A precipitation reaction is a test which is involved in the serology, for the detection of antigens and antibodies. It is the reaction, where the soluble reactants, i.e. antigen and antibody are converted into the aggregated form. The sensitivity of the precipitation reaction is comparatively less than the other immunoassay reactions.
For the precipitation reaction, polyclonal or multivalent antibodies and antigens are used because if it is not so, the formation of the lattice will not occur. For the appearance of a precipitin ring or band, the reaction may take a few hours to days.
Precipitation test is used to quantify both antigen and antibody, and therefore its specificity also depends upon the concentration of both the reactants. If the concentration of anyone either antigen or antibody is in excess, the Immunocomplex will not form.
So, for the reaction to occur, the concentration of both antigen and antibody must be equal as a result of which visible precipitate or lattice will form. The precipitate which is the end product of precipitation reaction can be measured by light scattering devices like turbidometer, nephelometer etc.
Content: Precipitation Reaction
- Definition of Precipitation Reaction
- Important Terms
- Principle of Precipitation Reaction
- Methods of Precipitation Reaction
Definition of Precipitation Reaction
Precipitation reaction can define as the immuno-serological test where the two reactants, i.e. both antigen and antibody diffuse either in one dimension or two dimensions in liquid or semi-solid media. The antigen and antibody react with each other to form an “Immunocomplex” or “Lattice” at the zone of equivalence where the concentration of both are equal.
The lattice formed by antigen and antibody is visible, either in the form of precipitin ring or in the way of precipitin line. Precipitin is the antibodies which react with their respective antigens to form a “Precipitate”.
There are specific terms, which we should know before moving into the theory of precipitation reaction.
Precipitin: It can define as the soluble antibodies which react with the soluble antigens to form an end product which is known as “Precipitate”.
Precipitinogen: It can define as the soluble antigens which induce the formation of precipitin, i.e. soluble antibody.
Principle of Precipitation Reaction
A precipitation reaction is based on the principle of “Antigen-Antibody Reaction”, which occurs at the zone of equivalence. At the region of equivalence, the ratio or concentration of both antigen and antibody are equal which bring out the formation of lattice or cross-linked structure.
In the zone of equivalence, cross-linkage occurs which results in the formation of the antigen-antibody complex as a visible ring or line of a precipitate. The least soluble antigens and antibodies will form a complex at the point of equivalence whereas, the free antigens and antibodies will remain as the “Supernatant”.
Methods of Precipitation Reaction
Precipitation in Solution
In this type, the precipitation method is performed in the liquid media or solution. It consists of two methods namely ring and flocculation test.
Precipitation ring test
In the ring test, the antibodies which are present in the serum is first taken in the test tube. After that, test antigen is added to the solution. Then, over incubation of the reactants, the precipitation reaction occurs between antigen and antibody and a white precipitate forms at the junction.
This test can be performed in either of the two ways:
- Slide flocculation test: In slide test first, the drop of antiserum is added onto the clean glass slide. Then over the antiserum, a drop of test antigen is added followed by thorough mixing. If clumping between antigen and antibody occurs, the slide test will give a positive result and vice versa.
- Tube flocculation test: In this, the procedure is the same as slide flocculation test, but the only difference is that it is performed in a test tube instead of slide.
The example of tube flocculation tests is RPR (Rapid Plasma Reagin) and VDRL (Venereal Disease Research Laboratory) test.
Precipitation in Agar
In this type, the precipitation method is performed in the ordinary media like nutrient agar media. In agar media, the antigen and antibody reaction occurs by two ways:
In this method, diffusion takes place through the intervening agar media whose rate is influenced or affected by the following factors like:
- The particle size of the reactants
- Gel viscosity
- Interaction between gel matrix and reactants
The preferred concentration of agar is between 0.3-1.5percent for the effective diffusion of the reactants for the formation of the lattice. In agar, diffusion takes place by two ways:
- Single diffusion
- Double diffusion
Single and double diffusion can occur both in one dimension and double dimension. One dimension diffusion involves the diffusion of either antigen or antibody on the agar gel. Double dimension diffusion consists of the diffusion of both antigen and antibody on the solid agar media.
