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Diluted patient serum is added to wells coated with purified antigen. IgG specific antibody, if present, bindsto the antigen. All unbound materials are washed away and the enzyme conjugate is added to bind to theantibody-antigen complex, if present. Excess enzyme conjugate is washed off and substrate is added. Theplate is incubated to allow the hydrolysis of the substrate by the enzyme. The intensity of the colorgenerated is proportional to the amount of IgG specific antibody in the sample.