be assayed will generally vary from about 10 2 to 10 13 m more usually from about 10 4 to 10 10 m higher concentrations of ligand may be assayed upon dilution of the original sample in addition to the concentration range of ligand of interest considerations such as whether the assay is qualitative semiquantitative or quantitative the equipment employed and the characteristics of the tracer and antibody will normally determine the concentration of the tracer and antibody to be employed while the concentration of ligand in the sample will determine the range of concentration of the other reagents i e tracer and antibody normally to optimize the sensitivity of the assay individual reagent concentrations will be determined empirically concentrations of the tracer and antibody are readily ascertained by one of ordinary skill in the art as previously mentioned the preferred tracers of the present invention are prepared from 5 4 6 dichlorotriazin 2 yl amino fluorescein or 4 4 6 dichlorotriazin 2 yl amino fluorescein and exist preferably as isomers of the formula str5 the following illustrative nonlimiting examples will serve to further demonstrate to those skilled in the art the manner in which specific tracers within the scope of this invention may be prepared the.