The precipitation reaction by immunodiffusion includes four methods which are as follows:
Oudin immunodiffusion method
It is a type of single immunodiffusion method, which occurs in one dimension.
In Oudin immunodiffusion first, add antibodies in the molten agar media and pour it into the petri dish. After solidification of the gel matrix, add a layer of soluble antigen. As a result, the incorporated antibodies that are present in the gel matrix will not diffuse, but the diffusion of antigen occurs. The antigen will diffuse towards the antibodies and form a line of a precipitate.
Oakley Fulthorpe immunodiffusion method
It is a type of double immunodiffusion method, which occurs in one dimension.
In Oakley Fulthorpe immunodiffusion first, add antibodies in the molten agar media. Over the layer of antibodies incorporated agar media, add a layer of plain agar media. Then on the top of plain agar media, add a layer of soluble antigen. On incubation, both antigen and antibody will move towards each other to form a precipitin line.
It is a type of single diffusion method which occurs in two directions.
In Radial Immunodiffusion first, the antibodies which are present in the serum is added in the molten agar media and poured into the glass slide. After the solidification of the gel, matrix creates wells and add test antigen into it.
Upon incubation, the antibodies which are incorporated in the agar gel will react with the specific antigens. A radial diffusion occurs exterior to the surface of well, refers as “Precipitin ring”. The diameter of the precipitin ring is directly proportional to the concentration of the antigen.
Ouchterlony Immunodiffusion method
It is a type of double immunodiffusion, which occurs in the two dimensions. In Ouchterlony Immunodiffusion first, add the serum into the agar gel and upon solidification create wells. The antigens are then added into the wells. This method is used for the comparison of antigens when are they are identical, non-identical and partially identical.
- When the antigens are identical, they will share the same antigenic determinants.
Suppose Antigen-‘A’ are the two types which are identical, so they will have a similar epitope suppose ‘a’. Therefore the antigen will react with the antibody incorporated in the agar gel and form an “Arc-shaped precipitin line” which will refer as the pattern of identity.
- When the antigens are non-identical, they will not share the same epitopes.
Suppose there are two antigens- ‘A’ and ‘B’ and epitopes ‘a’ and ‘b’ respectively. Therefore the antigen will react with the antibody and form a precipitin line that overlaps each other which will refer as the pattern of non-identity.
- When the antigens are partially identical, they will share one or more epitopes.
Suppose there are two antigens- ‘A’ and ‘X’ and epitopes ‘ax’ and ‘bx’ respectively. Therefore the cross-reaction will occur between the antigen and antibody, as a result of which incomplete precipitin line will form which will refer as the pattern of partial-identity.
This type of precipitation method involves the diffusion (Either in one or two directions) plus the technique of electrophoresis.
The immunoelectrophoresis includes the following two methods that are given below:
This method is performed on a glass slide which occurs in one dimension.
In Rocket immunoelectrophoresis, the agar is poured that is homogenized with the antiserum. Then a well is created on which antigens are added. Upon electrophoresis, antigens being negatively charged will migrate towards the positively charged antibody.
This migration of antigen on interaction with an antibody will form a complex appears as a visible precipitin line in the form of a rocket. The height at which the rocket move is directly proportional to the amount of antigen-loaded into the well.
It is a method of immunoelectrophoresis where the diffusion occurs in two dimensions.
In Counter immunoelectrophoresis, pour the agar gel onto the glass slide. Then create wells on both the edges of the glass slide. On one end, add antigens and on the other add antiserum.
Upon electrophoresis, the antigen will migrate towards the anode, and the antibody will migrate towards the cathode. On the interaction between antigen and antibody, a precipitin line will form.