The bsAb can be produced either as ascites or in culture supernatant. Ion-exchange chromatography using Bakerbond ABx columns under FPLC conditions is often successful in purifying the desired bsAb from parental antibodies and mispaired molecules. Chromatography conditions should be optimized by running the column with the parental hybridoma antibodies individually and as a mixture.
Ascites fluid or supernatant containing bsAb (unit 2.6)
25 mM 2-(N-morpholine)ethane sulfonic acid (MES; Sigma), pH 5.4
Bakerbond ABx column 7.75 x 100 mm (Baker Chemical) connected to an
FPLC system (Pharmacia Biotech) 1 M sodium acetate, pH 7.0 (or other buffer of choice) Sodium azide PBS (appendix 2)
Additional reagents and equipment for dialysis (appendix 3h) and quantitating protein (unit 2.11)
1. Dilute 1 vol ascites fluid or culture supernatant containing bsAb with 9 vol MES. Filter through an 0.2-|m filter.
Protein A may be used to prepurify fractions from ascites ( unit 2.7). Culture supernatants must be prepurified by Protein A chromatography using elution buffer with pH appropriate for the IgG subclass.
One milliliter ascites fluid may be purified per run under these conditions. Filtration removes debris and greatly enhances the life span of the column.
2. Load filtered sample on a Bakerbond ABx column connected to an FPLC system. Elute the column at 1 ml/min with a linear gradient of 0 to 1 M sodium acetate. Collect 3-ml fractions, add sodium azide to 0.02% (w/v) final concentration, and store up to 1 month at °C. Test for desired activity.
HPLC can also be used to fractionate immunoglobulins.
3. Pool active fractions and dialyze (appendix3h) against PBS.
4. Determine protein concentrations (unit 2.11).
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