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The protocol for the competitive ELISA is as follows:
Coat the wells of an enhanced binding 96 well polystyrene microtiter plate with the Ig fraction of the antiserum, diluted in 10mM Tris, pH8.5 (125ul/well).
Incubate the plate for 2 hours at 37 degrees C.
Wash the plate 4 times with Tris buffered saline containing Tween 20and tap dry.
Prepare standard solutions of the target drug in TBST at 0 and 10ng/ml and add to the appropriate wells (50ul/well).
Add 75ul of conjugate (derivative of target drug conjugated to HRP), diluted in Tris buffer (pH7.2) containing preservatives with stabilising agentsto each of the wells.
Incubate the plate for 1 hour at 25 degrees C.
Wash the plate 6 times over a 10 minute period with TBST.
Add 125ul of tetramethylbenzidine (TMB) substrate solution to each well of the plate.
Incubate for 15 to 20 minutes in the dark at room temperature.
Terminate the reaction by addition of 125ul 0.2M H2SO4 to each well.
Measure absorbance at 450nm using a microtiter plate reader.
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Answered on Apr 07 2012