反应详情

The wells of an enhanced binding 96 well polystyrene microtiter plate were coated with the IgG fraction of the antiserum raised to Immunogen A (Hapten A-BSA) (Example 21), diluted in 10 mM Tris, pH 8.5 (125 μl/well). The appropriate antibody coating dilution was determined using standard ELISA chequerboard techniques. The plate was incubated for 2 hours at 37° C., washed 4 times with Tris buffered saline containing Tween 20 (TBST) and tapped dry. Standard solutions of fentanyl and norfentanyl were prepared in TBST at 0, 1, 5, 10, 50 and 500 ng/ml and 25 μl of each was added to the appropriate wells (see FIG. 12). 100 μl of conjugate B (hapten B-HRP) (Example 25), diluted in Tris buffer containing EDTA, D-mannitol, sucrose, thimerosal and BSA, was added to each of the wells, as shown in FIG. 12. The appropriate dilution of conjugate was also determined using standard ELISA chequerboard techniques. The plate was incubated at 37° C. for 2 hours. The 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, which was then incubated for 15 to 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 generated in the assay is presented in Table 1 below.

1. temperatureraised to Immunogen A (Hapten A-BSA) (Example 21)
2. washwashed 4 times with Tris buffered saline
3. additioncontaining Tween 20 (TBST)
4. customtapped dry
5. additioneach was added to the appropriate wells (see FIG. 12)
6. additionwas added to each of the wells
7. waitThe plate was incubated at 37° C. for 2 hours
8. customThe excess unbound conjugate was removed
9. washby washing 6 times over a 10 minute period with TBST
10. addition125 μl of tetramethylbenzidine (TMB) substrate solution was added to each well of the plate, which
11. waitwas then incubated for 15 to 20 minutes
12. customdark at room temperature
13. customThe reaction was terminated by addition of 125 μl 0.2M H2SO4 to each well