Precipitation Reaction - Definition, Principle, Types

An antigen is any chemical that, when given into the body, induces the creation of an antibody with which it reacts in a precise and detectable manner.

Antibodies are distinct substances in vertebrate bodies that are released by lymphoid cells in response to stimulation by foreign substances (antigens) with which they respond specifically. The interaction between antigen and antibody is a unique chemical process.
Numerous noncovalent interactions between the epitope and paratope are involved in the response between an antibody and an antigen. The epitope is the Ag binding site, while the paratope is the Ab binding site.

Serology is the study of the response between antigen and antibody.
The classifications of serology include precipitation, agglutination, complement fixation, neutralisation, immobilisation, and intradermal response.
In addition, serology is a discipline of science that focuses on the study of serum.

An antigen-antibody reaction is a fundamental immunological reaction that describes the unique response between antigen and antibody.

Precipitation Reaction Definition

Immunology precipitation reactions are founded on the interaction between antigens and antibodies.
These are based on the combination of two soluble reactants to form precipitate, an insoluble product.

The interaction between antigen and antibody is a unique chemical process.
When antigen and antibody are present in appropriate proportions, lattices are formed (cross-links).
Intermolecular forces hold the molecules together, but they are only efficient when the antibody combining site and the antigenic determinant group are in close proximity.

Precipitation Reactions Principle

Antigens and antibodies are both composed of amino acid complexes with positive and negative polar groups arranged in particular but opposite patterns on their surfaces.
When the antigen and corresponding antibody molecules are mixed, electrical attraction and repulsion occurs, resulting in the orientation of the corresponding antigen and antibody molecules with respect to their molecular forms and electrical charges, thereby producing an absolute “fit” (mould and cast).
In precipitation reactions, soluble antigen reacts with IgG or IgM antibodies to generate massive, interconnecting molecular aggregates known as lattices.

Two separate phases comprise precipitation reactions. Antigens and antibodies generate tiny antigen-antibody complexes fast.
The antigen-antibody complexes create lattices that precipitate from solution in a slower reaction that may take minutes to hours.
Precipitation reactions only occur when the antigen-antibody ratio is appropriate.

No apparent precipitate arises when either component is in excess, as depicted in the diagram. When separate solutions of antigen and antibody are placed adjacently and allowed to diffuse together, the optimal ratio is achieved.
The equivalency zone indicates the antigen and antibody concentration at which complete precipitation occurs.

Antigen (soluble) + Antibody (soluble) → Ag-Ab complex (insoluble)

Prozone phenomenon

The ideal antigen-antibody reaction can only occur when the proportions of antigen and antibody are equal (zone of equivalence).
On each side of the equivalence zone, an excess of antigen or antibody prevents precipitation from occurring.
The zone of excess antibodies is referred to as the prozone phenomena, while the zone of excess antigens is known as the postzone phenomenon.

The lattice hypothesis was presented by Marrack in 1934 to explain the prozone phenomena. Each antibody molecule must have at least two binding sites, and antigen must be multivalent, according to Marrack’s hypothesis.
In the zone of equivalence where optimal precipitation occurs, the number of antigen and antibody multivalent sites is almost equivalent.
In this region, precipitation is caused by random, reversible processes in which each antibody attaches to many antigens and vice versa, establishing a persistent network or lattice.

As they join, a multimolecular lattice is formed that grows in size until it precipitates from solution.
In the interpretation of serological tests, the prozone and postzone phenomena are taken into account since false negative reactions might arise in either of these scenarios.
By diluting the antibody and rerunning the test, a false-negative result that may have been caused by prozone phenomenon can be corrected.

In the postzone phenomenon, the presence of minute amounts of antibodies may be obscured by an overabundance of antigen.
Typically, such a test is repeated a week later with an additional patient samples. This would allow time for the creation of more antibodies.
If the test is negative, it is improbable that the patient possesses this specific antibody.

