Techniques in Diagnostics
The precipitation reaction occupies an important position in immunology and
occurs when serum from a sensitized animal is mixed with the immunizing antigen.
The precipitate that forms represents a large complex of antigen and antibody
that have combined to form an insoluble lattice.
The quantitative precipitin reaction, provides a systematic aproach to determine
the amount of either antibody or antigen by defining the optimal proportions of
each reactant in the formation of the immune precipitate. The approach usually
followed is to prepare a series of test tubes, each containing a fixed amount of
antisera used for immunisation is added. Following addition of antigen, an
appropriate period of time is allowed for the reaction to occur and precipitate
to form. The amount of nitrogen or protein in the precipitate is then determined
by measuring the nitrogen. In appropriate propotions, all the antigen is
precipitated; therefore, the nitrogen of the precipitate due to antigen can be
substracted and a direct determination of antibody nitrogen in the immune sera
can be made.
Factors affecting the precipitin reaction, in addition to the relative propotion
of reactants, include, conditions of temperature, pH, ionic strength of medium
and certain characteristics of antibody known as avidity and affinity are
important in formation of the immune precipitate.
The sensitivity, simplicity and specificity of the precipitin reaction have
provided the basis for its importance as an analytical technique in the clinical
laboratory. Adaptation of the precipitin reaction to semi-solid media such as
agar gel and agarose greatly simplified the routine applications of the
technique. Investigators such as Preer, Oakley-Fulthorpe and Oudin redified the
use of precipitin reaction in gel deffusion systems for the quantitative
estimate of antibody content of immune sera. These investigators emphasized the
quantitative significance of the precipitin line formed in gels at the point of
equivalence.
In the early 1960s, investigators such as Macini, Carbonaea and Herman adapted
the immunoprecipitin reaction in gels to highly sensitive and specifically
quantitative technique of single radial immune diffusion which is now in common
use.
This involves incorpration of antibody into the agar gel at a temperature
suffciently low to prevent denaturation and yet allow subsequent filling of an
appropriate chamber - either small test tubes, petri-dishes or glass slide
surfaces. Direct application of the test antigen is then made either as an
overlay in the test tube or by diffusion from a well punched in the agar plates.
After a period of time to allow for diffusion and equilibrium, there occurs
formation of precipitin bands in the agar. Visual observation by inspection
confirms the presence of a precipitin line in the agar. This line is formed at
the point of optimum antigen/antibody ratio, which is the same as the
equivalence point earlier described in the precipitin reaction. The distance of
the precipitin line from the point of application of antigen has been shown to
be directly propotional to the concentration of antigen when using a defined
quantity of antiserum in the agar. By a graphic comparison with preparation of
antigen standards, the quantity of the unknown antigen can be determined.
The principle of the technique is that antigen diffuses through the agar
containing antibody until the point of equvalence is reached and a precipitin
line forms.
This incorporates an agar gel to act as a supporting medium which separates
the antigen and antibody. The type of chambers selected for the study can be a
test tube, a petri-dish or a glass slide containing agar. The antigen and
antibody are applied at separate points by punching wells in the agar or placing
antibody at the base of the test tube seperating it from the antigen by a layer
of agar gel. After diffusion of the reactants into the agar over a suitable
period. Usually 18-48 hrs., the reactants will contact at the interface of
diffusion, and at equivalence point there will be formation of a precipitin
line. This method can be used in a semiquantitative way by inspecting the
thickness of the precipitin line determining distance of migration from the
reactant wells. Comparison is made with a standard antigen of known
concentration on which it has reacted in a companion set up.
This is a method described by Ouchterlony which represents a variation of the
double immunodiffusion technique. This procedure is usually carried out in a gel
medium in a petri-dish or on a glass slide and it is used for comparing
different antgens or different antibodies. Those of complete antigenic
difference exhibit crossing of precipitin bands.
The electro immunodiffusion reaction is a variation of the double immunodifusion
reaction created by augmenting the diffusion of the reactants in agar gel by use
of an electric current. It is similar to that followed in the usual immuno-diffusion
reaction. Antibody is placed in the well favouring its migration in the
direction of the cathode, while the antigens that tend to be more negatively
charged are placed in the well favouring migration to the anode. The
elecrophoretic effect enhances mobility of the reactants and speeds up thier
movement towards each other. a precipitin line which occurs at point of
equivalence thus requires a much shorter time development owing to the
augmentation by electrophoresis. The size and position of the precipitin band
provides the same type of information regarding equivalence or antigen/antibody
excess as in the simple immunodiffusion system.
Immunoelectrophoresis couples electrophoretic seperation with the two dimensional reaction immunodiffusion reaction and is now used extensively of a wide range of antigens. The steps of the technique are out-lined diagrammatically. Te first step involves application of a macro-molecular sample such as human serum for electrophoresis in agar gel on a plastic or glass support. On completion of the electrophoresis, a trough is preapplied in the trough. Diffusion is then allowed to proceed. Notice that the relative thickness of individual bands is propotional to their relative concentrations. their respective positions in the electrophoretic migration also aid in their identity. By using this method homogenous populations of macro-molecules such as those which occur in monoclonal gammopthies of multiple myeloma or Waldenstrom's macro-globulinemia exhibit narrow localized areas of thickening of the precipitin bands. Indeed, this approach is the definitive method for identifying such monoclonal immunoglobulins.
technique has proved to be most useful method fo immunoglobulin and serum
protien quantitation in the clincal laboratory during the past few years. The
RID technique indeed has an advantage of operational simplicity as well as
sensitivity and specificity. The approach basically represents a variation of
the single immunodiffusion technique where antibody has been incorporated into
the agar which is poured into a plate or onto a glass slide. Wells are then cut
in the agar and test material is placed in the wells. Antigen standards of known
amount are placed in some wells along with unknown test material in other. After
allowing an adequate time for diffusion and formation of preciptin rings about
the wells, a standard curve is graphically drrawn by measuring the diameters of
the precipitin rings of the unknown are then measured and plotted on the
standard curve. By direct inspection test material can be made. The specificity
of this reaction quite obviously depends on the quality and mono-specificity of
the antiserum used as the reagent in the gel.
