Protocol 6

6. Analysis of Oligosaccharide Ligands by High Performance Liquid Affinity Chromatography

WeiTong Wang~GlycoTech Corporation, Rockville, Maryland 20850

High performance liquid affinity chromatography (HPLAC) is a useful technique for investigation of the interaction between a carbohydrate binding protein and its ligands. HPLAC operates in the physiologically relevant association constant range for carbohydrate binding proteins of 10^-2 to 10^-4M^-1. The technical requirements of this method are similar to conventional HPLC. One of the major applications of HPLAC is to screen and separate natural ligands from complex biological mixtures such as detecting, purifying and determining the binding constant of a carbohydrate ligand from bodily fluids. HPLAC is also useful in the rational design and synthesis of new carbohydrate drugs by providing a fast, sensitive, and simple method for the determination of K_D values.

The critical requirement to perform a successful HPLAC analysis is to construct an appropriate affinity column. There are many methods to construct such a column. The following protocol describes an effective and simple method including in situ coating an IgG or an IgG-chimeric protein to a perfusion chromatographic column of protein A followed by an optional cross-linking procedure.

Materials:

Boric acid-sodium borate buffer (0.2M), pH 8.2
Triethanolamine HCl (TEA), (0.2M), pH 8.2

Ethanolamine (0.1M)

Freshly made dimethylpimelidate dihydrochloride (DMP) (10 mM) in triethanolamine (0.2M)

Phosphate buffered saline (PBS, 20 mM sodium phosphate, 150 mM NaCl, pH 8.0)

Column of HPLAC A (Dynamic Coupling of IgG- HPLAC-A Column)

IgG or Ig chimeric protein

Protocol:

Coating IgG or IgG-chimera to HPLAC-A column

1. Dialyze IgG or IgG-chimera with PBS buffer. The protein concentration is adjusted to 0.5-1 mg/ml PBS. One ml of packed column binds about 10 mg of IgG or IgG chimera.

2. Condition the HPLAC-A column on a conventional HPLC system with the same PBS buffer at a flow rate of approximately 0.5 ml/min for 30 min at l0�C.

3. Pass the solution through the column at a flow rate of approximately 0.5 ml/min, l0�C. Monitor the eluant with a UV detector (280 nm).

4. Increasing absorbance at 280 mm indicates saturation of the column. At this time switch back to the PBS buffer.

5. The amount of IgG coupled to the column is obtained by subtracting the unbound IgG from the total IgG passed through the column.

Chemical Cross-linking IgG to the HPLAC A column (optional)

1. Keep HPLAC-A column at 10�C, and wash it with 10 column volumes of borate buffer at a flow rate of 0.5 ml/min.

2. Pass 10 column volumes of TEA solution through the column at a flow rate of 0.5 ml/min, 10�C.

3. Pass 15 column volumes of DMP solution through the column at a flow rate of 0.5 ml/min, 10�C.

4. Condition the cross-linked column with PBS buffer (0.5-1.0 ml/min) for 30 min. and the column is ready for affinity chromatography.

Run HPLAC and K_D Value Determination

1. HPLAC is operated in a conventional HPLC mode with the exception that physiologically compatible buffers are used (e.g. PBS or Tris buffer with pH>7). A relatively lower linear velocity (~0.04 cm/sec or 0.3 ml/min for 250 mm x 4.6 mm i.d. column) is highly recommended

2. The K_D value can be calculated according to the simplified equation:

where B_t is the total amount (moles) of immobilized ligand sites, V_m and V_o are the maximum retention volume (liters) and the void volume (liters), respectively. t'-t_o represents the time (minutes) that the elution of the peak of active material is retarded by the column, and v is the elution flow rate (liter/min).

References:

1. Zopf, D.A., and Ohlson, S. (1990) Weak affinity chromatography. Nature 344: 87-88

2. Wang. W., Lundgren, T., Lindh, F., Nilsson, B., Gronberg, G., Brown, J.P., Dibert, H., and Zopf, D.A. (1992) Isolation of two novel sialyl Lewis X-active oligosaccharides by high-performance liquid affinity chromatography using monoclonal antibody onc-M26. Arch. Biochem. Biophys. 292: 433-441.

3. Zopf, D.A., and Wang, W-T. (1993) Purification of oligosaccharide antigens by weak affinity chromatography. ACS symposium series 519, Carbohydrate Antigens, 80-91, American Chemical Society, Washington, D. C.

4. Arnold, F.H., Schofield, S. A., and Blanch, H.W. (1986) Analytical Affinity Chromatography. I. Local Equilibrium Theory and the Measurement of Association and Inhibition Constants. J. Chromatogr. 355Z:1-12.