The basic screening and identification basis for monoclonal antibodies
July 29 07:01:45, 2024
The first two screenings are at the cell level. In order to select the appropriate cell line, these two steps are very critical. If there is a problem, they will all be done in vain. The first screening was to select hybridoma cells. After the fusion of effector B cells and myeloma cells, it contains a variety of cells: hybridoma cells, myeloma cells fused with myeloma cells, effector B cells fused with effector B cells, unfused myeloma cells And effector B cells, and even multinucleated cells. Since the hybridoma cells inherit the genetic material of the parent cells, it not only has the ability to secrete specific antibodies to effector B lymphocytes, but also has the ability to proliferate myeloma cells in large amounts under in vitro culture conditions. The selection medium is usually used for selection, such as HAT medium (H is hypoxanthine, A is aminopurine, and T is thymidine). In this medium, only hybridoma cells can continue to multiply. The second screening is to select hybridoma cells that can secrete the required antibodies. After the fused cells are cultured in a selective medium, the viable cells are hybridoma cells. However, not all of these hybridoma cells are capable of secreting the required antibodies, and are usually selected by the "limited dilution method". The cells need to be cultured on a multi-well petri dish and cultured with only one hybridoma cell in each well, and then a cell population capable of producing specific antibodies can be selected. Since this cell population is cloned from a single hybridoma cell, the antibodies produced must be monoclonal antibodies with a single chemical nature and strong specificity. Then comes the identification of antibodies, which are basically common immunological antibody identification methods. 1. ELISA is used to detect soluble antigens (proteins), cells, viruses and other McAbs. 2. RIA is used for the detection of soluble antigen and McAb. 3. FACS (Fluorescence Activated Cell Sorter) is used to detect McAb detection of cell surface antigens. 4. IFA is used to detect McAb in cells and viruses. Source: Biotechnology Forum