Biochemical Techniques Biochemistry 22 Hybridoma Technology : Part A
Description of Module
Subject Name Biochemistry
Paper Name 12 Biochemical Techniques
Module Name/Title 22 Hybridoma Technology: Part A
1. Objectives
1.1 To understand concept behind production of monoclonal antibodies
1.2 To understand various steps in production of hybridomas
1.4 To understand method for preparation of hybridomas Part A
2.0 Introduction- Antibodies are produced in host in response to administration of molecule(s) which are foreign to blood circulation. These have typical Y shape structure and are proteins which can recognize administered molecule. Administered molecule is referred as immunogen. Besides immunogen, the synthesized Y shape protein molecule can also recognize related molecules which are referred as antigen. Proteins having Y shape structure are also normally present in blood and these are called immunoglobulins. If target of immunoglobulins is known, then these are referred as antibodies. Each Y shape antibody molecule is comprised of two identical light chains and two identical heavy chains. These chains are held together with the help of disulphide binds. Each light chain is about 220 amino acid long. Each chain is also comprised of variable region and constant region. These regions are about 110 amino acid long. Variable region is towards N-terminal end whereas constant region is towards C-terminal end. Heavy chain is about 440 amino acid long of which 110 amino acid towards N-terminal end is called variable region. Remaining
Biochemical Techniques Biochemistry 22 Hybridoma Technology : Part A
330 amino acids forms three constant regions of 110 amino acid each. There are two different light chains which are called kaapa (ҡ) and lamda (λ). These chains differ from each other in amino acid sequence in constant region. There are five different heavy chains and accordingly antibodies are classified into five different classes. N-terminal regions of light and heavy chains constitute antigen binding site. There are hypervariable regions within variable regions and amino acids in regions are involved in binding to antigen. For different antigen, different amino acid in hypervariable regions are required for providing specificity in binding. Antibodies are synthesized by differentiated B-lymphocytes (plasma cells). Each cell makes identical antibodies. In other words, antibodies produced by single plasma cell are homogeneous. This also means that sequences of all light as well as heavy chains will be identical. Different plasma cell make different antibody. Genetically identical plasma cells will make identical antibodies. Cloned cells are genetically identical and thus cloned plasma cells will make identical (homogeneous) antibodies. These identical antibodies produced from single clone of plasma cell are called monoclonal antibodies. MABs are antibodies that arise from a single clone of cells Antibodies produced by different clones of plasma cell are called polyclonal antibodies. These are obtained from the blood of animal immunized with antigen (immunogen). Plasma cells are mortal and die in culture, usually in 7-10 days. Thus, it is not possible to obtain monoclonal antibodies in reasonable amount unless these cells are made to be immortal. Kohler and Milstein (1975) revolutionized production of monoclonal antibodies against predefined epitopes. It was achieved by fusing spleen cells with myeloma cells. These fused cells can be continuously grown in culture and can also express immunoglobulin gene from spleen cells. Immortality is provided by myeloma cell to B-lymphocytes. Since hybridoma cells can be cloned, it is now possible to produce unlimited quantities of exquisitely specific antibodies against virtually any molecule, regardless of the purity of the immunizing antigen. Kohler and Milstein was awarded with Nobel prize in 1984.
Hybridoma technology is a method for producing large numbers of identical antibodies, monoclonal antibodies. The production of Hybridoma cells involves a number of well defined steps given below.
