The biological product known as an antibody is in high demand for academic and pharmaceutical research. When researching a particular target protein or working with immune system functions, you must consistently produce monoclonal and polyclonal antibodies.
Such antibodies are produced through several steps that require careful technical expertise, including protein purification, hybridoma generation, animal immunization, and antigen design. Screening and assays are also necessary between each step to guarantee quality.
This process can be made much simpler by outsourcing it to skilled service providers, freeing up time and energy that you can use for more insightful experiments. When selecting a service, consider all relevant elements, such as target species, starting material, conjugates, and turnaround time.
In this post, we will look at practical and valuable information on antibody production and development.
There are both broad and specific definitions for the phrase antibody production service. Generally, it refers to all the steps involved in producing a specific antibody, such as preparing the immunogen, creating a hybridoma, collecting samples, screening them, isotyping them, purifying them, and labeling them for immediate use in a particular procedure.
More importantly, antibody production refers to the processes involved in producing antibodies; it does not include various methods of purifying and labelling the antibody for specific applications.
Producing antibodies involves preparing antigen samples and safely injecting them into farm or laboratory animals to make significant levels of antigen-specific antibodies in the serum, which can then be extracted from the animal—direct recovery of polyclonal antibodies from serum (bleeds).
Monoclonal hybridoma cell lines express the particular antibody in the cell culture supernatant and are created by fusing immortal myeloma cells with antibody-secreting spleen cells from immunized mice.
Careful planning and implementing several crucial steps and factors are essential for successful antibody production.
- Synthesize or purify the target antigen
- Choose an appropriate immunogenic carrier protein
- Immunize animals using the appropriate schedule and adjuvant formula
- Immunize animals using right adjuvant formula
- Screen serum for antibody titer and isotype
The purification processes range from very basic to very specialized:
- Crude- the precipitation of immunoglobulins, a subset of the total serum proteins.
- General- affinity purification without consideration of antigen specificity of some antibody classes, such as IgG.
- Specific- only those antibodies in a sample that bind to a particular antigen molecule.
Depending on the application(s) for the antibody, a certain level of purification may be required to produce a functional antibody.
Three different types of tasks are involved in antibody characterization and are typically carried out at various stages of an entire antibody production and purification project:
- Screening- identifying antibody samples with specificity for antigen
- Titering- determining the functional assay titer and antibody concentration
- Isotyping- identifying the class and subclass of a monoclonal antibody
To determine which animals and hybridoma clones are producing a high level of antigen-specific antibodies, screening is first necessary during production. ELISA techniques are typically used to accomplish this general protein assay or a species- and immunoglobulin-specific approach, such as with specialized microagglutination assay kits, can be used to estimate antibody concentration.
While antibody titer and concentration are related, it more specifically refers to an antibody sample’s actual potency. Titer measurement typically entails figuring out the functional dilution of an antibody sample required for detection in a specific assay, like ELISA.
Finding an antibody’s class (IgG vs. IgM, for example) and subclass (IgG1 vs. IgG2a, for instance) is known as isotyping. This is crucial in producing antibodies because it determines the best way to purify and modify the molecule. The easiest way to perform isotyping is with commercial, ready-to-use antibody isotyping kits.
Purified antibodies can be altered for specific applications using various techniques, such as breaking them into smaller antigen-binding units, conjugating them with enzymes or other observable markers, or immobilizing them to solid supports.
Whole molecules of antibodies are used the majority of the time. However, using antibodies with non-essential portions removed can enhance the performance of some methods and experiments.
The term “antibody fragmentation” describes techniques for disassembling complete antibody molecules and removing parts that are not required for binding antigens. The most frequently produced and used antibody fragments of IgG by scientists are Fab and F(ab)’2.
Antibody labelling and immobilization
To be used as antigen-specific probes, antibodies are created and purified. However, the ability to secondary detects the antibody is necessary for them to be helpful in any given technique (ELISA, western blotting, cellular imaging, immunohistochemistry).
Mechanisms for attaching or immobilizing antibodies to chromatography media are necessary for immunoprecipitation and other affinity purification techniques (e.g., beaded agarose resin). The same factors and chemical processes that go into accomplishing this are considered.
Recognizing a problem exists is the first step in solving it. Since the problem with antibody validation came to light, many initiatives have been launched to find the best fixes. We are progressing, but there is still much to be done to determine the most effective ways to solve the issue.
Among the most important first steps is acknowledging the necessity of antibody validation standards. Given the significance of reproducibility for the development of science, it is in everyone’s best interest to take on the challenge of putting these standards into practice, including researchers and suppliers.