Too much antibody is present in the prozone phenomenon for efficient lattice creation. This is because antigen combines with a small number of antibodies, preventing the formation of cross-links. Small aggregates are surrounded by excess antigen in postzone phenomena, and no lattice network forms. Therefore, in order for precipitation reactions to be detected, they must be conducted inside the zone of equivalency.

Features of Precipitation Reaction

It is a type of antigen–antibody response wherein the antigen is soluble.
It is a test in which antibody reacts with soluble antigen in the presence of electrolyte at a predetermined pH and temperature to form a precipitate. Antigens and antibodies form a lattice; in certain instances, it is apparent as an insoluble precipitate.

Precipitins are antibodies that aggregate soluble antigens. When precipitate remains suspended as floccules rather than settling, the process is known as flocculation.
Formation of an antigen–antibody lattice is dependent on both the valency of the antigen and the antibody.
The antibody must be bivalent; monovalent Fab fragments will not create a precipitate.

The antigen must be either bivalent or polyvalent, with at least two copies of the same epitope or various epitopes that respond with different antibodies present in polyclonal antisera.

Types of Precipitation Reaction

Precipitation reactions often fall into three categories: 1. Precipitation in solution 2. Agar precipitation 3. precipitation using an electric field in agar

1. Precipitation in solution

Precipitation in solution is illustrated by the ring and flocculation tests.

a. Ring test

Antigen solution is placed over antiserum in a test tube for this procedure.
Precipitation between antigen and antibodies in antiserum solution is characterised by the formation of a precipitation ring at the interface between two liquid layers.
C-reactive protein (CRP) and the Lancefield method for categorising streptococci are examples of ring tests.

b. Flocculation test

The flocculation test can be conducted on a slide or in a tube. An example of a slide flocculation test is the VDRL test for detecting reaginic antibodies in syphilis.
In this test, a drop of VDRL antigen suspension is introduced to a drop of patient serum on a cavity slide, and the result is recorded when the slide is shaken on a VDRL shaker.
In a positive test, floccules are visible under a microscope, as is the case when the test is positive.

The syphilis Kahn test is an example of a tube flocculation test.
Another example is the tube flocculation test for standardisation of toxins and toxoids.

2. Precipitation in agar

The agar gel precipitation test is known as the immunodiffusion test.

In this test, reactants are introduced to the gel, and the antigen–antibody combination takes place through diffusion.
The rate of diffusion is influenced by the particle size, temperature, gel viscosity, quantity of hydration, and matrix-reactant interactions.
Agar concentrations between 0.3% and 1.5% permit diffusion of the majority of reactants.

Agar has a significant negative charge, whereas agarose has essentially none, hence interactions between the gel and reactants are limited when agarose is used.

Types of immunodiffusion reactions

Immunodiffusion reactions are characterised according to (a) the number of diffusing reactants and (b) the direction of diffusion:

a. Single diffusion in one dimension (Oudin Procedure)

Antigen diffuses in agar in a single dimension, as the term suggests.

It is also known as the Oudin procedure because this approach was pioneered by Oudin, who utilised gels for precipitation reactions for the first time.
In this technique, antigen solution is poured over agar gel containing antibody in a test tube.
In agar gel, the antigen diffuses downward toward the antibody over time, causing a line of precipitation to appear.

The number of precipitate bands indicates the variety of antigens in the antigen solution.

b. Single diffusion in two dimensions (Radial immunodiffusion)

Two-dimensional single diffusion is also known as radial immunodiffusion.
Antibody-containing antiserum solution is mixed into agar gel on a slide or Petri dish.

The wells are carved into the gel’s surface.
The antigen is then applied to a well that has been carved into the gel. When antibody already present in the gel reacts with antigen that diffuses out of the well, a precipitation ring forms around the wells.
Antigen concentration is exactly proportional to the diameter of the ring.

The ring will be farther from the well the more antigen there is in the well. Nephelometry and enzyme-linked immunosorbent assays are more sensitive and automated procedures that have lately superseded the test (ELISAs).

c. Double diffusion in one dimension (Oakley Fulthorpe procedure)

This surgery is also known as the Oakley–Fulthrope procedure. In this technique, the antibody is mixed into agar gel in a test tube, which is then covered with a layer of plain agar.
The antigen is subsequently put on this plain agar.

