Immunoprecipitation

Immunoprecipitation  is  the  basis  of a number  of analytical  techniques, which will be discussed below.  Many  of these  techniques  are  very ingenious, but they  are  now mostly  of historical  interest  only.  The reason is that  immunoprecipitation requires relatively  large amounts of antibody and antigen in order to form  a visible immunoprecipitate,  i.e.  it is not  a very  sensitive  technique,  and so it has largely  been  replaced  by techniques which involve  an amplification  step  to  make  the  Ab/Ag reaction more easily detected.

The paratope of an antibody and the complementary epitope on an antigen have a very specific stereo  relationship  with one  another.  If  an antigen contains at least two epitopes,  it may form  a precipitate  upon reaction with its  specific (polyclonal)  antibodies at  optimal  proportions. Monoclonal or peptide antibodies, which target only a single epitope,  will not form an immunoprecipitate, as they will not be able to form the extended network necessary.  The  mechanism of formation  of an immunoprecipitate is shown in Fig. 1.  Formation of the matrix required for immunoprecipitation requires at least two epitopes, each targeted by a different antibody.

Figure 1. The formation of an immunoprecipitate.

Formation of an immunoprecipitate requires optimal proportions of antibodies and antigen and if either the antibody or the antigen .

Figure 2. Formation  of soluble complexes in the presence of an excess of antigen.

Figure 3.  Formation of soluble complexes in the presence of an excess of antibody.

The dependence of immunoprecipitation on the proportions of Ab and Ag can be expressed graphically, as in Fig.  4.

Figure 4.  Immunoprecipitation at different proportions of Ab and Ag.

Often, with a novel combination of Ag and Ab, the optimal concentrations are not known.  This has led to the use of diffusion techniques, where the diffusion of either the  Ab or the  Ag generates  a concentration gradient of that molecule.  The optimal concentration  will occur somewhere  along the  concentration  gradient  and will lead to formation of an immunoprecipitate at that point.

Immunoprecipitation is also affected by pH and in general precipitation will not occur substantially outside of the range pH 5→9 .

1- Immuno single diffusion

Immunodiffusion is usually conducted in macroreticular agarose gels, which serve to stabilize the system against flow-induced disturbances,  such as convection, but do not  impede diffusion.  In immuno single diffusion, the one component is present throughout the gel at a constant concentration, while the other component diffuses into the gel from solution. In immuno single diffusion, the  precipitate  band moves  further  into the gel with time, due to the continual diffusion into the gel of the component originally present in solution.  The precipitate band also tends to be indistinct  as it  is spread over  an  area,  with the  precipitate band being formed on its leading edge and redissolving on the  trailing edge. This “fuzziness” of the bands makes it difficult to determine how many precipitate bands there may be.

Figure 5. Immuno single diffusion.

The Ag or the Ab is present throughout the gel at a constant concentration and theothercomponent is allowed to diffuse into the gel from solution, where it is present at a higher concentration. Immunoprecipitation will occur at the positions where the Ab and Ag are present in equivalent concentrations (indicated by the vertical lines in Fig. 5). As the one component will continue to diffuse into the gel over time, the position of the immunoprecipitate will move further into the gel with time and the precipitate will not form a sharp line, as it will be re-dissolving on one edge and precipitating on the other.

1.1 Mancini radial diffusion

A practical,  quantitative,  single diffusion system is Mancini  radial diffusion.  In this method the Ab is added to the gel and the  Ag to  a well cut into the gel.  The  Ag diffuses into  the  gel and forms a precipitate where it meets the Ab in optimal proportions,  forming  a circular precipitin line surrounding the central well.  With time, the circular precipitate will grow in diameter until the supply of Ag is exhausted, at which point  the  growth in diameter ceases.  The  method  gives  a quantitative measure of the amount of Ag, because the more  there  is, the larger will be the diameter of the circle of the  precipitin  band surrounding the central well.  A  standard curve can be  constructed  from  the  diameters obtained with known  concentrations  of a standard Ag, and this  can be used to  determine  the  concentration  of an  unknown,  from  the  diameter of its  precipitin  circle.

Figure 6.  Mancini radial diffusion.

2- Immuno double diffusion

The limitations of immuno single diffusion can be overcome  by using immuno double diffusion.  In this case the Ab and the Ag diffuse into opposite ends of the gel and form a precipitate  where they meet in equivalent concentrations.  With  time,  the  position  of the immunoprecipitate will not change, but simply more precipitate will form

at the  same position as the  Ab and Ag continue  to  diffuse into  the  gel (Fig. 7).  This is assuming that the Ab and Ag continue to diffuse at the same relative rate, which they will do if the temperature is kept constant. If there  is  more  than  one  antigen/antibody  couple  present,  then  each of these  systems  will  form  their  own  precipitate  band.  If the  antigens  are of different  sizes or  shapes,  they  will  diffuse  at  different  rates  and  will form precipitate bands, at different places.  (IgG antibodies are  all of the same size and gross shape and so will all diffuse at the  same  rate).

Because the precipitate band(s) formed by double diffusion are very sharp, different bands are easily distinguished from  one  another  and so  it is relatively easy to determine how many bands have been formed.

