The binding of IgG to protein A has been discussed in Sections 5.3.5 and 9.3.2. Rabbit antisera have the distinct advantage that all their IgG binds to protein A at pH 7.4, and all elutes at pH 4.0. This fact may be used to purify rabbit IgG, and to separate Fab and F(ab')2 fragments of rabbit IgG from Fc or undigested IgG (Goding, 1976, 1978). Affinity chromatography on protein A is the method of choice for rabbit antisera.
Sheep and goats have two IgG subclasses. The major subclass (IgGl) binds very weakly to protein A at alkaline pH, and virtually not at all at neutral or acid pH (Delacroix and Vaerman, 1979; Duhamel et al., 1980). The minor IgG2 subclass (10-20% of total IgG) binds firmly to protein A. Sheep, goat and bovine IgG will bind much more strongly to protein A in a buffer that combines high pH with high salt (e.g. 0.1 M Tris-HCl pH 8.5, with 1 M NaCl).
The subclass specificity of protein A for goat and sheep IgG2 has made this reagent somewhat unattractive for purification of IgG from these species. However, streptococcal protein G binds both IgGl and IgG2 from goats and sheep, and gives excellent recoveries from these species (Boyle et al., 1985; and Sections 5.3.5 and 9.3.3). Binding may be at pH 7.4 at physiological salt concentration, and elution at pH 2.7 in 0.1 M glycine-hydrochloride. The column should then be 'stripped' by washing with 0.1 M glycine-hydrochloride pH 2.0 to make sure that no trace of IgG remains, as it could contaminate subsequent purifications. Affinity chromatography on protein G may become the method of choice for purification of sheep and goat IgG.
15.4.2 Purification of Sheep and Goat IgG by Ion Exchange Chromatography
The purification of IgG from sheep and goat serum may also be performed by ion exchange chromatography (see Section 9.2.3). After precipitation in 40% saturated ammonium sulfate, the IgG is resuspended in PBS and dialysed overnight against 10 mM Tris-HCl, pH 8.0. It is then passed over a column of DEAE-Sephacel (or equivalent) equilibrated with the same buffer. Any dropthrough material will be pure IgG (those IgG molecules with the most basic isoelectric points). The remaining IgG (the majority) may be eluted with a linear salt gradient (0-250 mM NaCl in 10 mM Tris-HCl pH 8.0). The first peak to emerge will be IgG2, and it will be immediately followed by a peak containing IgGl. At this point, the IgG should be about 90% pure. If necessary, further purification may be achieved by gel filtration (see Section 9.2.4).
15.4.3 Production of Fab and F(ab')2 Fragments of IgG from Rabbits, Goats and Sheep
The production of Fab fragments of IgG from rabbits, goats and sheep is quite straightforward. The basic principles and procedures described in Section 9.5 may be applied with little modification (see also Mage, 1980). The IgG may be purified by affinity chromatography on protein G (see Section 15.4.1), or by careful ammonium sulfate precipitation (see Section 9.2.1), and dialysed against 0.1 M Tris-HCl, 1 mM EDTA, pH 8.0. Further purification is usually unnecessary, but may be performed by ion exchange chromatography or gel filtration (see Chapter 9).
Digestion is carried out using papain in 0.1 M Tris-HCl, 1 mM EDTA pH 8.0 (or PBS plus 1 mM EDTA) plus a small amount of reducing agent (25 mM mercaptoethanol or 1-2 mM dithiothreitol). An enzyme:substrate ratio of
1:100 is customary, but 1:1000 may be sufficient. After incubation at 37°C for 1 h, the reaction is terminated by addition of an excess of iodoacetamide. The Fab fragment may be separated from the Fc by ion exchange chromatography (see Sections 9.2.3 and 9.5.1; Mage, 1980).
The production of F(ab')2 fragments must be individualized depending on the species. Pepsin must be made up from the powder in 1 mM HC1 or pH 4 acetate buffer, as it is ireversibly denatured at neutral or alkaline pH. Rabbit IgG is easily digested in pH 4-4.5 acetate buffer at 37°C overnight, using an enzyme:substrate ratio of 1:100. Sheep and goat IgGs are rather resistant to pepsin, but adequate digestion may be achieved at pH 4.0-4.5 using an enzyme:substrate ratio of 1:50 at 37°C for 48 h. Alternatively, cleavage of sheep IgG with trypsin may be used (Davies et al., 1978).
Peptic digestion results in extensive degradation of the Fc, and simple dialysis against PBS at pH 7.4 will irreversibly terminate the reaction by denaturing the pepsin and remove most of the Fc products. A fragment of the Fc (pFc') consisting of a noncovalent dimer of the CH3 domains (total Mr 27 000) may be removed if necessary by gel filtration on Sephacryl S-200 or S-300.
Whichever method is chosen, it is strongly advisable to check the nature and completeness of digestion by SDS-PAGE.
15.4.4 Assessment of the Specificity of Polyclonal Antisera
The concentration of total IgG in serum ranges from 5-20 mg/ml. A very strong polyclonal antiserum might contain 1-3 mg/ml of specific antibody, or occasionally even more. More commonly, the antibody concentration may be 50-200 fig/ml, and an antiserum may be adequate for some purposes with even lower levels.
One cannot assume that because the antigen was 'pure', the antiserum will be specific. There are many reasons why this need not be the case (see Chapter 7). The principles of antigen analysis outlined in Chapter 10 may be applied with equal effectiveness to the analysis of antibodies.
If an antiserum is to be used in cell-binding assays, immunofluorescence or radioimmunoassay, the results of a crude test of specificity such as gel diffusion (Ouchterlony analysis) may be very misleading.
The specificity of an antiserum must be tested in a system that is at least as sensitive as the one in which it is to be used.
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