1. 0.05 M Sodium phosphate buffer is conveniently made by mixing solutions of 0.05 M Na2HPO4 and 0.05 M NaH2PO4 to obtain the correct pH.
2. This is most simply prepared by the addition solid NaCl to 0.05 M phosphate buffer pH 6.3. This will alter the pH and concentration of the phosphate, but in this case it will not affect the procedure because the critical factor is the sodium chloride concentration.
3. Gradient makers are available commercially, but instructions for making a simple but effective device are given by Flurkey (13).
4. Suitable matrices are available from a number of suppliers and include T-gel adsorbent (Pierce). Amersham Biotech offers "HiTrap IgM" and "HiTrap IgY" aimed at the purification of IgM and the chicken immunoglobulin, IgY. These are based on 2-mercapto-pyridine as the thiophilic adsorbent.
5. Prepare as recommended in Note 2. Any change in pH or phosphate concentration will not be relevant.
6. Use free cysteine rather than the hydrochloride salt. Cysteine solutions will oxidize readily and should be freshly prepared daily.
7 Glass containers are preferable as glass is more effective at promoting coagulation than plastic.
8. If the starting material is plasma rather than serum it is advisable to remove the fibrinogen as subsequent steps may remove the anticoagulant and initiate clotting. If the anticoagulant is EDTA or citrate add 0.1 mL of 0.5 M CaCl2 and 1 IU of thrombin per mL of plasma and incubate at 37°C for 15 min. If the plasma is heparinized then first add 10 ^L of 5 mg/mL protamine sulphate per mL of plasma before adding the calcium chloride and thrombin as previously explained.
9. Other salts, particularly sodium sulphate, can be used for the precipitation of immunoglobulins. Sodium sulphate can give a purer preparation of human and rabbit immunoglobulins, but the yield may be less than that obtained using ammonium sulphate.
10. In some instances 50% saturation may precipitate an unacceptable level of other serum proteins. If this is the case then reduce the amount of saturated ammonium sulphate added as the majority of the immunoglobulins are precipitated at less than 45% saturation.
11. Dialysis should be conducted at 4°C against a 50- to 100-fold volume excess of the appropriate buffer for at least 8 h with one change of buffer. If the volume of material to be dialysed makes this volume excess impracticable, then a smaller volume may be used and additional buffer changes incorporated.
12. It must be kept in mind that the immunoglobulin population is a heterogeneous one and although the values in Table 1 will precipitate the bulk of the serum proteins from the given species, it is advisable to test for the yield of the desired specificity and make minor adjustments to the volume added if necessary. Further information is given by McKinney and Parkinson (14).
13. This is achieved by washing the exchanger in a 10X volume excess of 0.1 M HCl followed by extensive water washing until the pH of the wash approaches 6.0. This can be carried out using a Buchner filter, by centrifugation or by decanting the supernatant after allowing the slurry to settle. The exchanger is then washed in a 10x volume excess of 0.1 M NaOH and the water-washing repeated until the pH of the wash falls below 8.0.
14. The immunoglobulin containing fraction can be recovered by cen-trifugation or filtration rather than using a column method. If a column is used, then its geometry is not important and a 'short fat' column will provide a better flow rate and thus speed up the process.
15. Fast-protein or high-performance liquid chromatography systems using columns such as Mono Q, Poros HQ or TSK DEAE 5PW are suitable for the purification of immunoglobulins following this protocol.
16. In methods involving a gradient elution, the column geometry has more of an influence and it is preferable to have a length to diameter ratio of at least 5.
17. The total volume of the gradient should be 10-20-column volumes. In this method, albumin elutes immediately following the immuno-globulin fraction. The separation can be improved if necessary by applying a shallower gradient.
18. The affinity is influenced by salt concentration in accordance with the Hofmeister series. It is increased in the presence of lyotropic anions such as sulphate and decreased in the presence of chaotropic anions at the other end of the series such as thiocyanate. Anions from the middle of the scale such as chloride have little effect on binding.
19. If the immunoglobulin is to be purified from a dilute source such as a tissue culture supernatant, use solid K2SO4. Add this slowly, with gentle stirring, to prevent localized high concentrations of the salt, which may lead to precipitation.
20. Protein G contains several binding sites for the constant region of the heavy chain of IgG and also a binding site for serum albumin (15). There are recombinant versions of protein G where the albumin-binding site has been removed and these are preferable for use in purification procedures.
21. Coupling at slightly alkaline pH is more efficient, but can result in a ligand that is more constrained and therefore reduced in its ability to bind its target protein. Coupling at a lower pH (e.g., using 0.1 M phosphate, 0.5 M NaCl, pH 7.0, or 0.1 M sodium citrate, pH 6.5) may result in a lower yield, but this can be offset by higher binding efficiencies.
