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Titer MAb supes at serial 5-fold dilutions to determine the concentration at which the titer begins to drop.
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Dilute the MAb to one-half the above dilution in PBT
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Dilute specific and non-specific peptides to 200 mg/ml in PBT.
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In a microtiter plate, mix equal volumes of the peptide dilutions and the MAb supe. Add an equal volume of PBT and MAb as the control.
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Incubate 1 hour at 37oC.
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Transfer 50 ml to an antigen coated plates, which has been blocked with PBS:2% BSA for 1 hour at 37oC.
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Incubate for 1 hour at 37oC.
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Wash 3 times with di-H2O.
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Add 50 ml/well anti-mouse IgG:HRP diluted in PBS:0.1% BSA:0.05% Tween 20.
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Incubate for 1 hr at 37oC.
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Wash 5 times with di-H2O.
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Add 50 ml/well working substrate solution
0.5 ml 4.0% OPD
50 ml 30% H2O2
1.0 ml 10X Substrate buffer
8.5 ml di-H2O.
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Incubate for 20 minutes at room temperature.
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Add 25 ml/well 4.5N Sulfuric Acid
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Read A490