Hybridoma Technology Protocol and FAQs

Hybridoma technology is a method for the preparation of monoclonal antibodies. It was first proposed by scientists Cesar Milstein and Georges Köhler in 1975, and thus won the Nobel Prize in Physiology or Medicine in 1984. The core idea of hybridoma technology is to fuse antibody-producing B cells with non-antibody-producing tumor cells (such as myeloma cells) to obtain hybridoma cells that can proliferate indefinitely and produce antibodies specifically.

The Principle of Hybridoma Technology

Hybridoma technology is a technique for obtaining monoclonal antibodies by cell fusion. The principle is to fuse the B cells of immune animals (usually mice) with myeloma cells (tumor cells, with unlimited proliferation) to obtain hybridoma cells that can proliferate indefinitely and produce specific antibodies. These hybridoma cells are capable of continuously producing a large number of single antibodies, called monoclonal antibodies.

1 B cells : derived from immune animals, responsible for the production of antibodies.
2 Myeloma cells : Tumor cells that have the ability to proliferate indefinitely.
3 Hybridoma cells : obtained by fusing B cells and myeloma cells, have both antibody production capacity and unlimited proliferation ability.

The Steps of Hybridoma Technology

Immunization

First, the target antigen (such as bacteria, viruses, tumor antigens, etc.) is injected into the body of experimental animals (such as mice), so that the animal 's immune system produces specific antibodies against the antigen. The purpose of immunization is to stimulate the immune response, so that B cells in animals produce a large number of antibodies.

Extraction of Spleen Cells

B cells (antibody-producing cells) were extracted from the spleens of immunized animals. After immunization, the number of B cells in the spleen increased significantly. Through cell separation technology, a large number of B cells can be isolated from the spleen.

Preparation of Myeloma Cells

Select a myeloma cell line that does not produce antibodies. These cells can proliferate indefinitely, but they cannot produce specific antibodies themselves..

Cell Fusion

B cells and myeloma cells can be fused together by chemical or physical methods (such as polyethylene glycol (PEG) or electric field fusion). The fused cells are called hybridoma cells and have the following two characteristics:

1 Specific antibody production ability of B cells : Hybridoma cells can produce the same specific antibody as the original B cells.
2 The unlimited proliferation ability of myeloma cells : Myeloma cells are malignant cells that can proliferate indefinitely, ensuring that hybridoma cells can continue to proliferate.

Selective Screening

When B cells and myeloma cells are fused, the obtained hybridoma cells will have some special properties, but this does not mean that all the fused cells are hybridoma cells. Most of the unsuccessfully fused B cells and myeloma cells will die. In order to screen successfully fused cells, HAT screening method (Hypoxanthine-Aminopterin-Thymidine) is usually used. This screening method takes advantage of the metabolic defects of myeloma cells. Unfused myeloma cells cannot survive in HAT medium due to the lack of specific enzyme functions, while B cells are restricted in this medium. Only the fused hybridoma cells can survive and reproduce in HAT medium. Through this screening method, hybridoma cells that can continuously proliferate (myeloma cell characteristics) and secrete specific antibodies (B cell characteristics) can be selected.

Cloning and Amplification

The successfully fused hybridoma cells screened out are usually single cells, which can be cloned and cultured after separation. A large number of hybridoma cells with the same clone were obtained by isolating single cells and culturing them step by step. These cloned hybridoma cells can continuously secrete a large number of specific antibodies.

Collection and Purification of Antibodies

The amplified hybridoma cells were cultured in a suitable medium, and the antibody was secreted into the culture medium through extracellular secretion. After that, the antibody was purified by affinity chromatography and ion exchange chromatography. The purified antibody can be further characterized and quality controlled to ensure its purity and activity.

Identification and Application of Antibodies

The obtained monoclonal antibodies were identified by various immunological methods, such as enzyme-linked immunosorbent assay (ELISA), immunofluorescence, Western blot, etc., to ensure their specificity and efficacy for the target antigen. The monoclonal antibodies successfully obtained can be used in various applications, such as diagnosis, treatment, vaccine development and scientific research.

Figure 1. A diagram of mAb generation by the hybridoma approach. (Reference source: Hybridoma Technology for the Generation of Monoclonal Antibodies.)

