immunoaffinity chromatography - blocking the resin (May/09/2007 )

Hi. I'm trying to purify monoclonal antibodies obtained by ascites fluid by affinity chromatography. I have the CNBr Sepharose 4B resine, do I need to blockunreacted affinity resin binding sites after coupling it with my antigen??? and is it affinity chromatography a good method to purify my MAbs vs. ammonium sulfate precipitation?

---

monoclonals from ascites fluid is cruelty of animals! it should not be supported by advisory service at this forum!

---

Dont worry we have specific regulations and we stick to them in order to avoid and in some necessary cases minimize discomfort, distress and pain on the animals, thank you any way

---

inmuno - in my opinion answer is 'yes' for both of your questions

---

Have you considered Protein A or G for purification? Of course it is not as specific as an affinity purified antibody, but the ones I have done have been quite good. But then again I guess I didn't compare the results of both methods.

---

hi!
It is usefull to block unreacted sites of activated matrix because of futher possible interaction of coupled antigen with activated matrix that may cause multiple site binding of antigen and prevent right epitope presentation. On the other side the possibility of non-specific binding of your targeted antiboby on modified resin will increase also. The blocking procedure is simple one. The most cheap reagent for this is glycine (0.1M) or ethanolamine. Incubate 2 hours at RT after coupling procedure and then wash your resin several times with acetate pH 4.5 and NaHCO3 pH 8.0. But concerning CNBr activated matrix - if you didn't do this, the active groups generated by CNBr-activation converse fast into non active carbamates so I think blocking procedure is not very critical for this type of activation if you will use affinity resin after 12 hours.

Affinity purification will be better if you want to get rid of non specific mouse Ig ( so you automatically increase activity of your purified Ab) which present in Ab purified by protein G or A.

Concerning AS precipitation I think that this step will decrease activity of antibodies. The main rule in Ab purification is minimal harsh purification steps.
The affinity purification on antigen with futher one step polishing by GF it will be a better choice to get active Ab but more expensive.

---