Serological reactions (Polyclonal) antisera lObtained a from animals (rabbits, goats, horses) after repeated immunisation by antigen. lMarkedly polyreactive – antibodies bind to many epitopes of the antigen but also with other antigens. lThis is advantageous in „classical“ serological reactions (agglutination, precipitation). clonal_selection Clonal selection theory Myeloma lTumor derived from plasma cell lThe tumor cells retain the capacity to secrete immunoglobulins lThe secreted immunoglobulin is a paraprotein - all secreted molecules have the same variable region (= react with only one concrete epitope) Monoclonal antibodies lPrepared by immortalization of B-cells from immunized mouse. lHybridoma is composed of an antigen-specific B- cell and mouse myeloma cell. lProduced antibodies are strictly monospecific and therefore cannot be used in several „classical“ serological reactions (agglutination, precipitation). l Preparation of monoclonal antibodies Laboratory use of monoclonal antibodies lHighly specific agent used for ELISAs, RIAs, determination of cells surface antigens… lBecause they react only with a single epitope, number of „bridges“ is to low to overcome repulsive forces in classical reactions like agglutination or precipitation. Clinical use of monoclonal antibodies lImmunosuppressive treatment (anti CD3, CD54, CD20) lAntinflammatory treatment – –Cytokine neuralisation (anti- TNFa, anti-IL1, IL6, IL-17) –Adhesion molecules blocade (anti-LFA-1….) lAnti-tumor treatment (anti-CD20, anti EGF..) lAnti allergic treatment – (anti-IgE, anti-IL15) lAnti aggregation treatment (anti- gpIIb-IIIa – blocks activation of thrombocytes) Two phases of serological reaction lPrimary phase – concrete antibody (with its variable region must be present) binds to a concrete epitope. = Specific phase of the reaction lSecondary phase – vizualization of the fact of previously occurred primary reaction. Serological reactions lAgglutinatin lPrecipitation lImmunoassays –RIA –ELISA –Immunofluorescence lReactions based on activation of complement cascade by complex-antigen-antibody lReactions based on neutralisation of some biologic effect of antigen Agglutination lReaction between antiserum and corpuscular antigen (erythrocyte, bacterium, latex corpuscle). The corpuscles are clumped together, which morfologically expressed as agglutinate. l Aglutinace II Aglutinace I lComplete antibodies: after reaction with antigen cause visible agglutination or precipitation reaction lIncomplete antibodies: despite the fact that the reaction between epitope and antibody occurred, the agglutinate or precipitate cannot be detected. l Cause: movalent antibody (IgA), low number of bridges between antigens, to intense repulsive forces between antigens… Coombs test Precipitation lReaction between polyclonal antiserum and soluble (molecular) antigen. A complex lattice of interlocking aggregates is formed. If performed in a solution the precipitate falls out of the solution. schema6a-01.png Immunodiffusion-I Gel Ags diffuse into gel setting up a concentration gradient Abs diffuse into gel setting up a concentration gradient schema6b-01.png Immunodiffusion - II Gel Large aggregates form at the place of equimolar concentrations of Ag & Ab Precipitate is formed only in the zone of equivalence l Imunodifuse IV ELISA ELISA chromagen Assay results ELISA READER Imunofluorescence - direct - indirect Imunofluorescence Unlabeled Ab DIRECT INDIRECT Tissue Section Tissue Section Fluorochrome Labeled Ab Fluorochrome Labeled Anti-Ig ANA Pozitive granular type IMM006 ANA – homogenous type