The procedure described in this chapter for the determination of
affinity constants and kinetic dissociation constants by band-shift
assay refers to an ideal antibody fragment (e.g., a scFv or an Fab
fragment) binding to a well-behaved protein antigen (pure, of
well-defined oligomeric state, migrating as a single band in
non-denaturing gel electrophoresis.
As mentioned in the introduction, either the antibody or the antigen
has to be labeled in these assays. In order to illustrate different
labeling methods, we will use radiolabeled recombinant antibodies in
the protocol for the determination of Kd, and fluorescently labeled
antigen in the protocol for the determination of koff.
Recombinant antibodies can conveniently be radiolabeled
site-specifically and without loss of immunoreactivity using
genetically introduced phosphorylation sites . This procedure is
described in this Chapter. However, other radiolabeling methods can be
used (e.g., radioiodination using Iodogen tubes), provided that
immunoreactivity is preserved and that a sufficiently high specific
activity is achieved.
The fluorescent antigen labeling method presented in this Chapter
features the use of N—hydroxysuccinimido ester derivatives of a
commercially available cyanine dye (Cy5—NHS; Amersham Pharmacia
Biotech). These reagents react with primary amino groups in the antigen
molecule, and may impair the antigen’s ability to react with the
antibody after labeling.
Phosphorylation of recombinant antibodies
Labeling the antigen with a red fluorophore
Native gel electrophoresis
Band-shift assay for the determination of Kd
In these experiments, it is convenient to work at antibody concentrations lower than the dissociation constant. This range of concentrations is typically not known a priori, and is determined by performing band-shift experiments at low antibody concentrations (e.g., 0.1 — 1 nM). The intensity of the bands is integrated with appropriate methods and fitted to the equation: Kd = [A][B]/[AB] (this procedure relies on the assumption that band intensity is proportional to the concentrations at the moment the sample is loaded on the gel). This fitting may yield an acceptable Kd value, or may indicate in which concentration range one should work to obtain more accurate results. Another important issue to consider is how long should antibody and antigen be allowed to react before analysing the reaction mixture on a native gel. In pseudo-first order conditions (e.g., when the antibody concentration is kept constant and << Kd) this reaction time is a function of the kinetic association constant kon and of the antigen concentration. The concentrations of antibody free and in complex will exponentially tend to a limit value, and one should wait at least t > 1 / (kon x [Antigen] total) before considering the reaction close to completion. Whenever the kon of the antibody is not known, it is important to measure affinity constants by band-shift with two different incubation times. If the reaction has reached equilibrium, the affinity values in the two experiments should not differ significantly. The protocol listed below is for a labeled antibody with Kd = 3 nM and kon = 106 s-1M-1.
Band-shift assay for the determination of koff
A prerequisite of this technique is that the antibody-antigen complex can be detected in a non-denaturing gel (if possible as a single band). If the antigen is fluorescently labeled, it is essential to be able to resolve the band of the complex from the band of the free antigen. It is also worth mentioning that only negatively charged proteins (or protein complexes) migrate towards the anode in a standard non-denaturing polyacrylamide gel with Tris/Glycine buffer, pH 8.3 (see above). If one wishes to study positively charged proteins (or protein complexes), one needs to work with reverse polarity electrodes and possibly with buffer systems different from Tris/Glycine (see manufacturer’s instructions of the Phast System, Amersham Pharmacia Biotech). The following protocol is written for the negatively charged complex of a recombinant antibody with a fluorescently labeled antigen. Although the reaction conditions are here given in the micromolar range, much lower concentrations can also be used if a high-sensitivity luminescence analyser is available (see for example .
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