It is sometimes advantageous to affinity purify an antibody preparation, in order to eliminate background "noise" in a given assay. In particular, it is frequently necessary to purify anti-phosphopeptide immunoglobulins in an anti-phophopeptide serum away from non-phosphopeptide immunoreactivity (see Note 2). With a ready source of immunogen in the form of a synthetic peptide, it is straightforward to purify anti-peptide antibodies by affinity chromatography. The peptide is covalently coupled to a matrix such as agarose and the crude antibody preparation passed down the column. Unbound material is washed away and the bound antibody eluted under denaturing conditions, for example, low pH (pH 2.5), high pH (pH 11.5), or 4 M MgCl2. Immunoglobulins are unusually resistant to permanent denaturation by pH extremes or chaotropic reagents, although there are always exceptions (especially with monoclonal antibodies). Low pH followed by rapid neutralization is, perhaps, most straightforward. A 4-M MgCl2 elution is milder and, therefore, should be used if low pH elution results in significant loss of activity.
The affinities of antibodies for their cognate peptides can be very high, so that it is sometimes difficult to quantitatively recover the higher-affinity antibodies in a polyclonal serum from the peptide-matrix. For this reason, it is important to use low concentrations of peptide on the matrix (typically 100-200 |ig of peptide per milliliter of agarose gel). In addition, elution of bound antibody in the reverse direction to which it was run into the column, so as not to drive eluting antibodies into a further excess of antigen, enhances recovery. If both of these criteria are adhered to, recovery is usually of the order of 60-80% (see Note 3).
Suitable affinity resins are CNBr-activated Sepharose and similarly activated V-hydroxysuccinimide ester-based gels with spacer arms. These react with free amino groups on the peptide. If the pep-tide contains several lysine residues, coupling via amino groups can adversely affect antibody affinity for the peptide (although this is not necessarily inevitable). For this reason, a cysteine residue added to the N- or C-terminus of the synthetic peptide affords the option of coupling to agarose via the sulfhydyl group (see Chapter 2).
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