Protein Protocols Protein Protocols

Affinity Purification of Monoclonal Antibodies 1123
classes or species. Furthermore, elution conditions are much milder, which might be
beneficial if the harsh elution conditions required for strongly binding antibodies to
protein A lead to their denaturation. Thiophilic gels also purify chicken IgY (4). A
general method is provided in Subheading 3.4.
The major disadvantage of thiophilic gels is the requirement to add high concentrations
of chaotropic salts to the adsorption buffer to facilitate binding. This requirement is
not a problem for small-scale purifications at the bench, but it is unattractive at larger scale.
Recently, a major improvement of thiophilic chromatography came about with
the utilization of very high ligand concentrations (9). By immobilizing mercaptoheterocyclics
such as 4-mercaptoethyl pyridine at very high concentrations, the need to
add chaotropic salts such as sodium sulfate was abolished. All antibodies investigated
are adsorbed at neutral pH, independent of the salt concentration. At acidic pH values,
the ligand becomes charged and through charge–charge repulsion, the antibody is
eluted. This variation of thiophilic adsorption chromatography is highly reminiscent of
protein A chromatography in its simplicity, but with the added advantage that it is
nonspecific for antibodies. Purities and recoveries obtained range from 90% for antibodies
from protein-free cell culture supernatant to approx 75% from ascitic fluid. The
method is outlined in Subheading 3.5.
1.3. Immobilized Metal Affinity Chromatography
Immobilized metal affinity chromatography (IMAC) is a general term for a variety
of different immobilization chemistries and metals utilized (10). The most commonly
used gel for IMAC is nickel-loaded iminodiacetic acid (Ni-IDA) gel, used, for
example, in kits for the separation of His-tail modified proteins. Under slightly alkaline
conditions, the interaction between the immobilized nickel and proteins is strongest
with accessible histidine residues. Ni-IDA binds to the Fc portion of the antibody (11),
and similar to thiophilic adsorption chromatography, it binds all antibodies without
discrimination between subclasses or species (11,12). Depending on the growth
medium used, some contamination is apparent, namely transferrin and traces of albumin.
The purity of monoclonal antibodies purified out of ascitic fluid is generally lower
than out of hybridoma cell culture supernatant. As described for protein A chromatography,
a shift in pH to acidic values leads to the generation of charges on the histidine
residues and consequent elution of the protein bound. Alternatively, competitive
elution with either imidazole or EDTA yields antibody in good purity. However, the elution
with EDTA delivers the antibody with the metal still bound to the protein, while imidazole
adsorbs at 280 nm, interfering with UV detection. A general protocol is outlined.
2. Materials
2.1. General Purification of Human IgG, Humanized IgG,
and Mouse IgG2a and Mouse IgG2b Using Protein A
1. Buffer A: 50 mM Tris-HCl, pH 7.5.
2. Buffer B: 50 mM Tris-HCl, 500 mM NaCl, pH 8.0.
3. Buffer C: 100 mM acetate buffer, 50 mM NaCl, pH 3.0.
4. Buffer D: 10 mM NaOH.