Antigen and antibody travel near one other over time through the layer of plain agar that separates them.
In this region of plain agar, the optimal concentrations of antigen and antibody react to generate a band of precipitation.

d. Double diffusion in two dimensions (Ouchterlony procedure)

This method is also known as the Ouchterlony method.

Antigen and antibody diffuse independently through agar gel in two dimensions, horizontally and vertically, using this approach.
The test is conducted by slicing wells into the agar gel that has been put onto a glass slide or Petri dish.
Antibody-containing antiserum is added to the central well, while other antigens are injected to the wells surrounding the central well. After 12 to 48 hours of incubation in a wet chamber, lines of precipitates appear at the sites of antigen and antibody interaction.

As follows, three sorts of reactions can be demonstrated:
- The presence of a precipitation line at the junction of two antigens, forming an arc, indicates serologic identity or the existence of a common epitope.
- A pattern of crossed lines suggests that the compared antigens are unrelated and do not have any common epitopes.
- Cross-reaction or partial identity is denoted by the fusion of two lines with a spur. In this final instance, the two antigens have a common epitope, but some antibody molecules pass through the initial precipitin line and join with extra epitopes on the more complex antigen.

Use of Double diffusion

Double diffusion in two dimensions has been utilised for:

Antibody demonstration in smallpox serodiagnosis

Antigen identification for fungi
Detection of antibodies to nuclear antigens that can be extracted.
The technique for demonstrating the toxigenicity of Corynebacterium diphtheriae is the Elek precipitation test in gel.

Advantages of Immunodiffusion

Immunodiffusion reactions are advantageous in the following ways:

The precipitation line is apparent as a band in this test, which can also be dyed for preservation.
The test can detect identity, cross-reaction, and nonidentity between distinct antigens in a combination of responding substances.

3. Precipitation in agar with an electric field

Immunoelectrophoresis

Immunoelectrophoresis combines immunodiffusion with electrophoresis.
Under the influence of an electric field, various antigens in serum are separated according to their charge.
A drop of antigen is placed in an agar well on a glass slide using this technique.

The agar is then put through an electric current.
Antigens travel in the electric field according to their charge and size during electrophoresis.
After electrophoresis, a trough is cut into the agar, filled with the antibody, and allowed to diffuse. As the antigen and antibody diffuse toward one another, a series of precipitation lines are formed.

The primary advantage of immunoelectrophoresis is the ability to identify several antigens in serum.
The approach is used to detect both normal and aberrant proteins in human serum, such as myeloma proteins.

Counter-current immunoelectrophoresis

Under an electric field, counter-current immunoelectrophoresis is dependent on the flow of antigen towards the anode and antibody towards the cathode through the agar.

The test is conducted on an agarose-coated glass slide with two holes cut in it.
One well contains antigen, while the other contains antibody. The gel is subsequently put through an electric current.
Under an electric field, antigen and antibody movement is considerably facilitated, and the precipitation line becomes apparent in 30–60 minutes.

Rocket electrophoresis

This method is a modification of Laurell’s radial immunodiffusion technique. At the conclusion of the reaction, the precipitin bands appear as cone-shaped structures (rocket look), hence the name.
This method involves incorporating antibody into the gel and placing antigen in wells cut into the gel.
The gel is then subjected to an electric current, which enhances the migration of antigen into the agar.

This leads in the creation of a precipitin line with a rocket-like conical shape. The amount of antigen in the sample is directly proportional to the height of the rocket, as measured from the well to its apex.
Rocket electrophoresis is mostly utilised for serum antigen quantification.

Two-dimensional immunoelectrophoresis

Rocket electrophoresis is a subtype of two-dimensional immunoelectrophoresis. It is a double diffusion technique utilised for both qualitative and quantitative examination of sera for a wide variety of antigens.

