Affinity techniques for immunoglobulins and other molecules

1.4.3 Isolation of IgG subclasses using protein A–agarose

Although in both human and mouse the IgG subclasses differ markedly from each other in their biological properties, they are structurally very similar. This similarity has made it almost im- possible to isolate single subclasses using physical chemical techniques. Fractionation of the IgG subclasses is possible using protein A affinity chromatography and pH gradient elution.

Isolation of mouse subclasses

IgG is common to mammalian species, but further evolution has occurred since subclasses of IgG are present in many animals but there is no clear relationship between subclasses in different

species. IgG 1 , IgG 2 , IgG 3 and IgG 4 are found in humans; IgG 1 , IgG 2a , IgG 2b and IgG 3 in mice; and

IgG 1 , IgG 2a , IgG 2b and IgG 2c in rats.

Mouse serum may be fractionated on protein A–agarose by:

1 allowing all the IgG to bind to the adsorbent; and then

2 eluting the separate subclasses with a stepped gradient of increasing acidity. MATERIALS AND EQUIPMENT

Mouse serum Protein A–Sepharose CL-4B Phosphate-buffered saline (PBS)

0.1 M phosphate buffer, pH 8.0

0.1 M citrate buffers, pH 6.0, 5.5, 4.5, 3.5

1.0 M tris (hydroxymethyl)-aminomethane(Tris)–HCl buffers, pH 8.5, 9.0 Chromatography column or 10-ml disposable syringe Antisera to the mouse IgG subclasses

20 C H A P T E R 1: Isolation and structure of immunoglobulins

METHOD

1 Swell 1.5 g protein A–Sepharose in 10 ml PBS for 1 h at room temperature and then pack it into a small chromatography column. Store and use this column at 4°C.

2 Equilibrate the column with 0.1 M phosphate buffer, pH 8.0.

3 Add 2 ml 0.1 M phosphate buffer, pH 8.0 to 4 ml mouse serum and adjust to pH 8.1 with

1 M Tris–HCl buffer, pH 9.0.

4 Apply the diluted serum to the column and wash through with 30 ml 0.1 M phosphate buffer, pH 8.0 (flow rate 0.4–0.5 ml/min throughout).

5 Elute the IgG 1 with 30 ml 0.1 M citrate buffer, pH 6.0.

6 Wash the column with 25 ml 0.1 M citrate buffer, pH 5.5.

7 To minimize the denaturation of the IgG 2a and IgG 2b antibodies, collect the eluates from steps 7 and 8 into tubes containing 1.0 M Tris–HCl buffer, pH 8.5.

8 Elute the IgG 2a with 30 ml 0.1 M citrate buffer, pH 4.5.

9 Elute the IgG 2b with 25 ml 0.1 M citrate buffer, pH 3.5.

10 Re-equilibrate the column to pH 8.0.

11 Determine the composition of each fraction with specific antisera, preferably using immunoassay.

Figure 1.7 shows the purity of fractions obtained from a protein A fractionation of mouse serum. An enzyme-linked immunosorbent assay has been used to examine the protein fractions dot blotted onto nitrocellulose.

TECHNICAL NOTE IgG 3 usually elutes with the IgG 2a fraction. Immunoaffinity chromatography on subclass-specific antibody affinity columns is required to remove this contamination.

Isolation of human IgG subclasses

Protein A may be used to obtain fractions of human IgG which, although not completely pure, are certainly much enriched for individual subclasses. IgG 3 does not bind to protein A, so if total IgG is filtered through a column of protein A–Sepharose, IgG 1 , IgG 2 and IgG 4 will bind to the adsorbent but IgG 3 will come straight through. IgG 1 and IgG 2 may be differentially eluted from the adsorbent with a pH gradient of increasing acidity. Although IgG 4 is a slight contaminant in the IgG 2 fractions, this problem may be reduced by starting with IgG prepared using DEAE– cellulose ion-exchange chromatography (see Section 1.3); this is relatively deficient in IgG 4 .

