Purification of Immunoglobulin by Precipitation

BACKGROUND

Glycoproteins termed immunoglobulin make up antibodies. A protein called immunoglobulin that binds to antigens is found on the B-cell membrane and is released into the serum by plasma cells. B-lymphocytes create immunoglobulins in response to external antigens.

Using the protease enzyme, Rod Porter and Gerry Edelman described the immunoglobulin structure (Papain and Pepsin). Two identical light chains form the immunoglobulin molecule, which is joined by a disulphide link. By weakening the S-S link and acidifying the solution, these chains can be broken. The Ig produce two identical pieces termed Fab fragments after being broken down by papain because they contain an Fc fragment.

Antibodies, which are protein moieties, precipitate at particular salt concentrations in solution. When there is a certain amount of salt in solution, divalent anions will precipitate. Due to effective salt denaturation, divalent anions precipitate out proteins more effectively than monovalent ions.

A significant number of divalent ions are present in ammonium sulphate [(NH4)2SO4]. The various components of the plasma protein solution precipitate in order as the ionic strength rises. IgG antibodies, both polyclonal and monoclonal, are made with caprylic acid. Precipitation of non-IgG proteins results in purification. Caprylic acid final concentrations for various species vary: human and horse serums need 6.1%; goat serum needs 8.0%; and rabbit serum needs 8.2%. For both polyclonal and monoclonal antibodies, polyethylene is helpful. PEG (Polyethylene Glycol) has the drawback of being difficult to remove from protein solutions.

Use the following equation to determine how much ammonium sulphate is needed for precipitation.

X = (Y×V) / 1-Y

Where V is the beginning volume of the sample undergoing the salt cut, X is the volume of saturated ammonium sulphate, Y is the desired ammonium sulphate concentration (given as a decimal fraction, where 1 = 100%).⁴

IgG, IgM, IgD, IgE, and IgA can all be found in a raw Ig fraction. The following are the fractions that precipitated at various ammonium sulphate concentrations.

• 25% (NH₄)₂SO₄ precipitate fibrinogen;
• 33% (NH₄)₂SO₄ precipitate IgG;
• 40% (NH₄)₂SO₄ precipitate IgG, IgA, IgM;
• 50% (NH₄)₂SO₄ precipitate α and β-globulins;
• 70% (NH₄)₂SO₄ precipitate albumins.

Most species’ IgG precipitates at a concentration of ammonium sulphate between 40% and 50%, which is why 50% is typically utilized. Ammonium sulphate should only be used as the initial stage in a multistep antibody purification technique since it does not provide a pure antibody fraction because other proteins can become “stuck” within the aggregated protein.²

These Ig fractions can be confirmed by various techniques (e.g.- immunodiffusion and electrophoresis).

REQUIREMENTS

Solutions to be used.

Solution	Preparation
Ammonium Sulphate Solution (NH4)2SO4	ammonium sulphate was dissolved in distilled water and kept at 4oc for a night. Extra salt settles down as crystals. This solution was used as saturated solution
Phosphate buffer Saline (PBS)	17.4 g of K2HPO4 and 13.6g of KH2PO­4 were added to 100 ml of distilled water and pH was adjusted to 7.2 and diluted to 10mM, to this 0.86% NaCl was added.

1. Serum Sample
2. Centrifuge
3. Centrifuge tube
4. Dialysed membrane bag and clamp
5. Pasteur pipette

PROCEDURE

1. In 3 ml of serum sample, 1 ml of PBS was added. Thus, 4 ml of mixture was obtained.²
2. In above mixture 1 ml of 100% (NH₄)₂SO₄ was added
3. Then this mixture was kept at 4^oc for 3 hr. and centrifugation was done at 10,000 rpm for 10 mins.¹
4. Pellet obtained was dissolved in 1 ml PBS, this solution was considered as 33% fraction of protein.²
5. Supernatant was collected and to it again (NH₄)₂SO₄ solution was added, so that the final concentration becomes 50%.
6. It was again kept at 4^oc for 3 hr. followed by a 10-minute centrifugation at 10,000 rpm.¹
7. The pellet obtained was dissolved in 1ml PBS and considered as 50% fraction of protein.
8. The 30% and 50% fractions were dialysed against PBS overnight with 4 to 5 changes in buffer to remove excess of salt.²

 

CONCLUSION

The immunoglobulins precipitated with ammonium sulphate is purified by Dialysis using PBS buffer and purified antibodies is stored at 4^oc.

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REFERENCES

1. Mark Page, Robin Thorpe and John M. Walker, “Purification of IgG by precipitation with sodium sulphate or ammonium sulphate”, The Protein Protocols Handbook, January 1996.
2. Jorden B. Fishman and Eric A. Berg, “Ammonium sulphate Fractionation of Antibodies”, Cold Spring Harbor Protocols,2018.
3. Asmaa Shawki, Nawal Abd El-Baky, Mohammed Ahmed, Mustafa H. Linjawi, Abdullah A. Aljaddawi and Elrashdy M. Redwan, “Simple Protocol for immunoglobulin G purification from camel ‘Camelus dromedarius’ Serum”, De Gruyter, May 4,2017.
4. Purification of IgG Antibodies with Ammonium Sulphate, vlab.amrita.edu,2011.

FAQs

1. We can use any other salts instead of ammonium sulphate?

 Ans:    Yes, ammonium sulphate is replaced by sodium sulphate.

2. There is any other method for precipitation instead of salt precipitation?

 Ans:     Yes, Caprylic acid can be used for precipitation instead of salt precipitation.