I assume you used the typical low pH elution into a neutralizing buffer? This is best done fast and cold in my experience.  High salt can also work if the Ab/Ag interaction is the right type, or even high pH.  Also competition sometimes works with peptide ~0.1M.  Really tight binders (the kind most want) are unfortunately difficult to separate.

In my experience, the high affinity antibodies bind so tightly to the hapten that eluting them without damage can be challenging.

Try eluting with MgCl2, even as high as 3M.

Hi Francisco, a question: which is your hapten? ok for bsa as a carrier during the immunization step, but why not to use the only hapten (cross-linked to beads, obviously) for the purification step?

Many thanks to everybody. Our hapten is aflatoxin M1, about 300 Da. In addition to the dificulties do bind covalently the molecule to the beads, its low molecular weight could make impractical direct binding because of the risk of steric hindrance with the binding of antibodies.

To purify monoclonal antibodies, we used small columns (15x80 mm) packed with 5 ml of immunoabsorbent (i. e. CNBr-activated Sepharose 4B coupled with the immunogen). Affinity chromatography was performed by passing overnight the IgG fraction of MoAb in continuous cycle in 3M NaCl. After recovering the effluent (50 ml) and washing the column, elution was carried out with 50 ml of 3.5 M MgCl2. During these processes, optical densities were monitored at 280 nm. The eluate was then dialyzed against PBS and concentrated.   

ok, so a new question (sorry). can you exclude a release of your hapten from the column? It means: maybe your Ab didn’t loose its activity but you eluted the Ab-Ag complex, and your Ab isn’t available for new bindings

Thank you Grabriela and Giuseppe. We can try now with MgCl2 following your suggestions, we work with NaSCN.  Actually detachment of ligand could be a good explanatíon for the lack of activity in the eluate, we can try to to rule it out in the experiments with peptides.

Francisco, do you have any binders in the flow through? How did you immobilize your antigen? Would you mind sharing some more details?

Immobilization on divynil sulphone activated agarose (Mini-Leak) works much better than CNBr activated sepharose. After immobilization, you can try the matrix with high or low pH or high salt concentrations without any troubles.

Thank you again. We are checking diferent affinity resins and immobilization method to try to improve performance. This is undoubtedly a good point to control to face our troubles. Nevertheless the recovery of total IgG in previous experiments was quite (maybe too) high, but very little specific activity was recovered. We are also checking absorption with "blanc" columns.

Is it conceivable that linking of the peptide to BSA "blocked" the area of the peptide, in which you are interested / your antibodies react? As an alternative, is there any reason for NOT using the classical approach as follows:

-- get your peptide of interest with an added Cysteine residue.

Here the same issue as above may apply. Of course, the additional Cysteine must be placed at the extreme of the peptide which is NOT involved in your antibody reactivity. As a matter of speculation, if one does not know which extreme is involved, one might get both Cysteine-extended peptides and see what happens. As a cautionary note, it might happen that for some difficult to predict reason "both ends" of the peptide (how large is it???) are somehow required for full antibody reactivity.

-- link it to the classical "sulpholink" gel

-- run the affinity purification according to one of the standard classical protocols you can find in the web. We did the same some time ago.

All best wishes,

Gian-Luca

[email protected]