Immunochemical assays

                                           Seminar No. 7

                                           - Chapter 23 -

              Immunochemical assays are based on antibody-antigen in vitro interaction

                                                 Q.

                                         What is antibody?

                                              Antibody

*      a defense protein synthesized by the immune system

*      immunoglobulins are plasma proteins produced by lymphocytes in response to some external
molecules

*      they are capable to bound specifically to antigen





                               The scheme  of immunoglobulin molecule

                                                 Q.

                       How many chains are there in immunoglubulin molecule?

                                      Immunoglobulin molecule

*      Y-shaped molecule

*      two heavy chains (H)  +  two light chains (L)

*      the chains are composed from series of domains

*      each chain contains variable (V) and constant (C) domains

*      each domain is closed by a single -S-S- bridge







                                                 Q.

                                          What is antigen?

                                  Antigen  =  antibody generating

*      a foreign macromolecule that provokes the formation of antibodies

*      proteins, glycoproteins, lipoproteins, polysaccharides ...

*      may be bound on the surface of cells or particles (viruses, bacteria, pollens, tissues from
another individual)

----------------------------------------------------------------------

*      hapten (= incomplete antigen), small molecule

*      can become antigen when bound to protein

                           Interaction of antigen (Ag) with antibody (Ab)

*       highly specific

*       based on non-covalent interactions (mainly hydrophobic)

*       Ag  +  Ab  (r)  immune complex  (r) other changes (in vivo, in vitro)



*       in vivo:  phagocytosis

*       in vitro: precipitate (insoluble in water)

                                   Immunochemical assays exhibit
                                   high specifity and sensitivity

                                     Immunoturbidimetry (p. 10)

*      turbid = not transparent

*      absorbance of light in colloid solution is measured

*      A ~ c (of immune complex)

                                     Immunonephelometry (p. 10)

*      nepheloid  =  turbid

*      intensity of scattered light is measured

*      I  ~  c (of immune complex)

                                   Single radial immunodiffusion

*      only one reactant diffuses

*      Ag is applied into a hole in gel medium

*      gel is impregnated with antibody

*      Ag diffuses in all directions (360°)

*      a circular immunoprecipitate is produced

                                       Double immunodiffusion

both reactants (Ag, Ab) diffuse in all directions

The proof of Ag identity

*      central well contains Ab, six Ag samples are places around

*      if the same antigens are in opposite wells   TH                                  
precipitation lines fuse (identity)

*      if antigens are completely different   TH                                     precipitation
lines cross each other (non-identity)

*      if two antigens are related but not identical   TH                       partially crossed
lines (partial identity)

                                Immunoelectrophoresis of Ag mixture

*      scheme on p. 139

*      Ag mixture is applied into a hole in agarose gel

*      simple electrophoretic separation afforded 5 fractions

*      then antiserum (= mixture of antibodies) is applied into  side troughs

*      antibodies and separated antigens diffuse

*      the result is a typical pattern of several precipitation lines



                                         Rocket immunoelpho

*      samples of Ag  move through the gel with antibody

*      precipitations are of rocket shapes (bec. of acceleration by electric field)

*      for a given Ab concentration there is a linear relationship between the height of rocket and
the Ag concentration

                                        Crossed immunoelpho

*      a more sensitive modification

*      produces higher resolution

*      1^st separation: elpho

*      2^nd separatation: elpho at 90° angle to the 1^st run through gel containing polyvalent
antibody





                                        Enzyme immunoassays

*      Ab or Ag/hapten is labelled with covalently attached enzyme

*      peroxidase (isolated from horseradish) catalyzes reduction of H[2]O[2] or organic peroxides

*      ALP (alkaline phosphatase, isolated from Escherichia coli) catalyzes the hydrolysis of
synthetic alkyl phosphates

                                           Homogenous EIA

*      free hapten and enzyme-labelled hapten compete for binding sites in antibody

*      enzyme in immunocomplex is not active, access to active site is blocked

*      activity of free enzyme-hapten conjugate (= quantity of product) is determined

*      the amount of product is directly proportional to the amount of hapten in sample

                                           ELISA methods

                                Enzyme  Linked  Immunosorbent  Assay

                                         Competitive ELISA

*      antibody is bound to solid phase (sorbent)

*      antigen to be analyzed (Ag) and Ag-E conjugate compete for binding sites in antibody

*      enzyme in immunocomplex is active and catalyzes                     the conversion of
substrate to product

*      the amount of product is indirectly proportional to the amount of Ag in sample