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56 Gallant et al.
10. Flush the column successively with 3 column volumes each of coupling wash
buffer, blocking buffer and coupling wash buffer at 2 ml/min (60 cm/h).
11. Store at 2–8ºC in PBS with 0.05% sodium azide (or in 0.2 M imidazole, 0.5 M
NaCl, pH 7) to prevent microbial growth.
12. Using the initial and final antisera titers measured by Bradford protein assay,
calculate the coupling efficiency as:
Coupling Efficiency =
Final Antisera Titer in Coupling Solution × Final Volume
Initial Antisera Titer × Initial Volume
Coupling efficiency should exceed 90%.
3.2. Immunoaffinity Chromatography
1. Remove storage buffer and replace with equilibration buffer at a flow rate of 1
ml/min (30 cm/h, see Note 5).
2. Load preparation (see Note 6).
a. Cell culture supernatant is clarified to 0.2-μm filtration using a combination
of centrifugation and filtration (see Note 7). Verify quantitative product yield
during clarification using activity assay.
b. Clarified cell culture fluid should be held at 2–8ºC until loading on the
antibody column. For extended storage, sterility of the load should be
maintained. Sodium azide, 0.05%, can be added to the harvest (as a protection
against microbial growth) provided that there is no loss of target protein
activity.
3. Pass the load of 0.03 mg of target protein per ml of resin (see Note 8) over
the antibody column at a flow rate of 1 ml/min (30 cm/h, see Note 5). Collect
the flow-through as fractions (20% of the load per fraction is convenient). The
flow-through fractions may be analyzed for activity to verify that the capacity of
the column has not been exceeded.
4. Wash the column with equilibration buffer until the A 280 nm trace returns to baseline
(∼20–30 column volumes). Retain the wash fraction for activity analysis.
5. Add 0.12 ml of neutralization buffer to each of 20 fraction collection tubes; these
will be required in the next step. It is important that the fractions be neutralized
as they emerge from the column to maximize the preservation of protein activity.
6. Elute using 48 ml of elution buffer at 1 ml/min. Collect the column eluate in
fractions of 2.4 ml (0.5 column volume) using the fraction collection tubes from
the previous step. The final volume of each fraction (including 0.12 ml of neutralization
buffer) will be approximately 2.5 ml.
7. Pass 25 ml (3–5 column volumes) of cleaning buffer at 1 ml/min.
8. Pass 25 ml (3–5 column volumes) of storage buffer at 1 ml/min and store column
at 2–8ºC until the next use of the column.