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 an 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

MAb



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)
–Adhesion molecules blocade (anti-LFA-1….)
lAnti-tumor treatment (anti-CD20…)
lAnti aggregation treatment (anti- gpIIb-IIIa)
lAntitoxins (digoxin)

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  agglutionation 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



Coombs



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.

Immunodiffusion-I
Imunodifise I


Immunodiffusion - II
l
Imunodifuse II

Immunodiffusion - III
Imunodifuse III


l
Imunodifuse IV


   ELISA



ELISA



Assay results






ELISA READER



   Imunofluorescence
- direct
    - indirect

•  Přímá



•  Nepřímá
Sérum


ANA
Pozitive granular type


IMM006
ANA – homogenous type