HA&HI is the agglutination reaction in immunology

HA&HI is the agglutination reaction in immunology

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  HAEMAGGLUTINATION Abhijith SP MVSc Scholar Departmentof Veterinary Medicine
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  WHAT IS AGGLUTINATION? •Agglutination is the visible expression of the aggregation of antigens and antibodies. • The antigen and antibody reaction in which the antigen-antibody complex formed is visible in the form of clumps is called agglutination. • Agglutination reactions apply to particulate test antigens that have been conjugated to a carrier. • The carrier could be artificial (such as latex or charcoal particles) or biological (such as red blood cells). These conjugated particles are reacted with patient serum presumably containing antibodies.
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  HAEMAGGLUTINATION • Agglutination isan antigen-antibody reaction in which a particulate antigen combines with its antibody in the presence of electrolytes at a specified temperature and pH resulting in the formation of visible clumping of particles. • It occurs optimally when antigens and antibodies react in equivalent proportions. This reaction is analogous to the precipitation reaction in that antibodies act as a bridge to form a lattice network of antibodies and the cells that carry the antigen on their surface. • Because cells are so much larger than a soluble antigen, the result is more visible when the cells aggregate into clumps. • When particulate antigens react with specific antibody, antigen-antibody complex forms visible clumping under optimum PH and temperature. Such a reaction is called agglutination. Antibodies that produce such reactions are called agglutinins.
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  AGGLUTINATION • Agglutination reactionis of two types: • Direct agglutination: It includes slide agglutination, tube agglutination, coombs’ test, and heterophile agglutination test. • Passive agglutination: It includes latex agglutination, hemagglutination test and, cooaglutination test.
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  HAEMAGGLUTINATION • Agglutination isthe visible expression of the aggregation of antigens and antibodies. • Agglutination test use visible clumping of cell or cellular antigen by binding of antibody. • The resulting clump or aggregate of cell is called agglutinate. • Because antibodies are bivalent, they can cross-link particulate antigens such as bacteria or foreign red cells, resulting in their clumping or agglutination. • Antibodies differ in their ability to cause agglutination; for example, IgM antibodies are more efficient than IgG antibodies. • If excess antibody is added to a suspension of antigenic particles, then, just as in the precipitation reaction, each particle may be so coated by antibody that agglutination is inhibited. this lack of reactivity at high concentrations of antibody is termed a prozone.
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  HAEMAGGLUTINATION • Hemagglutination useserythrocytes as the biological carriers of Viral/bacterial antigens, and purified polysaccharides or proteins for determining the presence of corresponding antibodies in a specimen.
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  The relationship betweenprecipitation and agglutination. Large particles agglutinate. Small particles and soluble molecules precipitate.
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  Hemagglutination test forNew castle Disease virus • All strains of Newcastle disease virus will agglutinate chicken red blood cells. This is the result of the haemagglutinin part of the haemagglutinin/neuraminidase viral protein binding to receptors on the membrane of red blood cells. • The linking together of the red blood cells by the viral particles results in clumping. This clumping is known as haemagglutination. • Haemagglutination is visible macroscopically and is the basis of haemagglutination tests to detect the presence of viral particles. The test does not discriminate between viral particles that are infectious and particles that are degraded and no longer able to infect cells.
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  MATERIALS REQUIRED • IsotonicPbs (0.01 M), Ph 7.0–7.2 • RBC taken from a minimum of three SPF chickens and pooled in an equal volume of Alsever’s solution. (If SPF chickens are not available, blood may be taken from unvaccinated birds monitored regularly and shown to be free from antibodies to NDV.) • Cells should be washed three times in pbs before use as a 1% (packed cell v/v) suspension. • Collect blood in Alsever’s solution --> wash 3 times at 1500 rpm for 5 minutes with pbs and then, for example 10 ml 2% RBC suspension, take 0.1ml blood and add 9.9 ml pbs and make 1% RBC suspension of 10 ml. • Positive and negative control antigens and antisera should be run with each test, as appropriate.
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  METHOD • 0.025 ml(25µl) of PBS is dispensed into each well of a plastic V-bottomed microtitre plate. • 0.025 ml (25µl) of the virus suspension (i.e. infective allantoic fluid) is placed in the first well. • Two-fold dilutions of 0.025 ml (25µl) volumes of the virus suspension are made across the plate. • A further 0.025 ml (25µl) of PBS is dispensed to each well. • 0.025 ml (25µl) of 1% (v/v) chicken RBCs is dispensed to each well. • The solution is mixed by tapping the plate gently. The RBCs are allowed to settle for about 40 minutes at room temperature, i.e. about 20°C, or for 60 minutes at 4°C if ambient temperatures are high, when control RBCs should be settled to a distinct button.
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  RESULTS • The settlingpatterns of single and agglutinated red blood cells are different. • Single cells roll down the sides of the V-bottom well and settle as a sharp button. Agglutinated cells do not roll down the sides of the well to form a button. Instead they settle as a diffuse film. • Negative HA result = a sharp button • Positive HA result = a diffuse film • Red blood cell control = a sharp button Definition of one HA unit • One HA unit in the haemagglutinin titration is the minimum amount of virus that will cause complete agglutination of the red blood cells. The last well that shows complete agglutination is the well that contains one HA unit.
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  REFERENCES • OIE TerrestrialManual 2018, Chapter 3.3.14 • Practical Immunology, Frank C. Hay, Olwyn M.R. Westwood, 2002 • Veterinary Immunology, 10th Ed, Ian Tizard