Protein Protocols Protein Protocols

1058 French
region disulfide bonds. Of the mouse IgG subclasses, IgG1 and IgG2a (three bonds), and
IgG3 (one bond) qualify, whereas IgG2b (four bonds) does not. F(ab') 2 derived from rabbit
Ig (one bond) and rat IgG1 (three bonds)can also be employed. However, rat IgG2a
and IgG2c both have two, and rat IgG2b has four and so cannot be used as the
maleimidated partner.
2. If F(ab') 3 derivatives are required, the number of SH-groups at the hinge of the
unmaleimidated partner should be at least two and preferably three, because this determines
the number of Fab'(mal) arms that can be conjugated.
3. We have found that a few antibodies give consistently low yields of BsAb when used as
the maleimidated partner. If large quantities of a derivative are required, it is worthwhile
performing small scale pilot preparations to determine which maleimidated partner gives
the optimal yield.
4. It is very important to avoid contamination of the Fab'-A(SH) with 2-ME. In order to
minimize the risk, stop collecting when the recorder has returned two-thirds of the way
back to the baseline. In order to ensure that Fab'-B(SH) is not contaminated with 2-ME
left over from the first run, make sure that it is not loaded until the chart recorder has
returned to the baseline.
5. Sometimes the mixture becomes slightly cloudy.
6. To avoid contamination with o-PDM/DMF, which elutes as a second large peak, stop
collecting when the chart recorder has returned halfway to baseline.
7. To avoid loss of product, slightly over concentrate, then wash the cell with a small volume
of chilled buffer.
8. It is very important to add excess iodoacetamide at this stage; otherwise the BsAb derivative
can precipitate.
References
1. Glennie, M. J., McBride, H. M., Worth, A. T., and Stevenson, G. T. (1987) Preparation and
performance of bispecific F(ab'γ) 2 antibody containing thioether-linked Fab'γ fragments.
J. Immunol. 139, 2367–2375.
2. Glennie, M. J., Tutt, A. L., and Greenman, J. (1993) Preparation of multispecific F(ab') 2
and F(ab') 3 antibody derivatives, in Tumour Immunobiology, A Practical Approach
(Gallagher, G., Rees, R. C., and Reynolds, C. W., eds.), Oxford University Press, Oxford,
UK, pp. 225–244.