Applications of Hybridoma Technology

Disease Diagnosis

Development of detection tools using monoclonal antibodies, such as immunochromatographic test strips, ELISA kits, etc.

Therapeutic Antibodies

For example, monoclonal antibody drugs for the treatment of cancer, infection, immune diseases, etc.

Vaccine Development

The use of antibodies against specific pathogens, the development of vaccines.

Basic Research

Monoclonal antibodies are widely used in immunology, molecular biology, cell biology and other disciplines to study the interaction mechanism between antibodies and antigens.

Alpha Lifetech provides proteins, peptides, small molecules, viruses, etc. as immunogens, mouse monoclonal antibody preparation services, as well as upstream antigen preparation and downstream antibody library construction and a series of services.

FAQ

1. Low cell fusion efficiency, resulting in a small number of hybridoma cells.

Solutions : (1) Optimization of fusion conditions : Adjust the chemical or physical conditions of fusion (such as PEG concentration, electric field strength) to improve the fusion rate. PEG or electrofusion is often used in the experiment, but the conditions need to be optimized according to cell type and experimental conditions. (2) Use more efficient fusion reagents : such as the use of new fusion reagents, or consider the use of special fusion equipment to improve the success rate of fusion.
2. Low survival rate of hybridoma cells.

Solutions : (1) Use appropriate selective medium : Ensure that the medium contains drugs such as HAT (Hypoxanthine-Aminopterin-Thymidine), which can effectively screen out well-grown fusion cells. (2) Optimization of culture conditions : for example, control the composition of the medium, temperature, pH value, etc., to ensure that cells can survive and proliferate under optimal conditions.
3. Low yield of monoclonal antibodies.

Solution : (1) Optimize the culture conditions : increase the nutrient concentration of the medium, use a special medium suitable for cell proliferation and antibody production, regularly replace the culture medium, and maintain the cells in a good growth environment. (2) Screening high-yield clones : The hybridoma cells were cloned and screened, and the clones with high antibody production capacity were selected. (3) Using engineered cell lines : Using genetically engineered myeloma cell lines, these cell lines may have higher antibody production capacity. (4) Optimize the harvest method : by increasing the cell density, expand the scale of culture and other methods to increase the amount of antibody collection.
4. Heterogeneity may occur in the cloning of hybridoma cells (that is, different clones of the same hybridoma cell line have different antibody production and affinity), resulting in difficulty in obtaining uniform quality antibodies.

Solutions : (1) Monoclonal cell screening : By monoclonalizing the fused cells, it is ensured that each hybridoma cell line is derived from a single cell, avoiding cell heterogeneity. (2) Stabilized cloning : For clones with unstable antibody production, selective culture or gene transfection techniques can be used to stabilize antibody production.
5. Sometimes, monoclonal antibodies produced by hybridoma technology may have low affinity or low specificity to antigens, resulting in their inability to effectively recognize target antigens.

Solutions : (1) Optimize the immunization program : By increasing the antigen dose and frequency of immunization or using appropriate adjuvants, the immune response is enhanced, thereby increasing the probability of B cells producing high-affinity antibodies. (2) Affinity maturation technology : affinity maturation of the obtained antibodies, such as affinity screening of antibodies by in vitro screening method, or optimization of antibody affinity and specificity by antibody humanization method. (3) Antibody optimization : To improve the affinity and specificity of antibodies by molecular biology techniques (such as single-chain antibody screening or antibody engineering).

reference

[1] Zhang C. Hybridoma technology for the generation of monoclonal antibodies. Methods Mol Biol. 2012;901:117-135. doi:10.1007/978-1-61779-931-0_7

[2] Hnasko RM, Stanker LH. Hybridoma Technology. Methods Mol Biol. 2015;1318:15-28. doi:10.1007/978-1-4939-2742-5_2

[3] Tomita M, Tsumoto K. Hybridoma technologies for antibody production. Immunotherapy. 2011;3(3):371-380. doi:10.2217/imt.11.4

[4] Hiatt AC. Monoclonal antibodies, hybridoma technology and heterologous production systems. Curr Opin Immunol. 1991;3(2):229-232. doi:10.1016/0952-7915(91)90056-7

[5] Yagami H, Kato H, Tsumoto K, Tomita M. Monoclonal antibodies based on hybridoma technology. Pharm Pat Anal. 2013;2(2):249-263. doi:10.4155/ppa.13.2