Immunology iv.serology
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Transcript of Immunology iv.serology
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Serological reactionsSerological reactions
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(Polyclonal) antisera(Polyclonal) antisera
Obtained a from animals (rabbits, goats, horses) after repeated immunisation by antigen.
Markedly polyreactive – antibodies bind to many epitopes of the antigen but also with other antigens.
This is advantageous in „classical“ serological reactions (agglutination, precipitation).
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Clonal selection theory
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MyelomaMyeloma
Tumor derived from plasma cell The tumor cells retain the capacity to
secrete immunoglobulins The secreted immunoglobulin is a
paraprotein - all secreted molecules have the same variable region (= react with only one concrete epitope)
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Monoclonal antibodiesMonoclonal antibodies
Prepared by immortalization of B-cells from an immunized mouse.
Hybridoma is composed of an antigen-specific B- cell and mouse myeloma cell.
Produced antibodies are strictly monospecific and therefore cannot be used in several „classical“ serological reactions (agglutination, precipitation).
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Laboratory use of monoclonal Laboratory use of monoclonal antibodiesantibodies
Highly specific agent used for ELISAs, RIAs, determination of cells surface antigens…
Because they react only with a single epitope, number of „bridges“ is to low to overcome repulsive forces in classical reactions like agglutination or precipitation.
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Clinical use of monoclonal antibodiesClinical use of monoclonal antibodies
Immunosuppressive treatment (anti CD3, CD54, CD20)
Antinflammatory treatment –– Cytokine neuralisation (anti- TNF)– Adhesion molecules blocade (anti-LFA-1….)
Anti-tumor treatment (anti-CD20…) Anti aggregation treatment (anti- gpIIb-IIIa) Antitoxins (digoxin)
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Two phases of serological reactionTwo phases of serological reaction
Primary phase – concrete antibody (with its variable region must be present) binds to a concrete epitope. = Specific phase of the reaction
Secondary phase – vizualization of the fact of previously occurred primary reaction.
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Serological reactionsSerological reactions
Agglutinatin Precipitation Immunoassays
– RIA
– ELISA
– Immunofluorescence
Reactions based on activation of complement cascade by complex-antigen-antibody
Reactions based on neutralisation of some biologic effect of antigen
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AgglutinationAgglutination
Reaction between antiserum and corpuscular antigen (erythrocyte, bacterium, latex corpuscle). The corpuscles are clumped together, which morfologically expressed as agglutinate.
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Complete antibodies: after reaction with antigen cause visible agglutionation or precipitation reaction
Incomplete antibodies: despite the fact that the reaction between epitope and antibody occurred, the agglutinate or precipitate cannot be detected.
Cause: movalent antibody (IgA), low number of bridges between antigens., to intense repulsive forces between antigens…
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Coombs test
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PrecipitationPrecipitation
Reaction 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.
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Immunodiffusion-IImmunodiffusion-I
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Immunodiffusion Immunodiffusion - - IIII
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ImmunodiffusionImmunodiffusion - - IIIIII
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ELISA
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ELISA
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ELISA READER
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Imunofluorescence
- direct - indirect
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• Přímá
Imunofluorescence
• Direct
Ag
FluorochromeLabeled Ab
Tissue Section
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• Nepřímá
Sérum
Imunofluorescence
Ag
FluorochromeLabeled Anti-Ig
Tissue Section
UnlabeledAb
• Indirect
Sérum
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ANA Pozitive granular type
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ANA – homogenous type