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Diluted patient serum (serum diluent contains sorbent to remove rheumatoid factor and human IgGinterference) is added to wells coated with purified antigen. IgM specific antibody, if present, binds to theantigen. 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 IgM specific antibody in the sample.