IsocRATic Method

1. Dialyse the serum, or preferably, the immunoglobulin-containing fraction resulting from one of the precipitation techniques previously described, against 0.05 M phosphate, pH 6.3 (see Note 11).

2. Wash the exchanger twice with a 10-fold excess of 0.05 M phosphate buffer pH 6.3 and pack into a suitable column (see Note 14). Use a volume of resin at least 2X the volume of Ig containing fraction. Equilibrate the column by passing through at least 10X the column volume of phosphate buffer.

3. Apply the sample and elute with 0.05 M phosphate, pH 6.3. The immunoglobulins should pass straight through; collect the eluate until the absorbance at 280 nm is less than 0.1.

4. Proteins bound to an agarose-based column can be removed by passing through 10 column volumes of 0.05 M phosphate, 1 M NaCl, pH 6.3, whereupon the column may be regenerated by passing through a further 10-column volumes of the phosphate buffer without NaCl.

Regeneration of cellulose-based columns is most reliably achieved by using the acid/alkali precycling procedure described (see Note 13).

3.3.12. Gradient Method

Immunoglobulins will bind to anion-exchangers if the pH is raised to between 8.0 and 9.0 and a higher degree of purity can often be achieved on subsequent elution using a salt gradient. Such methods can be readily adapted for use with high-resolution systems such as high-performance liquid chromatography or fast-proein liquid chromatography (see Note 15).

1. Dialyse the serum, or preferably, the immunoglobulin-containing fraction resulting from one of the precipitation techniques described earlier, against at least two changes of a 20- to 50-fold excess of 0.05 M Tris-HCl, pH 8.5.

2. Pack the exchanger (DEAE-agarose) into a suitable column (see Note 16) and equilibrate with 10-column volumes of 0.05 M Tris-HCl, pH 8.5.

3. Apply the sample and wash with two-column volumes of 0.05 M Tris-HCl, pH 8.5.

4. The majority of the immunoglobulins will bind to the column and can be eluted by the application of a NaCl gradient. The eluting buffer is progressively changed from 0.05 M Tris-HCl, pH 8.5, to 0.05 M Tris-HCl, 0.5 M NaCl, pH 8.5 (see Note 3). Collect fractions and monitor the absorbance at 280 nm to detect the protein containing fractions (see Note 17).

5. Finally elute the column with five-column volumes of 0.05 M Tris-HCl, 1 M NaCl, pH 8.5 This should remove any remaining proteins and allow the column to be re-equilibrated with 0.05 M Tris-HCl, pH 8.5, for re-use or washed with 0.02 (w/v) sodium azide for storage if reuse is not imminent.

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