MATERIALS AND EQUIPMENT Protein A–Sepharose CL-4B Human IgG, DEAE–cellulose purified or human serum

0.15 M citrate–phosphate buffers, pH 7.0, 5.0, 4.5

0.1 M citric acid, pH 2.2 Antisera to human IgG subclasses Chromatography column or 10-ml disposable syringe UV flow cell

1.4AFFINITY TECHNIQUES FOR IMMUNOGLOBULINS AND OTHER MOLECULES

4 Anti-IgG2a

5 Anti-IgG2b

6 Anti-IgG3

Mouse 3 pH 8.0

pH 8.0 pH 6.0

pH 6.0 Mouse 1 pH 4.5 pH 4.5

pH 3.5 pH 3.5 pH 8.0

IgA pH 6.0

Mouse 2 IgM pH 4.5

IgG1 pH 3.5

IgG2a IgG2b

IgG3

Fig. 1.7 Protein A fractionation of mouse IgG subclasses. Sera from MRL/lpr mice (an inbred mouse strain characterized by lymphoproliferation and high immunoglobulin levels) have been fractionated on protein A–agarose. The material coming straight through the column at pH 8.0 and fractions eluted at pH 6.0, 4.5 and

3.5 have been collected and dot blotted, in triplicate, on to six nitrocellulose membranes. Each membrane has been incubated with a biotinylated specific anticlass or subclass antibody. The blots (1–6) were then incubated with streptavidin labelled with peroxidase. Grid key to each blot shown on right of main figure.

Commercially obtained, purified myeloma IgA, IgG 1 , IgG 2a , IgG 2b , IgG 3 and HPLC-purified IgM were included on the blots as standards. The blots show that it is possible to considerably enrich fractions for single subclasses from whole mouse serum. Some slight cross-reactivity is seen with the antibodies reacting with the myeloma proteins. Here there is the problem of determining whether the myeloma proteins or the antisera (all obtained commercially) are impure, or even possibly both. This experiment cautions the reader against always taking manufacturers’ publicity at face value.

METHOD

1 Swell 1 g protein A–Sepharose with 10 ml citrate–phosphate buffer, pH 7.0.

2 Pack the swollen gel into a small chromatography column and equilibrate with citrate–phosphate buffer, pH 7.0.

3 Load either 5 mg human IgG or 0.5 ml human serum (premixed with 0.5 ml buffer, pH 7.0) onto the column.

4 Wash the column through with pH 7.0 buffer. If purified IgG was used on the column, pure IgG 3 will come out with the washing buffer. Otherwise, it will come out mixed with all the other non-IgG serum proteins.

5 Elute the IgG 2 and IgG 1 with a pH gradient of citrate–phosphate buffer. This is constructed

by using a gradient maker of three equivolume chambers connected in series. The first chamber should contain 6 ml 0.1 M citric acid, pH 2.2, the middle chamber 6 ml of

Continued

22 C H A P T E R 1: Isolation and structure of immunoglobulins 22 C H A P T E R 1: Isolation and structure of immunoglobulins

6 Use a flow rate of 12 ml/h and monitor the eluate with a UV flow cell. Two overlapping peaks will be obtained, the first being enriched for IgG 2 , the second for IgG 1 .

7 Each peak should be concentrated and recycled on the re-equilibrated protein A column to increase resolution.

8 To re-equilibrate the column, wash sequentially with 6 ml 0.1 M citric acid and 30 ml citrate–phosphate buffer, pH 7.0.

9 Check the purity of the IgG subclasses with specific antisera, preferably using immunoassay.

1.4.4 Preparation of human IgA 1 on jacalin–agarose

IgA is a comparatively difficult immunoglobulin to isolate by physicochemical methods. The lectin jacalin, obtained from the seeds of the jackfruit, Artocarpus integrifolia, binds human IgA 1 , but not IgA 2 . Binding is through O-glycosidically linked oligosaccharides containing galactosyl (β-1,3) N-acetylgalactosamine, in the presence or absence of sialic acid. This lectin is available conjugated to agarose for use in affinity chromatography methods.

An immunoglobulin fraction prepared with ammonium sulphate must be applied to the

column, as non-immunoglobulin serum proteins also bind to the lectin. Bound IgA 1 is then eluted with melibiose or galactose.