This test consists of two stages. In the first step, electrophoresis is used to separate antigens in solution. In the second stage, electrophoresis is performed perpendicular to the first stage in order to produce rocket-like precipitation.
In this test, a small trough is cut into agar gel on a glass plate and then the antigen solution is added.
Antigens migrate into the gel at a pace proportionate to their net electric charge once an electric current has been conducted through the gel.

In the second stage, following electrophoresis, the gel fragment containing the separated antigens is placed on a second glass plate and antibody-laden agar is poured around it.
A second electric potential is delivered perpendicular to the initial migratory direction.
Antigens that have been pre-separated migrate at a pace proportionate to their net charge into a gel containing antibodies, where they precipitate with the antibodies to form precipitates.

This approach is both qualitative and quantitative in that it recognises distinct antigens present in the serum solution and detects the amount of different antigens present in the solution, respectively.

Turbidimetry and nephelometry

Both turbidimetry and nephelometry rely on the phenomenon of light scattering by precipitates in a solution in order to detect and quantify precipitation reactions in serum.
Turbidimetry is the measurement of precipitate turbidity or cloudiness in a solution.

In this procedure, a detector is positioned in direct line with the incident light to gather light that has passed through the solution.
Thus, it quantifies the decrease in light intensity caused by reflection, absorption, or scattering. Light is scattered proportionally to the size, shape, and concentration of precipitates in solution.
Nephelometry is an advancement of this technique since it measures the light scattered at a specific angle from the incident beam as it passes through a suspension containing antigen–antibody precipitate.

The amount of light dispersed is an indicator of the solution’s concentration. A constant amount of antibody would result in an increase in antigen–antibody complexes as antigen concentration increases.
Therefore, the connection between antigen concentrations, as shown by the production of antigen–antibody complexes, and light scattering is close to linear.
Using a computer, this technique can estimate the exact antigen or antibody concentrations in the serum.

In order to increase the sensitivity of this system, laser beams have been utilised as the incident light source.

Nephelometry is presently the preferred technique in many laboratories for measuring plasma proteins, such as IgG, IgM, and IgA, complement components, RA (rheumatoid arthritis) factor, ASLO (antistreptolysin O), etc.

Applications

Countercurrent immunoelectrophoresis has numerous applications:

It is a quick and highly specific approach for detecting antigen and antibodies in serum, cerebrospinal fluid, and other body fluids for the diagnosis of a variety of infectious disorders, including bacterial, viral, fungal, and parasitic infections.
It is typically used to detect Hepatitis B surface antigen (HBsAg), -fetoprotein, hydatid and amoebic antigens in the serum, and cryptococcal antigen in the cerebrospinal fluid.

Limitations of Precipitation Reaction

Comparatively, precipitation is less sensitive than other procedures such as agglutination.

It may require additional time.
It cannot or will not occur in the absence of polyvalent antigens.
It will not occur without equal amounts of antigens and antibodies.

Precipitation in agar using electrophoresis is a technique that requires skill or specialists to execute.

Applications of Precipitation Reaction

It is commonly utilised in immunological diagnostics.
The VDRL (Venereal Disease Research Laboratory) test, the Kahn test, etc., are used to identify syphilis in patients.

Additionally, it can be used to separate individual proteins by precipitating them with their respective antibodies.
It can be used to classify different microorganisms, such as Streptococcus, based on the presence of distinct antigens.
It can be used to standardise the antitoxins with the corresponding toxins.

References

Ouchterlony, Ö. T. G. (1998). Precipitation Reaction. Encyclopedia of Immunology, 1999–2002. doi:10.1006/rwei.1999.0506
Deshmukh, Amol & Dighe, Pravin & Tiwari, Kundan & Garud, Supriya. (2020). PRECIPITATION REACTIONS IN IMMUNOLOGY. EUROPEAN JOURNAL OF PHARMACEUTICAL AND MEDICAL RESEARCH. 7. 214-216. http://ecoursesonline.iasri.res.in/mod/page/view.php?id=62004
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