then variable regions specific for antigen * Is very specific for its antigen * Several classes of antibody (IgA, IgE, IgG, IgM) all binding antigen, but differing in polypeptide compositionSTRUCTURE OF ANTIBODIES Basic Structure at Si re : “Bm, a* variable yy light chain heavy chainANTIBODY PRODUCTION * Purify antigen. * Immunize animal. * Bleed animal. Check antiserum for specific antibodies. Polyclonal! * Fuse spleen cells (antibody producers) with a cancer cell line to produce hybrids secreting antibody. Screen hybrids for specific antibody secretion. * Clone hybrids to ensure antibodies come from a single parent cell. Monoclonal!Immunization Blood collection ===> ne Antiserum Polyclonal antiserum productionae forming oa oO) fe \ ion Hybridomas xe@) = = @ Nes oclonal antibodies salnted for eulteraalans Monoclonal Antibody Productionclonal Single/Multiple Monoclo Single Require new animal when antiserum depleted Infinite. Clones can be frozen and revived when required Every batch of antiserum must be checked Specificity selected only once - at screening of clones Every batch of antiserum must be characterised Characterise original clone Intermediate HighThe Five Immunoglobulin (Ig) Classes lg IgG Secretory IgA Ig ‘gD pentamer monomer dimer monomer | monomer ‘Secretory component sie Y PES Heavy chains " y a € 3 Number of antigen 10 2 4 2 2 binding sites Molecular weight (Oaltons) 900,000 150,000 385,000 200,000 180,000 Percentage of total antibody in 6% 80% 13% 0.002% 1% serum Crosses placenta no yes no no no Fixes complement yes yes no no no ‘mast cells and Fe binds to phagocytes moons Main blood antibody of | Secreted into mucus, | Antibody ot | 8 call Function secondary responses, | tears, saliva, colostrum | allergy and | receptor antiparasitic IgM Serves as the activity receptorCopyright © The MoGraw-Hill Companies, Inc. Permission required for reproduction or display. Characteristics of the Immunoglobulin (Ig) Classes Number of Antigen Binding Sites Molecular Weight Percentage ot Total Antibody in Serum Average Half-Life in Serum (Days) Crosses Placenta? Fixes Complement? Fe Binds To Biological Function Dimer, Monomer 4 2 170,000-385,000 ¥¥ Monomer — Monomer 180,000 1% a 25 No No Receptor on BeellsAntibody Classes GENERAL STRUCTURE Monomer entamer Monomer Dimer Monomer LOCATION Free in plasma; about 80 percent of circulating antibodies Surface of B cell; free in plasma Surface of B cell Monomer found in plasma; polymers in saliva, tears, milk, and other body secretions Secreted by plasma cells in skin and tissues lining gastrointestinal and respiratory tracts FUNCTION Most abundant antibody in primary and secondary responses; crosses placenta and provides passive immunization to fetus Antigen receptor on B cell membra first class of antibodies released by B lls during primary response Cell surface receptor of mature B cell; important in B cell activation Protects mucosal surfaces; prevents attachment of pathogens to epithelial Found on mast cells and basophils; when bound to antigens, triggers release of histamine from mast cell or basophil that contributes to inflamma- tion and some allergic responsesIMMUNOMETRIC METHODS * Methods using antibodies * Require antigen and antibodyCOMPLEMENT FIXATION TEST Antigen ~ Aurbody ~ Complement Skeip RBC — Anibody te Shsxp REC oe (sentitizemoa of Esra]IMMUNOMETRIC TECHNIQUES PRECIPITIN REACTIONS ee copmalion Ewes comple. | 4. aL | 7 oe te y | ae ah > sal | ‘efo [9 Antibo body exces a Equivolence Soluble complexes : ce ote | oo —_ ~< a _ Antigen © PY Monovalent haptenSolution Supernate Suspension PrecipitatePrecipitation - Principles - The precipitation reaction is an older assay technique that originally relied strictly on visually detection to identity antigen-antibody interactions. Most older assays were performed in gels permeated with antibody. The antigens (in plasma or some other fluid) to be detected were placed into wells cut in the gels and allowed to diffuse into the gel containing the antibody. The antigen antibody complexes formed would then precipitate in the gel and be visible as a white precipitate. Assays for qualitative detection as well as quantitation were available using gel based assays.Well containing Line of precipitation Well containing antigen molecules antibodies against the antigen 1 } yo ~ Zone of Zoneof Zone of antigen optimal antibody (a) excess precipitation excessIMMUNOMETRIC TECHNIQUES PRECIPITIN REACTIONS 2. ImmunodiffusionSingle dimension double diffusion | __ Antigen b-— Two precipitate bands (two antigens) }— Agar +— Antiserum CTH Double dimension double diffusion (Ouchterlony) Identical Antigen are different Antiserum Agar plates Crossreactive Antigens are different Antiserum AntiserumIMMUNOMETRIC TECHNIQUES PRECIPITIN REACTIONS 5. Rocket electrophoresis Antibody in agarose| concentration — ayIMMUNOMETRIC TECHNIQUES Immuno-enzymatic Techniques ELISA Several variations Very sensitive Quantitative and qualitative systems User-friendly Stable reagentsIMMUNOMETRIC TECHNIQUES Labeled Antibody Techniques Western blottingWestern blot using UniVtg.IMMUNOMETRIC TECHNIQUES Labeled Antibody Techniques Immunohistochemistry Human Papillomavirus DNA demonstrated by In Situ Hybridisation (pink) in epithelial cells identified by indirect immunofluorescence using antibody against cytokeratin (green)