免疫技术

Peptide Inhibition ELISA Protocol

2025-06-19 免疫技术加入收藏

Peptide Inhibition ELISA ProtocolAuthor: Nanci DonackiSource: Contributed by Nan

Author: Nanci Donacki

Source: Contributed by Nanci Donacki

Reagents PBS PBS:0.1% BSA:0.05% Tween 20 (PBT) PBS:2% BSA 10X Substrate Buffer, pH 6.0 36.6 g Citric Acid, monohydrate 113.5 g Potassium dibasic phosphate Dissolve in 900 ml di-H2 O. Check pH and adjust to 6.0 if necessary. Qs. to 1 liter. 30% H2 O2 OPD Stock, 4.0% 4 g OPD in 100 ml di-H2 O. Aliquot and store at -20o C. Protect from light. 4.5N H2 SO4 12.0 ml Concentrated Sulfuric Acid 88.0 ml di-H2 O

Procedure

Titer MAb supes at serial 5-fold dilutions to determine the concentration at which the titer begins to drop. Dilute the MAb to one-half the above dilution in PBT Dilute specific and non-specific peptides to 200 mg/ml in PBT. 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. Incubate 1 hour at 37o C. Transfer 50 ml to an antigen coated plates, which has been blocked with PBS:2% BSA for 1 hour at 37o C. Incubate for 1 hour at 37o C. Wash 3 times with di-H2 O. Add 50 ml/well anti-mouse IgG:HRP diluted in PBS:0.1% BSA:0.05% Tween 20. Incubate for 1 hr at 37o C. Wash 5 times with di-H2 O. Add 50 ml/well working substrate solution 0.5 ml 4.0% OPD 50 ml 30% H2 O2 1.0 ml 10X Substrate buffer 8.5 ml di-H2 O. Incubate for 20 minutes at room temperature. Add 25 ml/well 4.5N Sulfuric Acid Read A490

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