MATERIALS Jacalin–agarose

Jacalin storage buffer: (4-(2-hydroxyethyl)piperazine-1-ethane sulphonic acid) HEPES 10 m M , pH 7.5, containing 150 m M sodium chloride, 100 m M calcium chloride, 20 m M galactose and 0.08% w/v sodium azide

175 m M tris(hydroxymethyl)-aminomethane (Tris)–HCl buffer, pH 7.5 Immunoglobulin preparation, e.g. 45% saturated ammonium sulphate precipitate of human serum Melibiose 0.1 M or galactose 0.8 M in 0.175 M Tris–HCl buffer Chromatography column or 10-ml disposable syringe

Note: Azide is a dangerous chemicalado not discard down the sink.

METHOD

1 Pour 2 ml of jacalin–agarose gel into a small chromatography column (or 5-ml disposable syringe barrel with the outlet covered by glass or nylon wool).

2 Wash the gel thoroughly with 50 ml of Tris buffer to remove the sugars used to stabilize the lectin during storage.

3 Slowly add 5 ml of human immunoglobulin (10 mg/ml) in Tris–HCl buffer.

4 Wash the column through with 20 ml Tris–HCl buffer (or until the absorbance returns to base line, if you are using a flow-through UV monitor).

5 Elute the IgA with 5 ml 0.1 M melibiose or 0.8 M galactose (if using a UV monitor, elute until the protein peak has been collected).

6 Collect fractions of 1 ml and determine their protein content by spectrophotometry at 280 nm.

Continued on p. 24

1.4AFFINITY TECHNIQUES FOR IMMUNOGLOBULINS AND OTHER MOLECULES

7 Pool the fractions containing protein and examine for IgA 1 content and purity by SDS-PAGE (see Appendix B.2.1) and Western blotting (see Section 4.11) with isotype-specific antisera, or alternatively by immunoelectrophoresis with antisera to IgA and whole human serum.

8 Regenerate the column by washing through with 20 ml storage buffer and store the jacalin–agarose gel at 4°C.

TECHNICAL NOTES • The binding capacity of the jacalin–agarose gel will vary between batches but is typically

4.0 mg monomeric IgA 1 per ml of gel.

• IgA 2 is lost with other immunoglobulins which do not bind to jacalin. Loomes et al. (1991) provide a method for the purification and characterization of IgA 1 and IgA 2 from serum that requires a series of column preparations including gel filtration, DEAE and affinity chromato- graphy on jacalin sepharose.

1.4.5 Preparation of IgM on mannan-binding protein (MBP)

Immobilized mannan-binding protein is commercially available from Pierce (Rockford, Illinois, USA) but can be prepared in-house by immobilizing mannan for isolation of MBP, then coupling the MBP to cyanogen bromide-activated Sepharose 4B (adapted from Nevens et al. 1992).

Preparation of immobilized mannan for isolating MBP

Activation of Sepharose 4B with cyanogen bromide

MATERIALS Sepharose 4B

Cyanogen bromide (this chemical is very toxic and must be handled in a fume cupboard ) Crushed ice

5 M sodium hydroxide

0.1 M sodium bicarbonate solution Yeast mannan

1.0 M ethanolamine, pH 9.0

1.25 M NaCl containing 20 m M CaCl 2 and 10 m M imidazole, pH 7.8

METHOD

1 Wash 100 ml Sepharose 4B in 1.6 l of distilled water, then remove the water by suction until it is dried.

2 Transfer the Sepharose 4B into a beaker and suspend in 100 ml of distilled water.

3 Put beaker containing the Sepharose 4B and water onto a magnetic stirrer and insert a pH meter probe. Next step (4) using cyanogen bromide must be performed in a fume cupboard (owing to possible release of cyanide gas). It is essential that the slurry is maintained at about 20°C and an alkaline pH.

Continued

24 C H A P T E R 1: Isolation and structure of immunoglobulins

4 Slowly add 20 g of solid cyanogen bromide over a 20-min time period, maintaining the temperature at around 20°C by adding crushed ice to the stirring slurry.

5 Keep the pH of the slurry between 10.5 and 11.0 by adding a concentrated solution of sodium hydroxide (dropwise).

6 Wash the cyanogen bromide-activated Sepharose using a glass Buchner funnel with around

2 l of ice-cold 0.1 M sodium bicarbonate, then dry by suction.

