Precipitation

Definition
Principle
Types
Steps
Uses

1. Presented By M.Kaviya

2. PRECIPITATION Definition Principle Seteps Types uses

3. Definition: Precipitation Reaction is a type of antigen antibody reaction In which the antigen occurs in a soluble form. When a soluble antigen reacts with its specific antibody, at an optimum temperature and PH in the presence of electrolyte antigen-antibody complex forms insoluble precipitate. Antibodies that aggregate soluble antigens are called precipitins.

4. Prozone Phenomenon Antigen and antibody reaction occurs optimally only when the proportion of the antigen and antibody in the reaction mixture is equivalent. On either side of the equivalence zone, precipitation is actually prevented because of an excess of either antigen or antibody. The zone of antibody excess is known as the prozone phenomenon and the zone of antigen excess is known as the post zone phenomenon .

5. Precipitation Reaction Principle Under suitable conditions of temperature and pH, the soluble antigens which are generally smaller in size react with a specific antibody, form an insoluble antigen-antibody complex called the precipitate. The complex is obtained due to the formation of antigen-antibody lattices. It occurs in presence of suitable electrolytes. When precipitate just floats on the surface of the solution, it is called floccule and the reaction is called flocculation. Antigen (soluble) + Antibody (soluble) → Ag-Ab complex (insoluble)

6. The proportion of Ag and Ab in the reaction must be equivalent for the precipitation reaction to occur. The zone at which there are equivalent numbers of Ag and Ab in solution is called the zone of equivalence . The zone above the zone of equivalence i.e where antigens are larger in number than antibodies is called the post zone phenomenon . This phenomenon in terms of lattice formation was explained in 1934 by Marrack.

7. Precipitation Reaction Types Precipitation in Solution Precipitation in Agar Precipitation in Agar in an electric field

8. A. Precipitation Reaction in Solution In this type of precipitation, a liquid medium is used. It is an initial technique that is not advanced in comparison to precipitation in agarose gel. 1. Ring Test It is one of the fast and simplest tests. It is performed in a test tube where the antibody solution to be detected in the sample is put at first and antigen solution is then poured. The test is confirmed as positive by the observation of a precipitate ring in the middle of the tube after a few hours. Ring Test Requirements Test tubes or capillary tubes, Serum containing reactant mainly antibody, Corresponding antigen solution, Chemicals such as glycerol. Note: Glycerol can be used in the ring test to avoid the intermixing of antigen and antibody solutions. Ring Test Result Interpretation An observable precipitate ring between antigen and antibody solution is seen after a few hours maybe about four which confirms the test to be positive. Ring Test Applications It is used in the Lancefield technique for grouping Streptococcus spp. It is also used in the detection of anthrax by Ascoli’s test.

9. 2. Slide test This test is performed on slides such as cavity slides in the case of the VDRL test. In this test, the serum sample of the suspected patient is kept in the cavity of the slide and antigenic solution(already known) is mixed with it and shaken properly. Floccules are formed after a while in the case of the positive test. Slide Test Requirements Glass slides (Cavity slides), A Serum sample, Known antigen or antibody solutions Slide Test Result Interpretation The mixture is shaken, and after a while, floccules can be seen in the suspension on the slide which confirms the test to be positive. Slide Test Applications It is mostly used for the detection of syphilis antigens in suspected antigens and the test is called the VDRL (Venereal Disease Research Laboratory) test.

10. 3. Tube Test It is a type of flocculation test in which antigens or antibodies solutions are mixed in a tube to observe the formation of floccules. It can also be used for quantitative along with the qualitative diagnosis of toxins. Tube Test Requirements Test tube, Toxins, and Antitoxins, Serum sample in case of antigen or antibody detection with specific known antibody or antigens respectively. Tube Test Result Interpretation In this also test tube is observed to have floccule formation and the test is confirmed to be positive only if floccules are formed. Tube Test Applications It is also used for the detection of syphilis but it was used before the VDRL test. The test is termed as Kahn test.

11. B. Precipitation Reaction in Agar It is performed on Agar or Agarose gel or polyacrylamide gel. It is also termed immunodiffusion. Agarose gel is generally preferred. Gels provide a medium for the diffusion of reactants through the pores. It is more advantageous than in liquid medium as clear observable bands are formed in this type of precipitation. These bands are generally easy to preserve for a longer time and further use. Another advantage is the differentiation of individual antigens from the mixture of antigens . It is of four types: 1. Single diffusion in a single dimension 2. Single diffusion in double dimension 3. Double diffusion in double dimension 4. Double diffusion in double dimension

12. C. Precipitation Reaction in Agar in an electric field It involves the precipitation in agar but with an addition of a current or electric field. It is advantageous because the use of electricity or current provides an additional force that increases the rate of movement of antibodies and antigens in the gel for their faster interaction. Sensitivity of this type is more than immunodiffusion without electrophoresis. Requirements Electrophoresis set with electric supply, Buffer, Agarose Gel, Antigen and Antibody solutions, Glass slides. 1. Immunoelectrophoresis (Immunodiffusion plus electrophoresis) 2. Counter Current electrophoresis 3. Rocket Electrophoresis

13. Precipitation Reaction Applications It is widely used in diagnostic immunology. It is used in the detection of syphilis in patients by VDRL (Venereal Disease Research Laboratory) test., Kahn test, etc. It can also be used in the separation of specific proteins by precipitating them using their specific antibodies. It can be used in the grouping of different microbes such as Streptococcus based on the presence of different antigens. It can be used for the standardization of the toxins with their respective antitoxins.

14. Precipitation Reaction Limitations The sensitivity of precipitation is comparatively less than other techniques such as agglutination. It can be more time-consuming. It can’t occur properly or won’t occur in absence of polyvalent antigens. It won’t occur in absence of equivalent numbers of antigens and antibodies. Techniques such as precipitation in agar with electrophoresis require expertise or professionals to conduct it.

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