Antibodies are protective proteins produced by B cells as part of the adaptive immune response to an antigen. Since they are aligned to an epitome, antibodies are widely used in research when identifying target proteins in different applications. For example, IgG is the most common antibody isotype used for exploration.
Custom antibody production entails injecting foreign antigens to determine the Vivo humoral response. Today, you can produce monoclonal or polyclonal antibodies in a laboratory for research use. This article provides a comprehensive overview of the steps needed in custom antibody production.
Polyclonal antibody production is done by immunizing an animal using different immune cells. Distinct clones of plasma B cells produce them without using hybridoma cell lines. The animal will have the affinity for the same antigen but different epitopes.
The custom production of polyclonal antibody probes is a relatively simple process. It involves immunizing an animal and relying on its immune system to produce antibodies against the injected molecule. There are several steps involved in this process.
1. Antigen preparation
This is the first step in making custom antibodies. It entails preparing the protein antigen samples before safely injecting them into an animal. Ensure you prepare antigens to deliver them in a form that maximizes the production of a specific immune response. The molecule should be able to solicit an immune response in the animal.
Injecting the antigen in a laboratory or farm animal evokes antigen-specific antibodies in serum, which you can recover from the animal. The antigen must have three characteristics: be foreign, high molecular weight, and chemical complexity. Foreignness means the immunized animal will recognize the antigen and evoke a reaction. This means the compound cannot come from the same animal.
It should have a high molecular weight greater than 6000 to facilitate crosslinking of the mLgM molecules. This crosslinking signals receptor-mediated antigen endocytosis. Finally, the antigen must have some high chemical complexity needed to generate an immune response.
2. Animal immunization
This is where you inject a protein antigen intramuscularly or intradermally into an animal. You can use a rabbit or goat when making custom polyclonal antibodies. Your choice of the animal should depend on how much antiserum you need during the laboratory production of antibodies. Rabbits are common choices since they are genetically different from human beings. When making custom antibodies with a rabbit, you can get as much as 25ml of serum from a single bleed without hurting the animal.
During animal immunization, use adjuvants to enhance the immune response. These are additives that boost immune response when mixed with an immunogen. For example, you can use Freund’s Adjuvant that comprises a water-in-oil emulsion that localizes the antigen for an extended period, allowing the mycobacterium to attract macrophages to the injected site.
Alternatively, use solutions of hydroxide during immunization. They don’t require emulsification, are powerful enough, and won’t elicit an immune response for a non-immunogenic compound. The concentration of the immunogen will determine the amount of conjugate to administer per injection. The polyclonal antibody production ends when the titer has reached your desired level, and a severe reaction occurs in the animal.
3. Antibody purification
Purification is part of the custom antibody production to get usable components. The process involves isolating the antibody from serum to enrich Immunoglobulin G in the raw antiserum and remove the bulk of unwanted proteins.
You can use affinity purification in polyclonal antibodies since it allows isolating specific antibodies from antiserum. It eliminates several non-specific IgG fractions while enriching the immunoglobulin fraction that reacts with the target antigen.
Antibody purification methods include specific or general practices. When performing general purification of the IgG serum component, use Protein A or Protein G affinity resin. This purification should be adequate for most applications, provided it doesn’t cause off-target binding and background.
If you want specific purification, you will need to purify hapten-specific antibodies. You can accomplish this by immobilizing the original hapten to a solid support to ensure it doesn’t contain the same carrier protein used in preparing the antigen. Use the ELISA assay method for specific purification.
4. Quality control
The last step after purification is to perform several quality control tests to ensure it has high quality. For example, you can evaluate the antibody concentration by absorption at 280nm and check the purity of the polyclonal antibody using SDS-PAGE.
The screening, tittering, and isotyping are crucial in quality control when performing custom antibody production. These tests provide all the information needed to determine which animal or cell line to select. For example, screening helps identity the antibody samples with antigen-binding specificity. Use tittering to measure antibody concentration and isotyping to determine an antibody class identity. The tests also help know-how whether the antibodies are effectively purified.
Monoclonal antibody production entails using identical immune cells cloned from a unique parent cell. You can use the same immunization protocol in producing monoclonals, except with a different animal. This means you follow similar steps from antigen preparation, immunization, purification, and quality control. Use a culture supernatant of a hybridoma cell to isolate antibodies from serum during antibody purification.
The Bottom Line
It’s possible to produce custom antibodies in a lab using these steps. Monoclonal antibody production is ideal for applications requiring large volumes of identical antibodies specific for a single epitope. For example, the method is perfect when developing therapeutic drugs. However, polyclonal antibodies are better for general research applications. Today, there are several organizations engaged in monoclonal and polyclonal antibody production around the world.