Antibodies are protective proteins B cells produce as part of an antigen’s adaptive immunity. Since they are aligned to an epitome, antibodies are widely used in research when identifying target proteins in different applications.
Custom antibody production is a valuable tool when needed to develop an antibody against a rare or unique antigen. Once you create a hybridoma cell line to produce an antibody, you can preserve that cell line for future use. This article looks at the stages needed in custom antibody production for research use.
Antigen preparation
This is the first step in making custom antibodies. It entails the preparation of antigen samples and the safe injection in the laboratory or farm animals to evoke antigen-specific antibodies in serum. Start by preparing an antigen into a form that maximizes the production of a specific immune response. The molecule should be able to solicit an immune response in the animal.
You can use a range of macromolecules as antigens. The antigen should have three characteristics: foreign, high molecular weight, and chemical complexity. A foreign antigen means the immunized animal will recognize the antigen and evoke a reaction. You cannot use a compound from the same animal as an antigen.
The antigen should have a higher molecular weight greater than 6000 to facilitate cross-linking of the monoclonal molecules. This cross-linking signal receptor-mediated antigen endocytosis. Use an antigen with a high chemical complexity to generate an immediate response.
Hybridoma development immunizations
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After antigen preparation, the next step is to immunize an animal with different immune cells. This is when you inject a protein antigen intramuscularly or intradermally into a farm animal. Distinct clones of plasma B cells produce the immune cells without using hybridoma cell lines. The animal will then have an affinity for the same antigens but with different epitopes.
Choose an animal depending on how much serum you need during custom antibody production. Rabbits are popular choices because they have a different genetic composition from humans. You can get as much as 25 ml of serum from a single bleed without hurting the animal.
Immunize animals and test their serum for antibody production. During immunization, use adjuvants to enhance the immune response. These additives boost the immune response when mixed with an immunogen. You can also use solutions of hydroxide that don’t require emulsification and don’t elicit an immune response for a non-immunogenic compound.
In monoclonal antibody production, you use identical immune cells cloned from a unique parent cell. You can use the same immunization protocol to produce monoclonals but with a different animal.
Hybridoma development fusions
Use an ELISA assay to screen the positive supernatants during the hybridoma development fusions. The process entails boosting animals with the best immune response to the antigen using the same antigen before harvesting their spleens for hybridoma fusion.
You then preserve these cell lines and store them during evaluation. Test and preserve these cell lines with increased longevity and productivity for further use. According to Elisa’s results, you can harvest spleen cells from 2 to 3 mice for hybridoma fusion.
Subcloning
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The step involves cloning the cell line until it is homogenous and consistently secreting antibodies. You will clone and subclone each positive hybrid producing the specific antibody of interest to maintain the stability and monoclonal character of the hybridoma. This step is essential for the long-term stability of clines and to ensure the cells maintain clonality over their production life.
Use the limiting-dilution technique in sub-cloning parental cell lines to obtain daughter cell lines. This is where you clone hybrids at one cell per well and sub-clone at 0.3 cells per well. Multi-cycle cloning involves multiple rounds of limiting-duration subcloning until all wells grow simultaneously. They should secret antibodies at the same concentration and have the same confirmed isotypes.
Antibody production and purification
Antibody application in biotechnology and sensor development demands highly pure sources of antibodies. Antibody purification involves isolating antibodies from serum in polyclonal antibodies or the culture supernatant of a hybridoma cell line. You can use several methods to purify your antibodies.
Purification methods range from general to highly specific. Use protein A or protein G affinity resin in purification, which should be adequate for most applications. For specific purification methods, you will need to purify hapten-specific antibodies. Immobilize the original hapten to a solid support to ensure it doesn’t contain the same carrier protein used in preparing the antigen.
You can use affinity purification in polyclonal antibodies since it allows isolating specific antibodies from antiserum. This method eliminates several non-specific IgG fractions while enriching the immunoglobulin fraction. Use the ELISA assay method for specific purification.
Antibody characterization
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After purification, antibody characterization is crucially essential in biopharmaceutical research. The characterization involves activities like screening, tittering, and isotyping. These tests help determine whether the antibodies are effectively purified.
Screening helps identify antibody samples with antigen-binding specificity using ELISA techniques. Use tittering to measure antibody concentration. You can estimate the antibody concentration using a general protein assay or a species and immunoglobulin-specific method. The production ends when the titer has reached the desired level, and several reactions occur.
Isotyping helps determine an antibody class identity. It involves specifying the class and subclass of a monoclonal antibody. The process helps when choosing an appropriate purification and modification method for the molecule.
Bottom line
Custom antibody production involves immunizing an animal and relying on its immune system to produce antibodies against the injected molecule. Follow antigen preparation, immunization, purification, and quality control steps to produce particular antibodies for your application. Successful custom antibody production depends on careful planning and implementation.
