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Factors Influencing Antigen-Antibody Reaction4. Factors Influencing Antigen-Antibody ReactionTemperatureMost antibodies have a thermal range, a range of temperatures at which they will combine with an antigen and a thermal optimum, a temperature at which the antibody when mixed with the antigen will react strongly. Generally IgM antibodies are more reactive at temperatures rnaging from 4°-27° C and IgG antibodies at temperature ranging between 30°C-37°C pH At pH levels outside the range of 5.5 to 8.5, antigen-antibody reactions may be adversely affected. Best reactions are observed at pH between 6.5-7.5. Buffered saline at pH 7.2 is recommended for use for all irnmunohaernatologic investigations. Incubation time Incubation time varies with different antibodies but most workers allow between 30 minutes and 60 minutes for antigen-antibody reactions to occur. Incubation time can be decreased to 5-15 minutes by reducing the ionic strength of the medium e.g. with LISS. Effect of ionic strength In normal saline NA and Cl ions cluster around and partially neutralize charges on antigen-antibody reactions to occur. Incubation time can be decreased to 5-15 minutes by reducing the ionic strength of the medium e.g. with LISS. Effect of centrifugation Centrifugation facilities agglutination by bringing the sensitized red cells together. The time and speed of centrigation should be adequate to produce a cell button with a clear supernatant but withoUt packing cells too tightly otherwise it would be difficult to dislodge the cell button. Overcentrifugation may lead to false positive reactions. Antigen-antibody ratio It is important to use correct ratio of antigen and antibody for optimal reaction. Usually 2 volumes of serum and 1 volume of 2-5% cell suspension is used. For cell suspended in LISS medium, use 1 1 ratio of cell and serum is used. Nature of antigen Different blood group antigens have varying number of antigen sites and even within a specific blood group system, a person may be homozygous or heterozygous. The homozygous individuals have considerably m9re antigen on the red cells than the heterozygotes and the antibodies may show different patterns of reaction, this is called the dosage effect of the antigen. The systems which show dosage effect are M,N,J,K and Rhesus blood group systems. Nature of the antibody The immunoglobulin class, size and strength of the antibody are of prime importance. As IgM molecules are much larger in size, these can bind antigen sites that are upto 35 nm apart and cause agglutination, while IgG antibodies can bridge gaps upto l4nm. Antigen-Antibody Reactions in vivo A red cell which has antibodies coated on its surface is liable to destruction by one of the following ways. 1. Intravascular haemolysis due to complement activation. 2. Extravascular haemolysis due to sensitization by complement components and subsequent engulfment by tissue macrophages. 3. Extravascular haemolysis due to sensitization by IgG antibodies and Fc receptor binding with tissue macrophages and phagocytosis 4. Non-specific antibody binding causing cells filtered during circulation through sinusoids of the spleen and liver.
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