1. Culturing of Myeloma cells. 2. Immunization of Balb/C mouse with antigen 3. Collection of myeloma cells and spleen cells on the date of fusion 4. Fusion of myeloma cells and spleen cells 5. Selection of fused cells in HAT medium
Biochemical Techniques Biochemistry 22 Hybridoma Technology : Part A
6. Culturing of fused cells 7. Screening of fused cells for production of antibodies 8. Cloning of fused cells 9. Freezing of Cells 10. Production of monoclonal antibodies
3.1 Culturing of Myeloma cells-
Myeloma cells are malignantly transformed plasmacytoma cells. These can be induced by administration of mineral oil into the peritoneum of Balb/C mouse. These cells are also called mineral oil plasmacytoma (MOPC). Myelomas have capability to make and secrete antibodies. Mouse myeloma cell line ‘P3-X63Ag8’ was generated by Kohler and Milstein (1975) and this cell line secretes IgG1 antibodies and thus it is not recommended for use. Other cell lines such as X63Ag8.653 (Kearney et al., 1979), Sp2/0-Ag14 (Kohler and Milstein, 1975) and NSO/1 (Galfre and Milstein, 1981) do not secrete their antibodies. These cell lines are accordingly divided into secretary and non-secretary cell lines. These myeloma cell lacks either HGPRT (hypoxanthine guanine phosphoribosyl transferase) or thymidine kinase (TK) and can be selected by culturing myeloma cells in presence of 8- azaguanine or 5-bromouracyl respectively. HGPRT- or TK- cell lines helps in selection of fused cells.
Myeloma cells can be cultured in RPMI-1640 or DMEM basal medium containing 10 to 15% fetal calf serum. These basal medium contains a number of salts, glucose, a number of L- amino acids, a number of vitamins and phenol red. Glucose acts energy source for cells. Tissue culture media are available in powered or liquid from commercial suppliers. Fetal calf serum is source for protein, hormones, vitamins and growth factors. Protein (albumin) provides protection mechanical damage of cells. For avoiding bacterial contamination, medium is supplemented with penicillin (100 U/ml) and streptomycin (100 µg/ ml). Myeloma or Hybridoma cells have simple growth requirements and therefore these can be grown in simple basal medium.
Myeloma and hybridoma are grown in tissue culture flask. These are rectangular in shape having tilted neck. Flasks are designed to provide maximum surface area for growth of cells and sufficient air space for exchange of gases with medium and also about layering of medium over the surface. Tilted neck assists in easy collection of spent medium, feeding of cells with fresh medium and collection of cells from suspension or surface. Flasks are of polypropylene or polystyrene and are transparent. Transparency or turbidity in flask can be
Biochemical Techniques Biochemistry 22 Hybridoma Technology : Part A
easily accessed from outside by naked eye. These are available in three different sizes viz. 25 cm2, 75cm2 and 225cm2 and accordingly named as 25 cm2 flask, 75cm2 flask and 225cm2 flask. Normally 10 ml, 30 ml and 90 ml of medium are fed in 25 cm2 flask, 75cm2 flask and 225cm2 flask respectively.
Myeloma cells can be revived from frozen stock or can be borrowed from other laboratory or can be purchased in suspension. Normally, cells on arrival are fed with fresh basal medium containing 15% FCS and placed in CO2 incubator maintained at 5% CO2 and at 37ºC. Cap of flask is kept loose for gas exchange. Phenol red indicator present in medium indicates pH of medium at a given time and colour of medium indicates whether medium is to be replenished or not. If medium turns yellow and is transparent, it indicates that medium is to be replenished. If medium turns yellow and is turbid, it indicates that bacterial or fungal growth has occurred. At times, poorly grown cells can also account for turbidity.
Cells are usually sub-cultured by splitting cells 1: 9 times with fresh medium and this is done when cells are growing in log phase. Confluency term is very commonly used in cell culture and refers to extent of surface occupied by growing cell mass. It is appropriate to sub-culture cells at 70-90% confluency. At 3rd or 4th day of culture, spent medium is removed and equal volume of fresh medium containing FCS is added.
Medium also supports growth of bacteria, fungus and mycoplasma. In fact these are called contaminants and these divide at much faster rate than myeloma or hybridoma cells. These also deplete nutrients very fast, turns medium turbid and change its colour from pink to yellow. Contamination in medium can come from environment, during its handling and
Biochemical Techniques Biochemistry 22 Hybridoma Technology : Part A
preparation. Thus, aseptic practices are to be followed preparation of medium and handling of cells. Prepared medium is filter sterilized after dissolving powered medium in distilled water and adjustment of pH with 1M HCl or 1M NaOH. Sterilized liquid media are also commercially available and are to be used before expiry date.
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Biochemical Techniques Biochemistry 22 Hybridoma Technology : Part A