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Undiluted urine mixed directly with HRP suppresses generation of enzyme product to less than 80% of that seen in buffer controls. Incubating dilutions of various urine preparations with HRP immobilized on concanavalin A coated microtiter plates reveals that the source of urine or HRP, and the type of HRP substrate used have minimal effect on the degree of HRP suppression; only dilutions of urine greater than 8-fold with buffer produce HRP activities equivalent to those in buffer. Treating urine with charcoal or C18 silica only partially reverses the HRP suppression. Inhibition of HRP in competitive assays biases results to the high side and in noncompetive assays biases results to the low side. The present findings suggest analysts should avoid immunoassay protocols that allow direct contact between undiluted urine and HRP reporter conjugates and should be cautious with quantitative results previously reported from assays that used undiluted urine with HRP reporters.