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University of Kufa Practical immunity College of Vet. Medicine Assist. Lect. Mohamed Talib Mohamed …………………………………………………………………............. Precipitation Reaction Precipitins can be produced against most proteins and some carbohydrates and carbohydrates- lipid complexes. Various system are available in which precipitation tests are performed in semisolid media such as agar or agarose, or nongel support medium such as cellulose acetate. Agar has been found to interfere with the migration of charged particles and has been largely replaced as an immunodiffusion medium by agarose. Agarose is a transparent, colorless, neutral gel. In the clinical laboratory several applications of the precipitation reaction are used. These methods include: A - Tube precipitation B - Immunodiffusion C - Electroimmunodiffusion A - Tube precipitation Solution of antibody is carefully layered on top of a solution of antigen, such that there is no mixing between the two. With time at the interface where the two layers meet, antigen antibody complexes form a visible precipitat. B - Immunodiffusion: These are of two type: single and double immunodiffusion. I. Double diffusion This technique also referred to as the Ouchterlony method, may be used to determine the relationship between antigen and antibodies. Principle: Antibody dilutions and specific soluble antigens are placed in adjacent wells. If the well size and shape, distance between wells, temperature, and incubation time are optimal, these solutions diffuse out, bind to each other, cross-link, and form a visible precipitate at the point of equivalence perpendicular to the axis line between the wells, the precipitation bands will be compared with a standard antigen. The precise location of the band depends on the concentration and rate of diffusion of antigen and antibody. In a condition of antibody excess, the band will be located nearer the antigen well. If two antigens are present in the solution that can be recognized by the antibody, two precipitin bands form independently. Antibodies associated with autoimmune disorders such as rheumatoid arthritis and systemic lupus erythematosus can be identified by double diffusion. Identity An identity reaction is indicated when the precipitin band forms a single smooth area. This precipitin is formed between the antibody and the two test antigens fuses indicating that the antibody is precipitating identical antigen specificities in each preparation. This does not mean that the antigens are necessarily identical; they are only identical insofar as the antibody can distinguish the difference. Nonidentity A non-identity pattern is expressed when the precipitation line cross each other. They intersect or cross because the sample contain no antigenic determinants in common. Partial Identity In a partial identity pattern , the precipitation lines merge with spur formation. This merger indicated that the antigen are non identical but possess common determinants. II. Single radial immunodiffusion This is a simple and specific method for identification and quantitation of a number of proteins found in human serum and other body fluids. Principle: Radial immunodiffusion is based on a technique using a precipitin reaction in which specific antibody is added to a buffered agarose medium, serum containing the test antigen is placed in a well centered in the agarose. The diameter of the resulting precipitin zone is related to the concentration of antigen placed in a well. C - Electroimmunodiffusion (EID) EID is a variation of the double immunodiffusion reaction in a support medium such as cellulose acetate or agarose through the use of an electric current that enhances the mobility of reactants and increase their movement towards each other. Antibody is placed in the well favoring its migration in the direction of the cathode; antigens that tend to be more negatively charged and placed in the well that favors migration of the anode. Precipitin bands form at a point of equivalence in a shorter periods of time. Electro immunodiffusion method, like immunodiffusion procedures, are classified into one-or-two-dimensional, singles, or double diffusion when a voltage is applied across the gels to move the antigens and antibodies together, immuno-double diffusion becomes counter current immune electrophoresis (CIE) radial immunodiffusion (RID) becomes electro immunoassay (EIA). Counter Current immunoelectrophoresis (CIE) is a variation of the classic precipitin procedure; it merely adds an electrical current to help antigens and antibodies move towards each other more quickly than in simple diffusion. The procedure takes advantage of the net electric charge of the antigens and antibodies being tested in a particular test buffer. Variables such as types of gel, amount of current, a concentration of antigen and antibody must be carefully controlled for maximum reactivity. The sensitivity of CIE is 10 to 20 times greater than in immuno-double diffusion, however, it is more expensive than other techniques such as immunodiffusion Electro immunoassay Antigens may be quantitiated by electrophoresis than in an antibody-containing gel electroimmunoassay. This technique combines the speed of electrophoresis with the accuracy and sensitivity of radioimmunoassay.