Immunoglobulins precipitation with ammonium sulfate

A classic preliminary step in antibody purification is precipitation with ammonium sulfate. With the use of this reagent, most immunoglobulins are precipitated at 35-40% saturation. Concentrations greater than this level seldom increase the yield of immunoglobulins but, instead, result in further increase of antibody contamination by other proteins. Following immunoglobulin precipitation, ammonium sulfate is eliminated commonly with dialysis. 

Serum, ascites, or hybridoma culture supernatant can, after filtration, be applied to the column directly. The crude antibody solution should then be diluted with 1/10 volume of IMTris-HCl, pH 8.0, and chromatography should be performed with 100 xaM Tris-HCl, pH 8.0. It is, however, advisable first to precipitate the immunoglobulins with ammonium sulfate see Chapter 2). This not only reduces the sample volume, but more importantly removes lipids, particularly from serum and ascitic fluid, extending the life of the column. 

Antibody sera obtained from immunized animals can in some cases be directly used for further experiments. Further purification and enrichmenL however, are often necessary to increase the specificity and overall affinity of fhe purified antibodies. Simple enrichment of fhe antibodies can be achieved by precipitation wifh ammonium sulfate [55] or by chromatography wifh protein A [56] or protein G, bacterial ceU waU proteins fhat specificaUy bind to fhe Fc portion of immunoglobulins. A more specific enrichment is achieved by affinity chromatography with an antigen column. 

Since ammonium sulfate fractionation will also cause precipitation of other proteins, antibody concentrations obtained from absorbance measurements at 280 nm are only estimates. Alternatively, a sample of the dialyzed solution can be resolved on a SDS-polyacrylamide gel alongside a series of known concentrations of IgG. Staining the gel with Coomassie blue can then be used to estimate the amount of immunoglobulin obtained and can also give an estimate of purity. 

Each sheep initially received 5 mg of protein in multiple injections at intervals of 5 weeks until a serum with a satisfactory precipitating potency was obtained. Generally, 2 booster injections were sufficient and blood was withdrawn 15 days after the last booster. The serum was purified by precipitation of the immunoglobulins with half a volume of a saturated ammonium sulfate solution and then dialysed for 4 days against a 0.9 % sodium chloride solution which was replaced every day. It was stored at -30 °C. 

Purification of Monoclonal Antibody. Immunoglobulins were precipitated from the pooled ascites by addition of an equal volume of saturated ammonium sulfate [50% (NH4)2S04]. The precipitate was collected by centrifugation (20 min 10,240 X g), dissolved in 0.01 M sodium phosphate (pH 6.8), and reprecipitated. After the second ammonium sulfate precipitation, the pellet was dissolved in a minimum volume of 0.01 M sodium phosphate (pH 6.8) and centrifuged for 10 min at 10,600 X g). The resulting supemate was applied to a P6G, gel filtration polyacrylamid, column (Bio-gel Biorad, Rockville Center, NY 1.5 X 40 cm). Fractions containing protein were pooled and applied to a hydroxyapatite column that had been equilibrated with 0.01 M sodium phosphate (pH 6.8). Proteins were eluted with a linear gradient of 0.01 to 0.3 M sodium phosphate. 

Ammonium sulfate precipitation (see Note 2) or fractional precipitation is based on the solubility of the particular immunoglobulin. Solubility is illustrated by salt precipitation. It is a convenient first step that allows the reduction of the large volume of the starting material and the precipitation of the desired protein. Contaminant proteins can be trapped or co-precipitated with the target protein, so other methods must follow the ammonium... 

The immunoglobulins and other proteins present in the rabbit serum sample are precipitated with saturated ammonium sulfate at a final concentration of 33% (see Chapter 3, Subheading 3.3.1.1, and Note 2). 

A number of other precipitating agents have been used in the purification of proteins. In some instances, the use of sodium sulfate can result in a purer antibody preparation, but generally it does not offer advantages over ammonium sulphate. Caprylic (octanoic) acid, however, offers a different approach and also has a long history of use (5). Conditions can be created where this short chain fatty acid will effectively precipitate the majority of serum proteins with the exception of the immunoglobulins. 

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