sprayed 7 8 mm from the bottom with polyclonal anti fluorescein isothiocyanate instead of anti human chorionic gonadotropin the control line is sprayed with a solution of avidin approximately 9 12 mm from the bottom of the membrane b preparation of fluorescein isothiocyanate alkaline phophatase conjugate gt 1400 u ml twenty five microliters of fluorescein isothiocyanate in 0 10m sodium borate ph 9 3 solution 3 mg 400 u03bcl is added to 5 mg of alkaline phoshatase in sodium borate at a total volume of 0 5 ml the resultant solution is allowed to stand for 3 hours at ambient temperature the solution is then separated on a g25 sephadex column and eluted with 0 1m sodium carbonate ph 8 5 to this is added 100 ml of a solution of 5 0 mg n hydroxysuccinimidyl biotin in 1 0 ml dimethylsulfoxide the resultant solution is stirred for approximately 12 hours free biotin is removed by separating the conjugate on a g25 sephadex column and eluting it with a buffer containing 10 mm tris 1 bovine serum albumin 150 mm sodium chloride and 0 1 sodium azide ph 7 4 the conjugate is then stored at 4 u00b0 c the conjugate solution is aliquoted into 10 tubes 75 ml and then lyophilized the tubes are stoppered and stored c substrate preparation into 10 additional tubes 75 mm is aliquoted 0 8 ml of 5 bromo 4 chloro indolyl phosphate nitroblue tetrazolium substrate solution.