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Agglutination is the process of clumping of antigens with their respective antibodies.
- Agglutination reactions are used to particulate test antigens that are usually conjugated to a carrier.
- The carrier can either be artificial (such as latex or charcoal particles) or biological (such as red blood cells).
- The agglutination process involving red blood cells is termed hemagglutination, and the process with white blood cells is termed leukoagglutination.
- These conjugated particles then react with the patient’s serum which might or might not contain antibodies.
- The result of this test can be collected based on the observation of clumps resulting from that antigen-antibody complex formation.
- The sensitivity and accuracy of the result depend on a number of factors like the duration of incubation with the antibody, amount and strength of the antigen conjugated to the carrier, and test conditions like the pH and protein concentration.
- Different forms of agglutination are used in diagnostic tests which include latex agglutination, flocculation tests, direct bacterial agglutination, and hemagglutination.
- Because the process is quick and straightforward, these diagnostic tests are preferred over other sophisticated tests.
- Agglutination has been used for the detection of antigens in bacteria which ultimately helps in the identification of those bacteria.
- A modified and more sensitive technique associated with agglutination is agglutination-PCR.
- In this technique, the antibodies bind and agglutinate with antigen-DNA conjugates which enables the DNA strands to ligate with the antibodies.
- The agglutination formed is then used to quantify the DNA strands by q-PCR.
Precipitation is a process where soluble antigens bind with their specific antibody at an optimum temperature and pH, resulting in the formation of an insoluble precipitate.
- The interaction between the soluble antigen and antibody results in the formation of insoluble lattice that forms precipitate out of the solution.
- The process of precipitation has some requirements involving the valency of both the antigen and the antibody.
- For precipitation to take place, the antibody must be bivalent, and the antigen must be either bivalent or polyvalent.
- Precipitation takes place in the zone of equivalence, where the concentration of antigen and antibody is equal. On either side of equivalence, precipitation doesn’t occur if the concentration of either antigen or antibody is in excess or deficient.
- Immunological techniques like immunodiffusion and electroimmunodiffusion utilize the principle of precipitation reactions.
- The principle of precipitation is also applied in analytical chemistry for the detection of various functional groups of chemical substances.
- If a lighter precipitate is formed, another process, termed flocculation, takes place. In flocculation, the precipitate floats instead of sedimenting.
- Precipitation reactions are commonly performed on semi-solid surfaces like agar media or non-gel support media like cellulose acetate.
- The precipitate formed in the reaction remains suspended until enough force of gravity can settle the precipitate towards the bottom of the surface.
Key Differences (Agglutination vs Precipitation)
Basis for Comparison
|Definition||Agglutination is the process of clumping of antigens with their respective antibodies.||Precipitation is a process where soluble antigens bind with their specific antibody at an optimum temperature and pH, resulting in the formation of an insoluble precipitate.|
|Antigen size||The antigen involved in agglutination is comparatively smaller.||The antigen involved in precipitation is comparatively larger.|
|Solubility||Insoluble antigens are used for agglutination.||Soluble antigens are used for precipitation.|
|Sensitivity||Agglutination reactions are more sensitive than precipitation reactions.||Precipitation reactions are less sensitive than agglutination reactions.|
|Principle||Agglutination is based on the principle of the clumping of particles.||Precipitation is based on the principle of the formation of lattices (cross-linkages).|
|Reactions involved||Agglutination involves complex-forming chemical reactions.||Precipitation involves chemical reactions between ions and salt molecules.|
|Media||No gel matrix is required for agglutination.||A liquid or semi-solid matrix is required for precipitation.|
|Resulting compound||Agglutination results in the formation of agglutinates.||Precipitation results in the formation of precipitates.|
|Nature of the complex formed||The agglutinins usually settle towards the bottom of the container.||The precipitins might either remain suspended or settle down towards the bottom. In flocculation, the flocculants float on the surface of the liquid matrix.|
|Nature of reactants||The starting molecules in agglutination are particles.||The starting molecules in precipitation are ions.|
|Requirements||Agglutination reactions are surface reactions, and thus the surface of the antigens must be exposed for the antibody to bind and form visible clumps.||The concentration of antigen and antibody should be equal. Any change in this equivalence prevents the formation of precipitins.|
|Reaction time||Agglutination reactions might require minutes to hours for completion.||Precipitation reactions might occur in hours to days.|
|Appearance||The end products of agglutination reaction appear as large visible aggregates.||The end products of precipitation reaction appear as large insoluble visible aggregates.|
|Applications||Agglutination reactions are useful in blood grouping.||Precipitation reactions are useful in quantitative analysis and pigment formation.|
Examples of Agglutination
- Haemagglutination assay is a diagnostic technique used for the detection of viruses, bacteria, and antibodies.
- The antigens present in various viruses or bacteria bind with the sialic acid receptors present on the surface of red blood cells, creating a network of RBCs and viral particles.
- The formation of these lattices depends on the concentration of viruses/ bacteria and RBCs.
- When the concentration of the antigen is too low, the RBCs are not arranged in lattices but settle down at the bottom of the container.
- Haemagglutination is based on the same principle as the one used by viruses during infection.
- Control is placed in one of the wells to compare the concentration of antigen present in the sample.
- Based on the amount of agglutination formed in the well, the concentration of the antigen can be determined. The antigen concentration can then be used to determine the concentration of the organism present in the sample.
Examples of Precipitation
Immunodiffusion precipitation test
- Immunodiffusion is an immunological technique used for the detection and quantification of antibodies and antigens, which are mostly immunoglobulins and nuclear antigens.
- In this technique, antigen and antibodies are applied simultaneously in two adjacent wells.
- As the antigen and antibody diffuse towards each other, precipitates are seen in the form of lines as the antigen and antibodies interact with each other.
- It is also possible to compare the concentration of different antigens by placing multiple antigens in multiple wells.
- Based on the formation of precipitation lines, the presence of different antigens and, in turn, the presence of viruses or bacteria can be detected.
References and Sources
- Judith A. Owen, Jenni Punt, Sharon A. Stranford (2013). Kuby Immunology. Seventh Edition. H. Freeman and Company.
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