In direct test solid phase (i.e., plastic plates) is sensitized with antigen. In another words antigen is coated in solid phase. It is washed to eliminate unattached antigens. The test antibody is added and permitted to react with the antigen and surplus un-reacted antibody is washed off. Then a ligand having enzyme which is added that binds to the antibody molecule.
The enzyme employed may be either alkaline phosphatase or Horse radish peroxidase. After washing, to eliminate the unattached ligand, the substrate for the enzyme employed is added. When the enzyme is attached via the ligand to antibody molecule, it will react and liberate a chromogenic compound (i.e., colored substance).
Figure: Indirect binding assay
The ultimate intensity of the color is read with the help of a spectrophotometer.
In indirect assay technique an antigen is coated onto the surface of polystyrene wells. The specimen having particular immunoglobulin for the suspected antigen is added followed by the enzyme labeled particular immunoglobulin (i.e., conjugate).In a positive condition, an invisible antibody-antigen complex is made. The complex is made visible by the addition of a substrate that the enzyme in the conjugate modifies to generate a color change. This reaction can be viewed by the naked eye or measured by electronic means like spectrophotometer.
In indirect technique the antibody to immunoglobulin is tagged with enzyme and not to the antibody which reacts with the antigen.
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