Making ELISA using good antibodies

Materials and Methods

Using Polyclonal Antibodies

You need the following materials and procedures.

1. Antibody purification: Protein G column is the best for this purpose. Please refer to the manufactures' manuals. Protein A column

2. Conjugate: This is critical for the ELISA . Making conjugate is the most important part. I will show making conjugate with horseradish peroxidase.

3. 96-well plate: Making the solid phase using the 96-well plate. I personally prefer the Corning's. Nunc's may also be applicable. Of course, you can try any company.

4. Buffers and other reagents:

Plate buffer: 0.1 M Sodium carbonate buffer, pH 9.5

Reaction buffer: 0.01 M Sodium phosphate buffer, pH 7.2, 0.15 M NaCl (PBS), 0.5% BSA, 0.05% thimerosal; You can also use Dulbecco's PBS or try others. Instead of BSA, you can use gelatin. Skim (0.5% to 1%) milk could reduce the non-specific reaction.

Washing buffer: 0.05% Tween-20, 0.01 M Sodium phosphate buffer, pH 7.2 or 0.05% Tween-20, 0.15 M NaCl

Developing buffer: 0.05 M Sodium acetate buffer, pH 5.5

TMB stock solution: Tetramethylbenzidine 1 mg/ml in DMSO

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