Question

How we can use chromatography in immune system and separate antibody or b cell , ?

Answer 1

The working principle behind Affinity chromatography relies on the reversible interaction between the immobilized specific ligand used in the chromatographic matrix and the protein. The sample of interest needs to be applied in favorable binding conditions to the ligand. Binding occurs due to electrostatic interactions, van der Waals’ forces, hydrophobic interactions and/or hydrogen bonding. After binding step, the unbound material is washed. After washing unbound material, the bound protein is recovered by using a buffer that causes desorption.

The basis for purification of antibodies (IgG) is the high affinity of protein A and protein G towards the Fc region of monoclonal and polyclonal IgG antibodies. Protein A is the cell wall protein from Staphylococcus aureus and Protein G is derived from Streptococcin groups C and G. Using these protein A or G sepharose columns immunoglobulin are separated and purified. The chromatographic column is buffer equilibrated and the antibody is added to a column with favorable binding conditions. After binding, the specific wash buffer is applied which will wash out the impurities and unbound protein. Using specific elution buffer the bound protein is desorbed from the matrix and eluted along with buffer.