7 Dissolve 1.78 g of yeast mannan in 100 ml 0.1 M sodium bicarbonate solution, add to the activated Sepharose beads and stir overnight at room temperature.

8 Wash the activated Sepharose beads with 1 litre of distilled water, then suspend in 160 ml

1.0 M ethanolamine, pH 9.0, at room temperature for 60 min.

9 Wash with 1 litre of distilled water and pack into a glass columnathe Sepharose is now ready for isolating the MBP.

10 Equilibrate the column with 10 column volumes of 1.25 M NaCl containing 20 m M CaCl 2 and 10 m M imidazole, pH 7.8.

Coupling of cyanogen bromide-activated Sepharose 4B and MBP

MATERIALS Sepharose 4B Solid cyanogen bromide (this chemical is very toxic and must be handled in a fume cupboard )

0.1 M sodium bicarbonate buffer, pH 8.5

1.0 M NaCl

10 m M tris(hydroxymethyl)-aminomethane (Tris) containing 1.25 M NaCl and 2 m M ethylene diamine tetra-acetic acid (EDTA), pH 7.4

Mannan-binding protein (MBP) solution Coomassie protein assay reagent

1 Activate the Sepharose 4B with cyanogen bromide by the same procedure as in steps 1–5 (above).

2 Wash the activated Sepharose with 200 ml of ice-cold distilled water followed by 100 ml

0.1 M sodium bicarbonate buffer, pH 8.5, then dry by suction.

3 Mix the MBP solution and activated Sepharose and stir overnight at 4°C.

4 Filter the gel suspension and wash with 200 ml 1.0 M NaCl, followed by 200 ml water.

5 Block the excess reactive groups on the MBP coupled to cyanogen bromide-activated Sepharose 4B column with 10 ml 1.0 M ethanolamine, pH 9.0, and stir at room temperature for 60 min.

6 Wash with 200 ml of distilled water followed by 200 ml 10 m M Tris containing 1.25 M NaCl and 2 m M EDTA, pH 7.4.

Note: Test the coupling of MBP with the activated Sepharose by mixing 200 µl of immobilized MBP suspension and 2 ml of Coomassie protein assay reagent into a test tube. If the gel remains blue and the supernatant is colourless it indicates there is no unbound MBP present.

1.4AFFINITY TECHNIQUES FOR IMMUNOGLOBULINS AND OTHER MOLECULES

Isolation of IgM using MBP coupled to cyanogen bromide-activated Sepharose 4B

MATERIALS Sample: serum or monoclonal antibody culture supernatant MBP coupled to cyanogen bromide-activated Sepharose 4B Hypodermic syringe and glass wool (Caution: Wear gloves when handling glass wool)

10 m M tris(hydroxymethyl)-aminomethane (Tris) containing 1.25 M NaCl with 0.02% w/v sodium azide, pH 7.4

10 m M Tris containing 1.25 M NaCl, 20 m M CaCl 2 with 0.02% w/v sodium azide, pH 7.4

10 m M Tris containing 1.25 M NaCl, 2 m M EDTA with 0.02% w/v sodium azide, pH 7.4 Note: Azide is a dangerous chemicalado not discard down the sink.

METHOD

Steps 1–8 are performed at 4°C; therefore all buffers must be ice cold.

1 Dialyse the monoclonal antibody supernatant or sample containing IgM overnight against two changes of 1000 ml 10 m M Tris containing 1.25 M NaCl with 0.02% w/v sodium azide, pH 7.4. (This step is to remove any phosphate ions that could form a precipitate with the calcium ions in subsequent steps.)

2 Dilute the sample with an equal volume of 10 m M Tris containing 1.25 M NaCl, 20 m M

CaCl 2 with 0.02% w/v sodium azide, pH 7.4.

3 Load the MBP coupled to cyanogen bromide-activated Sepharose 4B into a 100-ml hypodermic syringe plugged with glass wool.

4 Wash the column with 5 column volumes of 10 m M Tris containing 1.25 M NaCl, 20 m M

CaCl 2 with 0.02% w/v sodium azide, pH 7.4.

