after an incubation of 10 minutes at room temperature with shaking during which the goat anti mouse antibody u2014 hrp enzyme conjugate binds to 5 fu antibodies in the wells the plates were again washed three times to remove unbound secondary conjugate to develop a measurable color in the wells washing was followed by the addition of 100 u03bcl of tmb tmb liquid substrate sigma or biofx a substrate for hrp to develop color in a 10 minute incubation with shaking at room temperature following the incubation for color development 50 u03bcl of stop solution 1 5 sodium fluoride in di h 2 o was added to each well to stop the color development and after 10 seconds of shaking the absorbance was determined at 650 nm molecular devices plate reader the amount of antibody in a well was proportional to the absorbance measured and inversely proportional to the amount of 5 fu in the sample the absorbance of the color in the wells containing analyte was compared to that with no analyte and a standard curve was generated the ic50 value for a given analyte was defined as the concentration of analyte that is required to inhibit 50 of the absorbance for the wells containing no analyte the cross reactivity of a given analyte was calculated as the ratio of the ic50 for 5 fu to the ic50 for uracil thymine cytosine and tegafur expressed as a percent.