Sucrose → (4-methoxyphenyl)(1-pentyl-1H-indol-3-yl)methanone

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The wells of an enhanced binding 96 well polystyrene microtiter plate were coated with IgG fraction of antiserum raised to Immunogen VI, diluted in 10 mM Tris, pH8.5 (125 μl/well). The appropriate antibody coating dilution was determined using standard ELISA checkerboard techniques. The plate was incubated overnight at 4° C., washed 4 times over 10 minutes with Tris buffered saline containing Tween 20 (TBST) and tapped dry. Standard solutions of RCS-4 were prepared in TBST at 0, 0.3125, 0.625, 1.25, 2.5, 5, 10 and 20 ng/ml, and 50 μl of each was added to the appropriate wells (see FIG. 1). 75 μl of conjugate (appropriate hapten-HRP) diluted in Tris buffer (pH 7.2) containing EDTA, D-mannitol, sucrose, thimerosal and BSA, was added to each of the wells. The appropriate dilution of conjugate was also determined using standard ELISA checkerboard techniques. The plate was incubated at 25° C. for 1 hour. Excess unbound conjugate was removed by washing 6 times over a 10 minute period with TBST. 125 μl of tetramethylbenzidine (TMB) substrate solution was added to each well of the plate that was then incubated for 20 minutes in the dark at room temperature. The reaction was terminated by addition of 125 μl 0.2M H2SO4 to each well. The absorbance was then measured at 450 nm using a microtiter plate reader. The data are inputted to a computer program called ‘KC Junior’. It is a 4 parameter fit curve and allow the calculation of concentrations between the standard runs. This program is used to calculate the IC50s by dividing the 0 ng/ml OD value by 2 and obtaining the concentration value from the curve for this OD. The data generated in the assay and inputted to a computer program called ‘KC Junior’ are presented in Table 3.

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