5 Apply the diluted sample, allow to flow completely into the column, collect the eluate and re-apply to the column.

6 Repeat step 5 around five times for the best yield of IgM antibodies.

7 Allow the sample to incubate on the column for about 30 min by clamping the eluate tubing, and keep the top of the gel from drying by adding 200 µl of 10 m M Tris containing

1.25 M NaCl, 20 m M CaCl 2 with 0.02% w/v sodium azide, pH 7.4.

8 After the incubation, wash the column with 10 column volumes of 10 m M Tris containing

1.25 M NaCl, 20 m M CaCl 2 with 0.02% w/v sodium azide, pH 7.4, monitoring the fractions spectrophotometrically at 280 nm. Steps 9 onwards are performed at room temperature; therefore buffers must not be ice cold.

9 Take the column into room temperature and allow to stand for 60 min (do not allow the column to dry out).

10 Wash with 3 column volumes of 10 m M Tris containing 1.25 M NaCl, 20 m M CaCl 2 with 0.02% w/v sodium azide, pH 7.4.

11 Elute the IgM with 10 m M Tris containing 1.25 M NaCl, 2 m M EDTA with 0.02% w/v sodium

azide, pH 7.4, monitoring the fractions spectrophotometrically at 280 nm. An absorbance of 1.18 at 280 nm is equivalent to an IgM concentration of 1 mg/ml.

12 Store the IgM-containing fractions at –20°C.

13 Wash the column with 3 column volumes of distilled water followed by 3 column volumes

of 10 m M Tris containing 1.25 M NaCl, 20 m M CaCl 2 with 0.02% w/v sodium azide, pH 7.4, and store at 4°C.

26 C H A P T E R 1: Isolation and structure of immunoglobulins

1.4.6 Purification of IgD on Griffonia simplicifolia I lectin

MATERIALS Monoclonal antibody culture supernatant derived from an IgD-secreting hybridoma 5-ml hypodermic syringe Glass wool (Caution: Wear gloves when handling glass wool) GS-I lectin–Sepharose (purchase coupled or linked as above) Phosphate-buffered saline (PBS) containing 1 m M CaCl 2

PBS containing 1 m M CaCl 2 and 0.1 MD -galactose

UV spectrophotometer Dialysis membrane tubing

METHOD

1 Centrifuge monoclonal antibody culture supernatant at 10 000 g for 30 min at 4°C or room temperature.

2 Save the supernatant and discard the cell debris.

3 Dilute the supernatant with 10 volumes of PBS containing 1 m M CaCl 2 and centrifuge at

15 000 g for 30 min at 4°C and save the supernatant for step 7.

4 Prepare a mini-column such as a 5-ml hypodermic syringe plugged with glass wool (use gloves when handling glass wool).

5 Pour in 3 ml of GS-I lectin–Sepharose and maintain at a temperature of 4°C.

6 Wash GS-I lectin–Sepharose with the following ice-cold buffers:

12 column volumes PBS containing 1 m M CaCl 2 ;

12 column volumes PBS containing 1 m M CaCl 2 and 0.1 MD -galactose;

12 column volumes PBS containing 1 m M CaCl 2 .

7 Load the supernatant and allow to flow into the GS-I lectin–Sepharose.

8 Wash the column with sufficient ice-cold PBS containing 1 m M CaCl 2 , monitoring the fractions spectrophotometrically at 280 nm, until a baseline is reached.

9 Elute the IgD antibodies with ice-cold PBS containing 1 m M CaCl 2 and 0.1 MD -galactose. Around 3–5 column volumes should be enough to elute the IgD, but it is advised that the eluate is monitored spectrophotometrically at 280 nm.

10 The column may be regenerated with 2 column volumes PBS containing 1 m M CaCl 2 and

0.1 MD -galactose, then 10 column volumes PBS containing 1 m M CaCl 2 . To preserve the column add 0.02% w/v sodium azide to the final wash and store at 4°C.

11 Dialyse the IgD sample against 500 ml ice-cold PBS, pH 7.3, at 4°C with five changes of buffer.

12 Estimate the IgD concentration of the dialysed sample by measuring at 280 nm with a UV spectrophotometer.