The use of custom antibodies as agents in biology and diagnostics has entirely restructured and transformed life and medicinal science. Monoclonal antibodies (mAbs), polyclonal antibodies (pAbs), recombinant antibodies, and antibody fragments are employed in various purification techniques.
Moreover, recombinant procedures have increased the potential of antibody production, which has extensively enhanced the purification requirements to around 100kg batches. Therefore, the process now needs indigenous methods and strategies of purification.
To meet the industry standards of purification, researchers and investigators employ affinity chromatographic ligands. Presently, proteins can also be used as bio-affinity ligands thanks to restricting elements such as Protein A leakages, high costs, low resin reusability, and assumed antibody aggregation when there is pH desorption. This post will discuss the production of custom antibodies and everything you need to know about them.
Antibodies Have Different Sizes and Shapes
The first antibody structure was Y-shaped, and Rodney Peter and Gerald Edelman presented the shape. These scientists later won the Nobel Prize for Medicine and Psychology. The Y-shaped antibody structure is called IgG, and it comprises four polypeptide chains linked to noncovalent bonds and disulfide bridges.
The primary custom antibody elements are the Fc and antigen-binding platforms. Consequently, the region is influenced by variable light and heavy parts, and the Fc section is linked to constant H1, H2, and H3 regions. Antibodies come in different shapes and sizes in modern medicine, depending on animal species.
The combinatorial deoxyribonucleic acid technology has played a significant role in the growth of recombinant antibodies, such as fragment variable (Fv), disulfide-stabilized Fv antibody fragment, single-chain fragment variable (scFv), fragment antigen-binding (Fab), and divalent antibody formats.
Custom Antibody Production – Monoclonal Antibodies Vs. Polyclonal Antibodies
Antibodies are typically generated by the B-cells of the adaptive immune system to respond to offending pathogens. An antibody has a unique lock and critical recognition for invading viruses, bacteria, or other molecules. They are collectively referred to as antigens. Antibodies are proteins that recognize the binding place of a specific antigen.
The specificity of the antigen-antibody recognition can be applied in a wide variety of sensor and biotechnology developments. All these applications require the use of pure sources of antibodies. The antibodies are conjugated to sensor surfaces and detect antigens or can be used together with nanoparticles or other detection platforms.
Purified antibodies that possess specificity to other antigens are often available commercially. These antibodies are divided into one or two segments depending on their mode of production and harvest – monoclonal and polyclonal. Monoclonal antibodies are generated from one B-cell line and can only identify a single epitope on a particular antigen. In contrast, polyclonal antibodies are caused by several B-cell lineages and can identify multiple epitopes on a specific antigen.
When an antibody is required to recognize a particular antigen but unavailable from the supplier, antibodies can be generated through custom antibody production. A sample that is 90% purified is all that is required to generate a custom antibody and harvest it against the antigen.
Moreover, if the antigen sample is unavailable, custom proteins or peptides can be formed against them. Both monoclonal and polyclonal antibodies can be generated through custom antibody production procedures. Here are the processes of creating these custom antibodies.
Production of Custom Monoclonal Antibodies
It takes about six months to complete the production of custom monoclonal antibodies from start to finish. The entire process takes place in five key steps, namely:
- Preparation of antigens. The customer could provide a very pure peptide or protein. Alternatively, the company can design and produce a custom protein or peptide.
- Immunization. Here, the animal is immunized, and the serum is tested for the production of antibodies.
- Fusion. The animals with the best immunity response to a particular antigen are boosted with the antigen again, and then their spleens are harvested to conduct hybridoma fusion. Hybridoma is the cell line of myeloma cancer cells and immortal B cells. These cells tend to have increased reproductivity and longevity. Here, the cell lines undergo testing and preservation.
- Subcloning. The cell line is cloned until it becomes homogenous and secretes antibodies consistently.
- Production and purification. Single-cell culture can generate around 10 mg to 30 mg of antibodies. Purification is then carried out using protein C or protein A resin.
Production of Custom Polyclonal Antibodies
The process of custom polyclonal antibody production takes place in six key steps, namely:
- Designing peptides
- Synthesizing peptides
- Immunizing animals
- Collecting serum
- Analyzing the titer
- Purifying the final antibody
The first two phases aren’t necessary if the customer provides a 90% pure antigen sample. Custom polyclonal antibodies can be generated from a mouse, rabbit, chicken, guinea pig, or even a goat, with an average production period of about 80 days.
Rabbits are the most preferred option because they respond to most antigens. Depending on the employed protocol, they also have high yields and adaptive immune systems that can rapidly produce antibodies in about 70, 90, or 120 days.
Rats and mice can only be used in smaller amounts of the desired antiserum. Goats have the highest yield; however, they respond the slowest to antigen (at least 120 days). Chicken can only be considered if the available rabbits don’t generate antibodies or react to antigens. Guinea pigs can be used as a secondary, but they have a shallow background in drosophila research.
Final Thoughts
With the recent advancements in bioscience and biochemical technology, researchers and investigators have established more and better mediums to promote their experiments. When it comes to custom antibody production services, most institutions and companies provide protein purification and expression services, gene synthesis, peptide synthesis, and protein production services. They do so while considering all the factors involved.
Custom antibody production is quite a handy tool when researchers or investigators want to develop an antibody for a unique or rare antigen. The production process, especially for the monoclonal custom antibody production, can also be helpful for the science realm. This is especially the case because when a hybridoma cell line has been developed to generate a particular antibody, the cell line can be preserved for future purposes.
