Researchers can harness the ability of an animal immune system to produce antibodies that can bind specifically to antigens. The antibodies are protective B cells aligned to an epitome, which means you can use them in research to identify target proteins in different applications. Besides, the B lymphocytes make antibodies as a part of an antigen’s adaptive immunity. This article looks at how antibodies are produced for research.
What is antibody production?
It refers to creating a usable-specific antibody. Antibody production also describes the steps leading to antibody generation. The success of custom antibody production depends on careful planning and implementation of all the steps. There are several steps involved in custom antibody production. They include immunogen preparation, immunization, hybridoma creation, screening, isotyping, purification, and labeling.
The first part of custom antibody production is antigenic preparation. This is where you prepare an antigen sample and inject it into a farm animal to elicit antigen-specific antibodies in serum. You can recover polyclonal antibodies directly from serum or use antibody-secreting spleen cells from immunized animals for monoclonal antibodies. Start by preparing an antigen in a form that can help produce a maximum immune response.
You can use different macromolecules as antigens during the preparation stage. Three qualities should characterize the antigen: foreignness, high molecular weight, and chemical complexity. This means you cannot use a compound from the same animal as an antigen. The immunized animal will recognize the antigen and elicit a reaction when you use a foreign antigen.
The antigen you use in custom antibody production should have a molecular weight higher than 6,000 to facilitate cross-linking of monoclonal molecules. Focus on achieving cross-linking for receptor-mediated antigen endocytosis. For example, an antigen with a high chemical complex helps generate an immediate response.
After preparing an antigen, the second step is to immunize an animal with different immune cells. The immunization involves injecting a protein antigen intramuscularly or intradermally into a lab animal. During this step, you can use a rat, mouse, rabbit, or any other animal, depending on how much serum you need for antibody production. Rabbits are popular for injecting protein antigens because their genetic makeup differs from that of humans. You can also get more serum from a single bleed on a rabbit without hurting the animal.
You can use adjuvants to enhance the immune response during the immunization process. Adjuvants are additives that boost the immune response when mixed with an immunogen. If you are conducting monoclonal antibody production, you can use identical cells from a parent cell and the same immunization protocol.
Hybridoma development fusions
The next step is to use Elisa’s assay to determine the positive supernatants in hybridoma development fusions. You will need to preserve the cell lines when performing the antibody production. Ensure you test and preserve the cell lines to increase their longevity and productivity. The Elisa kits can harvest spleen cells from over one lab animal for hybridoma fusion.
In antibody production, subcloning involves creating homogeneous and consistently secreting antibodies. This is an essential step in custom antibody production, where you clone homogeneous antibodies. When you clone and subclone the positive hybrid, you can produce an antibody that maintains the stability and monoclonal character of the hybridoma.
You can use several sub-cloning techniques, such as limiting diluting to sub-clone parental cell lines. This method involves cloning hybrids at a set of one cell per well and sub-cloning at 0.3 cells per well. Another technique is the multi-cycle clonality method, where you perform multiple rounds of limiting-duration sub-cloning. Run the process until all wells grow simultaneously and release antibodies at the same concentration. The goal is to have similar isotypes for antibody production.
During antibody production for research, you need highly pure antibodies. This explains the importance of antibody purification during production to ensure you achieve high-purity results. Antibody purification involves isolating an antibody from a serum or hybridoma cell line.
There are various purification methods available, ranging from highly specific to general. The specific methods require purifying the hapten-specific antibodies using protein A or protein G affinity resin. During this method, you can immobilize the original hapten to ensure it doesn’t have the same carrier protein as the one used to prepare an antigen.
The affinity purification method isolates specific antibodies from the antiserum, removing non-specific IgG fractions and enriching the immunoglobulin fraction. You can use an Elisa assay to achieve a specific purification during affinity purification.
The characterization involves screening, tittering, and isotyping. It is one of the most crucial steps you can take in biopharmaceutical research to determine whether antibodies are pure. During the screening process, use Elisa techniques to identify which animals and hybridoma clones produce higher antigen-specific antibodies. Researchers can estimate antibody concentrations using a general protein assay or an immunoglobulin-specific method.
You can measure and estimate the antibody concentration using a general protein assay or an immunoglobulin-specific method in tithing. Isotyping is when you determine the class and subclass of monoclonal antibodies. This is an important step in antibody production because it allows you to choose purification and modification methods for the molecule.
You can modify purified antibodies for different users. One way is through fragmentation, breaking the antibodies into smaller antigen-binding units. While antibodies are mostly whole, you can divide them to improve the performance of some techniques and experiments. The fragmentation process entails cleaving a whole antibody molecule to remove unnecessary portions for binding antigens. The last custom antibody production step is labeling and immobilization, attaching them to chromatography media.
Antibody production for research involves several steps, from antigenic preparation to immunization, purification, and quality control. Since its inception in the 1970s, the procedures for generating, purifying, and modifying antibodies for research developed have remained the same. Follow the steps to produce quality antibodies for research.