Hi Everyone, we are trying to purify a mouse monoclonal IgG1/λ for a p24 sELISA system. From several experiments we have determined this antibody to be the bottleneck in increasing our sensitivity. Because the system is very sensitive (pg/ml range), we are trying to obtain antibodies with the highest quality and purity as we can. We are currently using Sepharose 4B Protein A columns from Invitrogen with the following conditions:
Binding Buffer: 3.3M NaCl + 0.1M Sodium Borate, pH 8.9
Wash Buffer A/B: 3M NaCl + 50mM/10mM Sodium Borate, pH 8.9
Elution Buffer: 0.1M Glycine + 0.1% Triton X-100, pH 2.5
Elutions are balanced with 5% 1M Tris, pH 9.5
Also, the antibodies are purified from mouse ascites after delipidation and fractionation.
What are some things that we can do to ensure high purity and quality? We are currently looking at either optimizing the binding buffer or try using Protein G.
Thanks in advance!
Dear Jason,
Definitely you should change to Protein G. The relative affinity of Protein A for mouse IgG1 is verry low (+). Better is Protein G (++++). In addition you can use commercial optimized Elution buffer.
Jason, Another option might be to try a mouse lambda specific resin. I have used CaptureSelect LC-lambda mouse successfully. It's available through Life Technologies. Elution can be anywhere from pH 3.5 down to pH 2.2, though. I have found that I can use a pH 2.8 citrate/phosphate buffer for most applications, and the Abs look just fine. Good luck, Steve.
Dear Jason
I agree with Mrs. Sabine Witt, in certain cases Protein G could be an excellent election for purifing Ab. This could be one, but strong interaction involves stronger elution conditions. Elutions at pH 2.7 could be enought, but it is not recomended less because the denaturation induced. Capto system sell by GE are excellent for monoclonal purification as I had observed in practice. Other option could be immovilization of the target protein in Sepharose activated with cyanogen bromide or other function. Purity are latter verified by electrophoresis techniques and functional characteristics by Dotbot, Westernblot or even indirect ELISA (coating with the protein and using a secondary conjugated Ab).
Good results and luck for you
Carlos