Immunoglobulin (Ig), a type of globulin with antibody activity, can specifically bind to corresponding antigens. It is produced by lymphocytes in response to antigenic stimulation in vertebrates and is widely present in the blood, tissue fluid, lymph fluid, and external secretions of mammals. With the development and demands of immunology, the purification of immunoglobulin and the refinement of its components have become indispensable methods. The following introduces several commonly used methods for purifying immunoglobulin.
I. Salting Out Method
Salting out, as an important precipitation method, is often used in laboratories to separate and purify proteins, especially for the initial separation of raw materials. The solubility of proteins in water depends on the degree of hydration of protein molecules. By adding inorganic salts to change the degree of hydration of proteins, protein precipitation can be caused. Due to the different numbers and distributions of polar groups on the surfaces of various proteins, as the amount of inorganic salts added increases, different proteins have different precipitation sequences. Therefore, by controlling the amount of inorganic salts added, proteins can be separated and purified in a stepwise precipitation manner.
II. Ion Exchange Chromatography Method
Ion exchange refers to the reversible exchange reaction between ions in the liquid phase and dissociable groups on the stationary phase. By utilizing this reaction, the mixture to be separated is first dissociated in a solution of a certain pH, and then flows through the stationary phase, where it undergoes ion exchange with the dissociable groups on the stationary phase and gets adsorbed. Components that cannot undergo exchange adsorption flow out of the chromatography column. Then, based on the differences in the dissociation degrees of the groups adsorbed on the stationary phase, solutions of different pH values or different salt concentrations are used as the mobile phase to flow through the chromatography column to exchange and elute the components respectively. In this way, the components of the mixture are separated, achieving the purpose of separation. This is known as ion exchange chromatography.
III. Gel Filtration Method
Gel particles have a reticular structure. When a mixture of molecules of different sizes passes through a gel column, the larger molecules come down first, while the smaller ones get embedded in the reticular structure of the gel particles and come down later, thus separating substances of different molecular sizes. This is the function of a molecular sieve. Commonly used gels include dextran gel, polyacrylamide gel, and agarose gel. G150 is often used for the separation of Ig.
IV. Affinity Chromatography Method
Taking advantage of the specificity and reversibility of antigen-antibody binding, purified antigen is first cross-linked to a carrier (agarose beads) to make an affinity chromatography column. When Ab (Ig) passes through, it specifically binds to the antigen on the column, while impurities in Ab flow out. Then, by changing the pH and ionic strength of the buffer, Ab is eluted.
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