Precipitation (Immunology)- Definition, Principle, Steps, Types, Uses

What is Precipitation?

Precipitation or immunoprecipitation is the non-covalent interaction between soluble (small) antigens and soluble antibodies that bind to form an insoluble precipitate.

  • It can occur both in vivo and in vitro.
  • Antibodies involved in this reaction are called precipitins. Precipitation won’t occur with monovalent Fab fragments.
  • The precipitins need to be bivalent for precipitation.
  • Antigens involved need to be bi or polyvalent so that they can bind with multiple antibodies which aid in the formation of the lattice. They can be called precipitinogens.

Note: Lattice is a net-like structure formed by the interaction of multivalent antigens with multi antibodies cross-linked with each other.

Precipitation Reaction

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)

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

A precipitation reaction is interfered below or above the zone of equivalence due to obstruction in a lattice formation. The zone below the zone of equivalence i.e. where antigens are lesser than antibodies is called the prozone phenomenon.

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.

Immunoprecipitation Protocol
Figure- Immunoprecipitation Protocol.

Precipitation Reaction Types

They are of mainly three types:

  • Precipitation in Solution 
  • Precipitation in Agar 
  • Precipitation in Agar in an electric field

A. Precipitation 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.

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.

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.

B. Precipitation 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

This technique was invented by Oudin hence it is also named after him. In this type, only one of the reactants which are mostly antigens is allowed to diffuse in a single direction towards their respective antibody.

One of the reactants i.e. antibody is mixed with the gel medium and kept in a test tube with a temperature of around 60-degree Celcius. After cooling it for a while the antigen solution is kept. The antigens diffuse below(one direction) and form a precipitate ring with antibody.

Requirements of Single diffusion in a single dimension

Test tube, Optimum temperature of 60-degree Celcius, Agarose Gel, Known Antibody, and Solution or sample with antigens.

Result Interpretation of Single diffusion in a single dimension

After a while, the antigens and antibodies interact as antigens move downwards and form a line or ring of precipitate which confirms the test to be positive. 

Also, multiple rings or bands can be observed which indicates the presence of different types of antigens in the solution.

2. Single diffusion in double dimension

This technique was invented by Mancini. It is also termed radial immunodiffusion.

In this, agarose gel is mixed with antibody solution. It is then poured on a Petri plate. Well is made in the medium and antigen solution is poured into the well.

Requirements of Single diffusion in double dimension

Petri plates or Glass slides, Antibody solution, Well borer, Sample or Solution Containing antigens

Result Interpretation of Single diffusion in double dimension

As antigen diffuses through the well in all directions from the well, a precipitate ring around the well is formed which confirms the test to be positive. Hence it is termed radial immunodiffusion.

If the concentration of antigens is higher in the solution, it would make a ring of larger diameter and vice-versa. It helps in qualitative as well as quantitative estimation.

Applications of Single diffusion in double dimension

It can be used in the estimation of immunoglobulins and detection of transferrin in serum, influenza infection, etc.

3. Double diffusion in a single dimension

This technique is also termed as Oakley-Fulthrope technique.

In this technique, the antibody is at first mixed with agar and kept in the test tube. Another layer of plain agar is laid over it and antigen solution is then poured over it. The antigens and antibodies both move(double diffusion) towards the plain agar layer between them(one dimension) to interact.

Requirements of Double diffusion in a single dimension

Test tubes, Agar or Agarose Gel, Buffering Chemicals, Antibody, and Antigen solutions

Result Interpretation of Double diffusion in a single dimension

At the equivalent concentration of reactants, a band of the precipitate can be observed in the plain layer between them which confirms the test to be positive.

4. Double diffusion in double dimension

This technique is also termed as Ouchterlony technique.

In this technique, the gel is at first poured on a Petri plate and one well is made at the center, and around the central well, multiple wells are made. In the center, antibody solution is poured and antigen solutions are poured in other wells. It is then kept for incubation for one or two days in a moist chamber.

The antigens and antibodies both diffuse towards each other in two directions i.e horizontal and vertical. 

Requirements of Double diffusion in double dimension

Petri plates or glass slides, Agar or Agarose gel, Well borer, Antigen, and Antibody Solutions, Moist Chamber 

Result Interpretation of Double diffusion in double dimension

At the point of equivalence of reactants, precipitate lines can be seen. 

If the wells contain similar antigens, the precipitate lines get fused.

If the wells contain different antigens, the precipitate lines don’t fuse but cross.

If the wells contain partially similar antigens, spur formation can be seen.

Applications of Double diffusion in double dimension

It is used in the detection of smallpox infection, Corneybacterium diphtheriae infection, fungal infections, etc.

C. Precipitation 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. Immuno electrophoresis (Immunodiffusion plus electrophoresis)

It can be performed on agar gel in which precipitate lines are formed by the faster movement of reactants towards each other on the slide in presence of the electric field. The movement of antigens is influenced by their size or charge.

Immuno electrophoresis Application

It is used in the detection of antibodies such as myeloma(abnormal) which can be present in the serum of humans.

2. Counter Current electrophoresis

It is performed with agarose gel kept on a slide and two wells are made, one for antigen and another for antibody. It is based on the attraction of antigens to the anode and antibodies to the cathode. Formation of precipitate line occurs rapidly within half or an hour.

Counter Current electrophoresis Application

It is used in the detection of antigens of amoeba, on the surface of the Hepatitis B virus, etc. 

3. Rocket Electrophoresis

It is an extension of radioimmunodiffusion. In this, agarose gel is mixed with antibodies at such pH that they won’t move or diffuse. The antigens are kept in wells made in the gel and only antigens diffuse in the gel under the influence of an electric field to form precipitate bands. It is named so because the precipitate bands appear in the shape of cone-like or rocket shape after the reaction.

Rocket Electrophoresis Application

It is used in the estimation of the number of immunoglobulins, Anti-streptolysin o proteins, etc.

Precipitation Applications

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

Precipitation Limitations

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

Precipitation vs Agglutination

              Precipitation Reaction            Agglutination Reaction
In this type, the binding of soluble antigens with soluble antibodies takes place.In this type, the binding of insoluble antigens with soluble antibodies takes place.
The complex formed is termed as precipitate or floccule.The complex formed after the reaction is termed a clump.
It takes more time to occur.Comparatively, it takes less time to occur.
Its sensitivity is less.Its sensitivity is higher than the precipitation reaction.
Comparatively larger size antigens are involved in this.Smaller size antigens than in precipitation reaction.
Eg. Ring test, VDRL test.Eg. ABO Blood Grouping, Widal test, etc.

References

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  2. Mujtaba MG, Baliban T, Bhagu J, Herrera M. A Laboratory Exercise Simulating Antibody and Antigen Reactions of the Ouchterlony Double Immunodiffusion Assay Using Inorganic Salts. J Microbiol Biol Educ. 2021;22(2):e00103-21. Published 2021 Jun 30. doi:10.1128/jmbe.00103-21.
  3. Han S, Wang G, Xu N, Liu H. Quantitative Assessment of the Effects of Oxidants on Antigen-Antibody Binding In Vitro. Oxid Med Cell Longev. 2016;2016:1480463. doi:10.1155/2016/1480463.
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  7. Alhabbab R.Y. (2018) Precipitation and Agglutination Reactions. In: Basic Serological Testing. Techniques in Life Science and Biomedicine for the Non-Expert. Springer, Cham. https://doi.org/10.1007/978-3-319-77694-1_3.
  8. Parija S.C., (2009), Textbook of Microbiology and Immunology, 2nd edition, Elsevier, a division of Reed Elsevier India Private Limited.
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