Antibodies are essential components in fighting against viruses and bacteria. Antibodies are protein building blocks. To successfully produce an antibody, a sequential procedure must be followed. Two types of antibodies can be produced. The difference between the two antibodies is the process of producing them. For example, antibodies are directly separated from the serum when producing polyclonal antibodies.
On the other hand, monoclonal antibodies are produced by combining the antibody synthesis spleen cells collected from the immunized animal with the myeloma cells. The combination results lead to the formation of monoclonal hybridoma cells lines. In this article, we will concentrate on producing monoclonal antibodies.
Process of producing an antibody
Producing a monoclonal antibody starts with the generation of cells producing cells formed through the fusing of myeloma cells with the desired antibody that produces splenocytes. There are two methods of producing monoclonal antibodies; the Vivo or in vitro method. The two methods can be used simultaneously during antibody production. Before the production process begins, the hybrid cells have to be produced first regardless of the method to be used. To ensure the successful production of custom antibodies, the following steps are used in the process.
Immunization of the animal and isolation of splenocytes
The first step in antibodies production is to immunize the chosen animal with the specific antigens that will trigger the production of certain antibodies. The antigens used to inoculate the animal must be in a form that optimizes the production of particular responses from the animal immune system. The antigens have to be prepared first before being used for the immunization process. For example, mice are used primarily to produce monoclonal antibodies.
Therefore, it has to be immunized with a specific antigen that will ensure a positive response from the immune system and, in turn, gives maximum production of the antibodies. For animal immunization, various techniques can be applied to ensure the successful synthesis of antibodies. The method applied in the immunization depends on the nature of the antigen and the type of antibody required by the researcher. Once the animal is immunized with a specific antigen, they are isolated for several months, and the immunization process can be done repeatedly.
Screening of animal blood
After a few weeks or months of immunization, blood samples are collected from the immunized animal. The collected blood undergoes a thorough test for serum antibodies. Numerous humane techniques have been developed for a thriving collection of small amounts of blood from immunized animals such as mice. The enzyme-linked immunosorbent test and flow cytometry measure the serum antibody titer.
Cell blending often occurs when the titer is higher. However, in cases where the titer is lower, the mice are raised till they have an adequate response, which is indicated in the repeated blood samples. In incidents where the antibody titer is more robust, the mice can be boosted a few days before the blending.
Myeloma Cell Preparation
When the spleen cells are responsible for producing combined antibodies, they create a limitless growth potential hybridoma. After a cell fusion, immortalized myeloma cells are designed with an 8-azaguanine to ensure high sensitivity. Only the merged cells can survive in the HAT media.
Merging of myeloma cells
Single immunized mouse spleen cells are combined with previously generated myeloma cells. When freshly collected spleen and myeloma cells are centrifuged together in polyethylene glycol, a substance that stimulates cell membranes to fuse, fusion occurs. Only fused cells will develop in the unique selection medium, as mentioned in step 3. The cells are later transplanted to a particular plate using feeder cells found in saline washes of a mouse.
These cells are usually hypothesized to give growth factors that help in the proliferation of hybridoma cells. Custom preparations created from several factors favoring the growth of cultured cells and integrating growth influences can be used instead of feeder cells. Macrophages derived from mouse bone marrow could also be used as feeder cells.
Cloning of Hybridoma cells
Antigen bindings provide a variety of tissue cultures that can give rise to small clusters of hybridoma cells. At this stage, cloning through restricted dilution will ensure that each cell is well contained by one clone. During this stage, the hybridomas cells with the ability of expansion vis a vis the total loss of the cell fusion product is selected. However, underpopulation or inefficient vitro development at high dilution leads to a lot of judgment. Secreted antibodies can be harmful to fragile cells kept in vitro in some cases.
At this time, optimizing the mouse ascites expansion strategy can help save the cells. Many others have also found that a brief time of growth using the mouse ascites approach results in cell lines with improved resilience and excellent antibody production later in vitro and in vivo phases. Guidelines have been issued to help investigators apply the mouse ascites procedures in these ways.
Once the antibodies are produced, they have to undergo a purification process. The purification process helps eliminate any unnecessary elements that may have been accrued through the process and eliminate the antigens that the animal may have had before immunization. In addition, the produced antibodies can be classified or characterized through screening, isotyping, and tittering. To successfully produce quality antibodies, prior preparations and implementation of every step effectively must be observed.