A precipitation reaction is a very important antigen-antibody reaction in which the soluble antigen reacts with its antibody in the presence of electrolytes at a suitable temperature and pH.
It leads to the formation of antigen-antibody complexes or immune complex formation are called precipitates. They can either in the form of insoluble precipitate or in form of floccules it remains suspended.
Conditions required for precipitation reaction:-
- The antibody must be bivalent I.e. having two antigen-binding sites.
- The antigen must be soluble. It can be bivalent or multivalent.
- For the formation of lattice, the formation of antigens and antibodies must be equal.
- It can take place in liquid media or in gels.
Mechanism of a precipitation reaction:-
The mechanism of precipitation reaction lies under the lattice hypothesis proposed by Marrack in 1934.
According to this hypothesis when the multivalent antigen reacts with bivalent antibodies then precipitation occurs due to the formation of the large lattice.
Precipitation can occur in the zone of equivalence where the amount of antigen and antibody must be equal and lattice formation takes place by which insoluble precipitate can be seen.
In the case of excess antibodies, so no cross-linkage will form, many antibodies are there but due to insufficient amount of antigen no lattice formation takes place.
In the case of excess antigens, no cross-linkages occur now antibodies are insufficient to the formation of lattice formation.
Various types of precipitation reaction:-
1.) Precipitation reaction in liquid
- Ring test – in this test we layered the antigen solution over the column of antibody in a narrow tube and then we see the precipitate at the junction of two liquids.Example – C-reactive protein (CRP) and Lancefield grouping of streptococci.
- Flocculation test – This test can be done in the slide and in the tube .
– Slide flocculation – The drop of antigen and antiserum on a slide and mixed it by shaking then floccules will appear. Example VDRL slide test for detection of antibodies against syphilis
-Tube flocculation- Antigen and antiserum mixed in a test tube by shaking floccules will appear. Example Kahn test which is previously used diagnostic test of syphilis.
2.) Precipitation reaction in gel
Immunodiffusion– it is generally done in 1% agar gel. There are various advantages of this type of precipitation reaction –
- Reacting band easily visible, stable and can be stained for preservation.
- Different antigens can be observed.
- Cross reaction and non-identity between different antigens can be detected by this reaction.
There are different types of immunodiffusion tests which are listed below:-
- Single diffusion in one dimension – Oudin procedure
- In test-tube we mixed the antibody in agar gel and layered the antigen solution over it. The antigen diffuses towards the agar gel and the formation of precipitation line takes place. And the number of the precipitate bands directly proportional to the number of different antigen presents.
- Double diffusion in one dimension – Oakley -fulthorpe
- In this reaction antibody added to agar gel in the test tube and above that we put a layer of plain agar then we layered the antigen solution on top of this. The antigen and antibody move towards each other where the precipitate band is formed.
- Single diffusion in two dimension – Radial immunodiffusion
- In this test, we take a petri dish or side and add antiserum in gel and cut wells in it. Antigens are added to wells then antigen diffused radially and ring-shaped bands of precipitation are formed around the well. The diameter of the ring indicates the concentration of the antigen .
- Double diffusion in two dimension -Ouchterlony procedure-
- Most widely Immunodiffusion technique .we take a slide and put agar gel on it and cut the wells using a template add antiserum in the central well and antigen in surrounding wells They move toward each and form cross to each other which means they both are unrelated to each other but if they form a spur it means they are partially identical. Example the Elek’s test for toxigenicity in C.Diphtheria.
It is particularly useful for detection and abnormal serum proteins like myeloma protein
- Electroimmunodiffusion – it is an Immunodiffusion reaction that is driven by electricity .there are three types of Electroimmunodiffusion i.e. counterimmunoelectrodiffusion, rocket electrophoresis, and Laurell’s two-dimensional electrophoresis.
Application of precipitation reaction:-
- In the identification of bacteria for example Lancefield grouping of streptococcus
- In the detection of antibody
- In the identification of human blood and seminal stains.
- In Food adulterants testing
- To standardize the toxins and antitoxins.