Figure 7.  Immuno double diffusion.

2.1 Ouchterlony double diffusion analysis

Although the concentration  gradients formed  during immuno  double diffusion enable the Ab and Ag to “find” one another at optimal proportions, sometimes the starting concentration of either the Ab or the Ag is out of range,  i.e.  is too  high or too  low, and  so it is necessary, with an unknown system, to repeat the experiment using different Ab and Ag concentrations.  An efficient way of doing this  was devised by Ouchterlony.  In this method the gel is cast as a layer,1 →1.5  mm thick, on a support (a Petrie  dish is convenient)  and a pattern  of wells (Fig. 8) is cut into  it using a die and template.  Ab and Ag are added to appropriate  wells; typically, Ag may be added to  the  central  well and a serial dilution of Ab added to the surrounding wells.

Fig. 8 shows a single central well surrounded by six circumferential wells, but the  Ouchterlony  technique  is not  limited to  this  arrangement. The pattern  can be extended  ad infinitum,  to accommodate different Ab/Ag concentrations.

The serial dilution series is conveniently  constructed  in the  wells of a microtitre plate.  A constant volume, say 100 µl, is added to 5 wells of the plate,  100 µl  of antiserum is added to the first well and mixed in,  100 µl of this  mixture  is transferred  to  the  next  well,  mixed  in,  and  100  µl transferred to the next well etc.,  until the  last well contains  200  µl of mixture containing 1/32 of the original concentration of Ab.

Figure 8.  Ouchterlony double diffusion.

Three practical points must be borne in mind:-

•  Development of  the  precipitin  bands  must  be  done  at  a  constant temperature, usually 37C,  to prevent changes in diffusion rates, which can give rise to indistinct bands.

• To  prevent  the  gel from  drying out,  it  must be incubated  in  a  sealed container with an atmosphere  saturated with water vapour.  A Tupperware-type container,  containing several sheets of filter paper, saturated with water, is suitable.

•  The gel and the Ag and Ab solutions must contain a preservative,  such as merthiolate, to prevent microbial growth.  A moist environment, at 37C, in the presence of protein  (the  Ab and Ag) and carbohydrate (the agarose gel) is ideal for microbial growth.

Ouchterlony double-diffusion can  be used in  a test  of identity  of two antigens. If these  are  identical,  their  immunoprecipitin  lines  will  fuse into a single line (Fig. 9 a) whereas if they are non-identical their precipitin lines will cross (Fig. 9 b).  Partial  identity is indicated by a fused arc, with a spur (Fig.  9 c).

Figure 9.  Test of identity using immuno double diffusion.

2.2 Determination of diffusion coefficients

As mentioned above, the development of different  precipitin  bands in double diffusion is due to  differences in the  rate  of diffusion of different antigens, which is reflected in their different diffusion coefficients. Diffusion coefficients give information  about the  size and shape  of molecules. Because the position of the precipitin band is a function of the diffusion coefficient of the antigen, immunodiffusion can be used to measure diffusion coefficients.

One such method, devised by Polson8, uses a device which Polson refers to as his “mouth organ” apparatus (Fig. 9). This consists of four Perspex blocks,  marked A, B, C and D, with holes  drilled through  three of them and part way into D.  The blocks are able to slide  relative  to  one another on joints greased with petroleum jelly.

With the blocks aligned, a serial dilution of Ab can be introduced into the wells in block D after which block D is  moved  to  one  side to  seal  the wells.  Molten  1% agarose is introduced into  the  wells in block C and sealed off by moving blocks A and B to one  side, before the  agarose sets. Finally, Ag is added to  the  wells in block  B and sealed off by moving block A to one side.

Figure 10.  Polsons mouth  organ  apparatus for the determination of diffusion coefficients by  immunodiffusion.

The apparatus is incubated at 37C and  Ab and Ag diffuse through the column of agarose,  of known  precise  dimensions, to  form  a sharp precipitin band where they meet in optimal  proportions.  Where  the proportions are not optimal the bands are relatively fuzzy (Fig.  10).

Figure 11.  Quantitative immunodiffusion in Polsons apparatus.

From the  column in which the  precipitin  band is most  sharp,  the measurements Xg and Xb are taken (Fig.  10b).  From these  the  diffusion coefficient of the Ag can be calculated by substitution in the equation:-

Where,

 Dg = diffusion coefficient of the antigen,

Db = diffusion coefficient of the antibody

Since all IgG molecules have essentially the same gross structure, they will all have the same diffusion coefficient.  Db is thus a constant  (4.6 x  10^-7 cm² sec^-1.

Diffusion coefficients  can also be measured by molecular exclusion chromatography, since the separating mechanism in this technique is  also diffusion dependent.  A  standard  curve  of  1/D  vs  K^av, constructed using proteins of known diffusion coefficient (D), can be used to  determine  the diffusion coefficients of unknowns. It is useful to have two independent measures of the  diffusion coefficient -  in  this  case  by immunodiffusion and MEC - as the results from the  different  methods  serve as a check  on one another.

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 .