What is antibody purification? Antibodies which are made by various methods are always mixed with a variety of hybrid proteins. It needs to do antibody purification when get antibodies with uniform composition or with certain purpose. From the perspective of component analysis, antibody molecule is a kind of protein molecule. Thus it has an isoelectric point , solubility, charge and hydrophobicity properties and it can be used inelectrophoresis , salting out precipitation and other Tomography including separation and purification. Antibodies purification are usually used to test antigens directly. A marker of the antibody can be bind and detect the antigen. The following are the tips on purification of antibodies of different resources
Tips on antibodies of different resources. Clarifying serum, ascites and tissue culture supernatant, centrifugation and filtration are basic equipment technologies. These technologies can remove lipids and small particulate matter which will block column. In addition, buffer exchange and desalting are necessary steps in dealing with antibody samples. As the further pretreatment step, ammonium sulfate precipitation is usually used in concentrating immunoglobulin of ascites.
How to deal with antisera? Some monoclonal antibodies are not purified and these antibodies are called serum or antisera. Antisera is the immune host’s blood whose clotting proteins and red blood cells has been removed. All kinds of antibodies and other serum proteins are included in antisera. They contain both antibodies which can recognize target antigens and antibodies that fail to recognize, so non-specific binding happens sometimes in immunoassays. Therefore, antisera needs to remove serum protein by purification. Then specificity and targeted combined ratio of immunoglobulin can be increased.
For tissue culture serum, polyclonal antibodies can be produced by developing hybridoma and getting culture supernatant of hybridoma.
Tips on ascites. Polyclonal antibodies can be produced by developing hybridoma in abdominal cavity of mouse. After injecting hybridoma in mouse, it will multiply and produce ascites in hybridoma in abdominal cavity of mouse. This kind of ascites contains antibodies with high concentration. Antibody production of this technology are far more than that of hybridoma culture.