| | Re: Antibody Purification: Ammonium Sulfate precipitation
Ammonium sulfate precipitation is a method used to purify antibodies by altering their solubility from ascites ,serum or hybridoma supernatant. Immunoglobulins precipitate out of either sodium or ammonium sulphate especially when non refined fractions of immunoglobulin are required. Further refinement can be done by ion exchange or other forms of chromatographic technique. Ammonium sulphate is useful for polyclonal and monoclonal IgG isolation. A solution with 35% saturated will produce a pure IgG precipitation, but not all IgG will be contaminated with other proteins(eg., albumin). Precipitation at 45% saturated produces an ideal starting material for other purification techniques. Sodium sulphate is used for rabbit or human polyclonal IgG. Immunoglobulins precipitate at 40-50% ammonium sulphate saturation depending on species and sub class. The desired saturation is brought about either by addition of solid ammonium sulphate or by addition of a saturated solution. Although the use of solid salt reduces the final volume, this method has a number of disadvantages. Prolonged stirring, required to solubilize the salt can lead to denaturation of protein in the solution at the surface/air interface. Localized high concentration of ammonium sulphate salt may cause unwanted proteins to precipitate. Since ammonium sulphate is slightly acidic in solution, pH of the protein solution requires constant monitoring and adjustment if solid salt is added and is advisable to add a buffered solution of saturated ammonium sulphate. A saturated ammonium sulphate is considered to be 100% and the ascites or serums are mixed with an equal volume of saturated ammonium sulphate to give a 50% solution. For to calculate the volume of ammonium sulphate required for precipitation using the following equation : X = (Y×V)/1-Y; where X is the volume of saturated ammonium sulphate, Y is the desired final concentration of ammonium sulphate (expressed as a decimal fraction, where 1 =100%), and V is an initial volume of sample undergoing the salt cut. Precipitated immunoglobulins can be solubilized in a minimal volume of buffer. Proteins of very high molecular weight usually precipitate below 25% ammonium sulphate saturation.
Centrifuge fluid antibody containing at 2500 rpm for 15-30 min to clarify the starting material. Transfer the supernatant fluid to a clean beaker with a stir bar and place on a magnetic stirrer. Add a volume of (4.1 M) acqueous saturated Ammonium Sulfate (sAS) solution equal to the volume of the starting material fluid for to obtain an 50 % sAS. Slowly add the ammonium sulfate while it is blended into the material fluid. Allow precipitates to redissolve before adding more AS. When you have added nearly all of the AS, irreversible precipitation will occur. Continue to add the AS until all of it has been added to the preparation. Allow the solution to continue to mix for 6-24 h at 4°C. When precipitation is complete, centrifuge the solution at 3000 g for 30 min. Decant the supernatant and save a sample of the discard for later testing. Slowly add 150 mM PBS to the pellet and gently stir with a pipette. Add sufficient PBS equal to 25-50% of the original volume of the starting material. Dialyze the dissolved antibody preparation against PBS in a 20:1 (v/v) ratio of PBS to antibody preparation. Because of the high salt concentration of the AS, at least three changes of buffer for a minimum of 2 h each are recommended. Protein concentration determination, zonal electrophoresis, densitometry scan, and specificity testing of the final preparation can then be performed for to know level of purity and specific activity of the antibody. The antibody preparation should be divided into aliquots and stored at –70 °C.