Immunoglobulin (Ig) is a kind of globulin with antibody activity and specific binding reaction with corresponding antigen. It is a kind of protein produced by lymphocytes in vertebrate immune response to antigenic stimulation and generally exists in mammalian blood, tissue fluid, lymph and in vitro secretion. With the development and demand of immunology, the purification of immunoglobulins and their components has become an essential means. Below, we will introduce several commonly used methods for purifying immunoglobulins.

1、 Salting out method

Salt precipitation, as an important precipitation method, is often used in the laboratory to separate and purify proteins, especially for the preliminary separation of raw materials. The solubility of proteins in water depends on the degree of hydration of protein molecules. Adding inorganic salts to change the degree of hydration of proteins can cause protein precipitation. Due to the different number and distribution of polar groups on the surface of various proteins, as the amount of inorganic salts added increases, the precipitation order of different proteins varies. Therefore, by controlling the amount of inorganic salts added, protein separation and purification can be achieved through graded precipitation.

2、 Ion exchange chromatography

Ion exchange refers to the reversible exchange reaction between ions in the liquid phase and dissociative groups on the stationary phase. Using this reaction, the mixture to be separated is first dissociated in a solution of a certain pH, and then flows through the stationary phase to exchange ions with the dissociatable groups on the stationary phase, adsorbing them onto the stationary phase. Any components that cannot be exchanged or adsorbed are then discharged from the chromatographic column. Based on the differences in the dissociation degrees of each group adsorbed on the fixed phase through exchange, solutions with different pH values or salt concentrations are used as mobile phases. Each component is then exchanged and eluted through a chromatography column, and the mixture components are separated to achieve separation. This is called ion exchange chromatography.

3、 Gel filtration method

Gel particles are a network structure. When mixtures with different molecular sizes pass through the gel column, the larger molecules come down first, and the smaller molecules are embedded in the network structure of gel particles and then come down, so as to separate substances with different molecular sizes, which is the role of molecular sieves. Common gel: dextran gel, polyacrylamide gel, agarose gel, and G150 is commonly used to separate Ig.

4、 Affinity chromatography

By utilizing the specificity and reversibility of antigen antibody binding, the purified antigen is first cross-linked onto the carrier (agarose beads) to create an affinity chromatography column. When Ab (Ig) passes through, it binds specifically to the antigen on the column, and impurities in Ab flow out. Then, Ab is released by changing the buffer pH and ionic strength.

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