22. Do not allow the pH to rise higher than 11.5 because the activated groups are not stable above that pH, and the binding capacity of the matrix will be severely reduced.
23. Glassware that has been in contact with cyanogen bromide should be decontaminated before reuse. It is advisable to take advice on local procedures, although soaking overnight in 2 M NaOH has been suggested as has the use of hypochlorite, although care should be exercised in using hypochlorite with protein containing solutions.
24. Avoid the use of magnetic stirrers because damage to the resin beads can result. Rotary ("Ferris wheel")-type mixers are ideal.
25. Incorporate 0.05% (w/v) sodium azide for long-term storage.
26. Yield can be increased by recirculating the unbound material back through the column two or three times.
27. This procedure should be performed with purified IgG.
28. The absorbance at 280 nm of a 1 mg/mL solution of human IgG is 1.36, and the values for other mammalian IgG are similar (16).
29. It can be beneficial to perform a trial digestion because the optimum time can vary with different Ig preparations.
30. A fragment with very similar properties to Fab, Fab' can be prepared by gentle reduction of (Fab')2 with 10 mM cysteine in 0.1 M sodium bicarbonate, pH 8.2 at 25 °C for 2 h. The sulfhydryl groups are then alkylated by the addition of iodoacetamide to a final concentration of 12 mM and incubation at 25°C in the dark for 2 h.
31. Gel filtration matrices are based on agarose, dextran, polyacrylamide or combinations of these. The most extensive range is supplied by Amersham Biosciences under the trade names of Sepharose, Superose, Sephacryl, Superdex, and Sephadex, and there are several versions of each brand with a variety of pore sizes and therefore a range of molecular weights for which they are most applicable. Sephadex has an upper limit of around 100 kDa, and thus its usefulness in immunoglobulin purification is as an alternative to dialysis for desalting applications. Superdex and Superose have better flow properties, permitting much faster separations and higher resolution, but are significantly more expensive than Sepharose or Sephacryl and require a pump capable of dealing with back pressures beyond the capabilities of a peristaltic pump. If such equipment is available, then Superdex 200 is a suitable choice for the purification of (Fab')2. Prepacked Superdex columns are available commercially, but columns can be packed in house. If more modest equipment is all that is available and self-packing is to be attempted then Sephacryl S300 HR, or Sepharose 6B (or CL-6B) may be used.
32. Gel filtration can also have a role in a final "polishing" step in the purification of IgG for which the matrices suggested earlier for (Fab')2 purification would be suitable. It has can also be used as a main step in the purification of IgM, which has a significantly greater molecular weight than the majority of other proteins. For IgM purification, the matrices, which would be applicable would be Superdex 200, Sephacryl 400HR, or Sepharose 4B.
33. The amount of matrix required is approx 110 to 120% of the final column volume.
34. In gel filtration the buffer has little effect on the elution of the proteins in the sample and the limitations are imposed by the stability of the matrix. Sepharose is stable within the pH range of 4.0-9.0, CL-Sepha-rose, between pH values of 3.0 and 14.0, and Sephacryl from pH 3.011.0. There is the possibility of some ionic interaction between the matrix and the protein at very low ionic strength and therefore it is usual to maintain a salt concentration of approx 0.1-0.15 M to prevent this.
35. Column length has a significant influence on gel filtration whereas column diameter does not influence the resolution but, does affect the amount of protein that can be loaded. In this instance, column lengths between 30 and 60 cm and diameters between 1 and 3 cm would be suitable. Some columns are supplied with flow adaptors, which eliminate the dead volume above the gel bed.
36. Operating pressure is the height differential between the level of liquid in the reservoir and the end of the outlet tube. Sephacryl has better flow properties than Sepharose and can be packed at higher pressures. A peristaltic pump may be used to create a flow rate of 1 mL/min in a column of 1.6 cm diameter or 2.5 mL per min in a column of 2.5 cm in diameter.
37. It is important to ensure that the column does not "run dry," that is, that no air should be allowed to enter the packed gel bed.
38. The volume of sample applied should be 1-2% of the column volume; greater than 5% the resolution is adversely affected. The concentration of the sample is less critical unless this increases the viscosity, but this is unlikely to be a consideration unless the concentration exceeds 20 mg/mL.
39. Use of a flow adaptor can facilitate sample application. The sample may also be layered under the buffer after increasing the density by the addition of sucrose or glycerol.
40. Separation by gel filtration depends on the lateral diffusion of the solute molecules into the pores of the gel, and thus the resolution can be impaired by high flow rates.
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