Immunoelectrophoresis

Immunoelectrophoretic methods combine electrophoresis with subsequent immuno-detection, and the  conditions  used are  a compromise of the  optimum  for  each  of  the  two  steps.  A  number  of immunoelectrophoresis methods  will be discussed below, as it is useful to have an appreciation of the  principles  of each method,  but it must be emphasized that  methods  involving  immunoprecipitation  have  today largely been replaced  by  methods  involving  amplification,  most commonly with enzymes.

1. Cross-over electrophotesis

One of the  limitations  of the  Ouchterlony double diffusion system is that a large proportion  of the Ab and Ag diffuse in non-productive directions and are thus wasted.  Only that proportion  of the  Ab that diffuses towards the  Ag, and  vice versa,  will productively  form immunoprecipitate.

It will be  recalled from the discussion of paper electrophoresis that due to Electroendosmosis the γ-globulin  which are the  antibodies  migrate towards the  cathode,  whereas all of the  other  serum  proteins  migrate towards the anode.  For the  analysis  of blood  proteins, this provides a way of ensuring that all of the  Ag meets  all of the  Ab, by a process known as cross-over electrophoresis. In this process, the Ab and Ag are placed in two  wells in an  agarose  gel and subjected to electrophoresis as illustrated in Fig.  1.  In this  way all of the  Ag encounters all of the Ab  and wasteful diffusion is obviated.  This may be useful when the  amount  of Ag  available  is  limited,  for  example  in forensics.

Figure 1.  Cross-over electrophoresis.

2. Rocket electrophoresis

A limitation  of Mancini  radial diffusion  is that differences in the  diameters of the precipitin  rings may  be small.  The reason for this is that  diffusion is in all directions, and a consequence is reduced sensitivity In the “rocket”  electrophoresis  method  of Laure, diffusion of the Ag is replaced by electrophoresis of the Ag into the gel containing the Ab.  Electrophoretic  migration  of the  antigen  occurs  in one direction only so that immunoprecipitation, instead of occurring in a circle. occurs in a rocket shape (Fig.  2). hence the name.

Figure 2.  Rocket electrophoresis.

An advantage of rocket electrophoresis  is that  it is more  sensitive than Mancini radial diffusion. because, being “pulled out” in one direction only, differences in the length of the  rockets are greater and are more easily measured.  A standard curve of rocket  length  vs antigen concentration can be constructed and used to determine the concentration of unknowns analyzed under the same conditions.

3.  Grabar-Williams  immunoelectrophoresis

Grabar-Williams immunoelectrophoresis is a binary method in  which an Ag  mixture  is  first  separated  by electrophoresis and the  separated  components are subsequently detected by immuno-diffusion. It may be considered as a development  of the  Ouchterlony  technique,  with  better resolving power because of the  separation  of the  Ag mixture  in the electrophoresis step.

Conditions used have to be a compromise between the requirements  of the two stages.  For example;-

•  Electrophoresis is  best  performed  in  a  sieving  gel,  such  as polyacrylamide, partly because this restricts diffusion. However, immunodiffusion is dependent upon diffusion and so agarose, a non-sieving gel, is used as this does not impede diffusion.

• The  requirement  for  immunoprecipitation  restricts  the  buffer  pH, which must not be too far from physiological pH.

•  Both electrophoresis and  immunoprecipitation  require a buffer of low ionic strength.

The sample is introduced into a well cut into an agarose gel, supported on a glass slide,  and  is separated  by electrophoresis.  To  prevent  protein from migrating off the end of the gel, a sample of bromophenol blue may

be run in  parallel  with  the  protein  and the  run  stopped  when the bromophenol blue reaches the end of the gel.  A trough is then cut, parallel to the direction of electrophoresis and a few millimeters from the well (Fig.  3).  Ab is introduced into the trough and the gel is incubated as described for the  Ouchterlony technique for several days to allow formation of the immunoprecipitin bands.

Figure 3.  Grabar-Williams immunoelectrophoresis.

Optimal proportions of Ab and Ag may be established by a prior Ouchterlony analysis, or replicate runs, covering a range of Ab and Ag concentrations, may be carried out.

4. Clarke-Freeman 2-D immunoelectrophoresis

Rocket electrophoresis   is a method for the quantitation of Ags by electrophoresis into an Ab-containing gel.  In  the rocket method different Ags are placed in different wells and either the Ag must be pure  or,  more  usually,  a mono-specific antiserum is used.  In Grabar-Williams immunoelectrophoresis    impure Ag mixtures are first separated by electrophoresis, before being analyzed by immuno-precipitation. The Clarke-Freeman technique combines aspects of Grabar-Williams  immunoelectrophoresis  and of rocket electrophoresis. The sample is first separated by electrophoresis in one dimension. as in the Grabar-Williams technique, and then  at right-angles to the first electrophoresis   is electrophoretically  drawn  into  an Ab-containing gel, as in the rocket technique.  Like rocket  electrophoresis, the latter stage is a form of cross-over electrophoresis  and so an advantage over the Grabar-Williams technique  is that all  of the  Ag  meets Ab and there is no wasteful, unproductive, diffusion.

Figure 4. Clarke-Freeman immunoelectrophoresis.

Sample in the well is separated by electrophoresis  in the  first dimension. A gel containing Ab is then cast adjoining the first gel along one of its edges.  The  separated sample components  are then  drawn into the Ab-containing gel by electrophoresis in the second dimension.

References

Dennison, C. (2002). A guide to protein isolation . School of Molecular mid Cellular Biosciences, University of Natal . Kluwer Academic Publishers new york, Boston, Dordrecht, London, Moscow .