Cocaine Immunoassay Case Study

Direct competitive cocaine immunoassay was performed in a transparent high binding 96 well microtiter plate. Each well was coated with 200 µL of 1mg/L anti-mouse IgG in PBS (phosphate buffered saline, 10 mM sodium dihydrogen phosphate, 70 mM sodium hydrogen phosphate, 145 mM sodium chloride, pH 7.6). The microtiter plate was left over night for shaking at 750 rpm. Therefore, the plate was washed three times with automatic 96-channel plate washer by washing buffer (45 M potassium dihydrogen orthophosphate, 0,375 mM dipotassium phosphate. 1.5 M Potassium sorbate, Tween 20, pH 7.6). Afterwards, 200µL of benzoylecgonine (BEC) antibody diluted in PBS (0.005 µg/mL) was added to each well and incubated for 1 hour with shaking. This step was preceded by three cycles of washing and 100 µL of standards were added to each well.
Immediately, 100 µL of BEC-HRP conjugate diluted using PBS (1:15000) was added to the standard solution. The microtiter plate was incubated for another hour while shaking at 750 rpm. Consequently, another three cycled washing step was performed, 200 µL of the freshly prepared HRP substrate solution was added to each well. The HRP substrate solution25 was 21 ml citrate buffer (220 mM sodium citrate monobasic, pH 4.0) + 8.1µL H2O2 (30%) + 525 µLTMB solution (40 mM TMB, 8 mMtetrabutylammonium borohydride, in N,N-Dimethylacetamide). The microtiter plate was incubated for 30 minutes. Eventually, the color development was stopped by adding 100µL of 1 M H2SO4. And therefore, the absorbance was measured using plate spectrophotometer SpectraMax Plus384 (Molecular Devices, Ismaning, Germany) at 450 nm with 620 nm as a reference. Four parameter fit (4PL) was used for measuring Cocaine ELISA calibration curve; A (upper asymptote), B (slope at the inflection point), C (the inflection point on the calibration curve), and D (lower