Antibodies bind specifically to the antigens. When antigens are multi-valent, their interaction with antibodies lead to crosslinking and formation of precipitation. When the antibodies precipitate soluble antigen, large and insoluble immune complexes are formed (fig 1).
The precipitation reaction forms the basis of a large number of different assays. These assays can be used to detect either of the reactants in the unknown sample. If the antibody are labelled, the assays can be made more sensitive and used quantitatively or qualitatively.
As is clear, there are two components of the reaction: the antibodies and the antigens.
• The antibodies:
The antibodies can be either polyclonal or monoclonal.
– Polyclonal antibodies:
The polyclonal antibodies are a mix of antibodies, specific to different sites on the antigen. The antisera, containing polyclonal antibodies, can be obtained by immunization of the animals, commonly used in rabbits. To increase the amount of the antibodies, the rabbit may be given a booster dose. The blood is then collected from the posterior marginal ear vein of the rabbit. The serum is separated and incubated at 56oC for 45 mins to inactivate the protease and the complement components and stored at -20oC. Sheep, goat and horses used for large scale antiserum production.
– Monoclonal antibodies:
The monoclonal antibodies, which are homogenous or identical synthesized from a single B-cell clone. They recognize same epitopes or binding sites, on the antigen. The monoclonal antibodies can be generated by a technique pioneered by Georges Kohler and Cesar Milstein in 1975, already discussed in one of our previous posts.
• The antigens:
Antigens are molecules causing an immune reaction in an individual. Two important properties of an antigen are;
– Immunogenicity: the capacity of an antigen to induce an immune response
– Antigenicity: the capacity of an antigen to be recognized by the antigen-specific receptors and antibodies.
Therefore, every immunogen is also an antigen but not all antigens are immunogens. Antigens are constituted by antigenic determinants or epitopes, to which the antibodies bind.
Antibodies bind specifically to multi-valent antigen and can result in crosslinking and forming insoluble immune complexes, which eventually cause precipitation or immunoprecipitation. Immunoprecipitation is also known as precipitin reaction.
The precipitation of the immune complex occurs at a particular concentration of both antibody and antigen. If the increasing amount of antigen is added to the solution of antibody (or vice versa) and the graph is plotted for the same, the graph can be divided into three zones:
- 1. Zone of antibody excess
- 2. Zone of equivalence
- 3. Zone of antigen excess (fig 3)
The formation of immune complex occurs in the zone of equivalence. Usually the precipitate, can be easily detected with the bare eyes or microscope. However, in some cases, in antigen or antibody excess, small soluble complexes are formed, which are called as microprecipitates and remain in the solution. These microprecipitates are invisible to naked eye, but can be detected by nephelometer. Nephelometer is an instrument which measures the turbidity of the solution based on the scattering of the light passed through the particles.
(Just for info: Here’s an old paper on the use of laser nephelometry for a simple quantitative assay of circulating immune complexes.)
Many immunoprecipitation assays are used regularly in the healthcare sector wherein detection of the immune complexes are involved. These assays can be performed in a particular medium, either liquid or semi-solid, wherein the antigen and the antibodies are allowed to interact with each other. The antibodies will precipitate the soluble antigens when the zone of equivalence is obtained.
As mentioned, the precipitation reactions can be divided into two broad classes, depending on the state of medium used:
1. Precipitation Reactions in Solution.
2. Precipitation Reactions in Gel.
The diffusion in the liquid is relatively faster than that in the gel, i.e. the diffusion in liquid medium may take around 10 minutes while that in gel may sometimes take upto 72 hours.
Let us see the different techniques in each the medium:
1. Precipitation Reaction in Solution.
The immunoprecipitation assay in solution can be of two types depending on the purpose, they are;
A. Qualitative analysis:
The qualitative analysis will give the information of whether a particular antibody or antigen is present in the sample or no. (It will answer the question yes or no.) These analyses cannot be used to determine the amount of the components present in the sample. Qualitative immunoprecipitation analysis in liquid medium is done by adding varying amount of antigen to fixed amount of antiserum (containing antibody). At the equivalent concentration the presence of antigen can be detected by the presence of whitish precipitation ring, hence is also known as precipitin ring test.
The disadvantage of this test is that it may give false negative result. If the concentration of either component do not fall in the zone of equivalence, then there is no precipitation despite the presence of both the antigen or the antibody.
B. Quantitative Analysis:
The quantitative analysis can be used to find the amount of the analyte present in the sample. Hence, a quantitative immunoprecipitation assay will answer ‘how much of antigen or antibody is present in the sample’. The quantitative immunoprecipitation reaction can be carried out once the zone of equivalence is determined. Optimum concentrations of antigen and antibody are added to give precipitation. The excess is washed off and the amount is determined by sensitive assays like:
– Micro-kjeldahl method for total N2
– Folin estimation of phenolic hydroxyl groups (tyr, phe)
– UV absorption at 280nm for tyr, try and phe
– Biuret reaction for proteins
– Liquid scintillation with antibody labelled with negatron emitter.
– Gamma counting with antibody labelled with a gamma-emitting isotope.
The graph is plotted with samples with known concentration of the antigen and the concentration in the test sample can be found using the standard curve.
C. Hapten inhibition test
Hapten inhibition test is a test which is carried out in solution but is carried out to determine the presence of haptens. Haptens are small-molecular weight compounds, which is foreign but cannot initiate immune response or antibody production.
The hapten are small compounds with a single antigenic site, and hence unable to form cross linking structures. They can be made immunogenic by coupling it to an immunogenic protein or the carrier. Therefore, a free hapten is ‘antigenic’ and not ‘immunogenic’, as they can bind but not crosslink antibodies to form immune complex or precipitation.
(Just for Info: Here is a paper in which they synthesis protein conjugates of an hapten for the insect growth regulator Fenoxycarb and design immunoassays.)
The presence of a hapten can be determined in a sample by the Hapten inhibition test. Here, the antibodies are added to the sample solution, and the hapten binds to the antibodies. In the next step, the antigens are added to the mixture. If on addition of antigen, there is no precipitate, then it can be concluded that the sample solution contains hapten and the haptens have occupied the antigen binding sites of the antibodies and hence no precipitation is formed on addition of multivalent antigen (fig 6A).
If precipitation occurs, it means that the hapten are not bound to antibodies which are hence free to form crosslinking immune complexed (fig 6B). It can lead to the conclusion that the haptens are absent or do not have common determinants as the antigens.
Hapten inhibition test can be used to study the chemical nature of complex antigen.
These were the different methods of determining the presence of antibodies, antigens or haptens in the liquid medium. The reaction in liquid requires proper ratio of antigen to antibody to be in the region of equivalence, or else false negative result is achieved. The technique may hence become lengthy and may require large quantities of valuable antisera or antigens. To solve these issues the precipitation reactions are preferably carried out in gel.
2. Precipitation Reaction in Gels:
The different type of assays in gels, that is immunodiffusion and immunoelectrophoresis and their subtypes will be discussed in the next post.
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Read other posts by The Biotech Notes:
High Pressure Liquid Chromatgraphy (HPLC).
Adorini (1998) Immunodominance. Encyclopedia of Immunology (Second Edition, 1290-1292.