The availability of commercial antibody providers has dramatically expanded the scope of research in immunoassays and immunohistochemistry. Thousands of antigen-specific products are just a click away and can be delivered the following day.
Today, we will discuss the production of polyclonal antibodies. This procedure can be intimidating because of the upfront expenses and potential risks, but it is an excellent approach employed by commercial antibody providers.
In addition, there is only a little difficulty involved. If you are intelligent enough to manage your thermocycler's warranty, you can order polyclonal antibodies and learn about the procedures involved. Ultimately, the animal performs most of the work.
Let's begin by addressing basics, such as what a polyclonal antibody is.
Polyclonal antibodies: What Are They?
A polyclonal antibody is a collection of several distinct immunoglobulins, each produced by a different clone of B cells. These antibodies target multiple epitopes, or binding sites, on a single antigen. Polyclonal antibodies distinguish themselves from monoclonal antibodies because they originate from various B-cell clones and subsequently focus on multiple epitopes.
The Secret to Polyclonal Antibody Production
Polyclonal antibodies (pAbs) are created by introducing a particular antigen into laboratory animals, such as rabbits or goats. The animal is immunized several times to produce higher titers of antigen-specific antibodies. These polyclonal antibodies can be extracted and separated from the antiserum in weeks.
Polyclonal antibody production in rabbits is simpler and less expensive than monoclonal antibody synthesis. In addition, polyclonal antisera can be produced in a shorter time (between four and eight weeks), whereas the production of monoclonal antibodies might take anywhere from three to six months.
How to Make Polyclonal Antibodies?
All antibody production, whether polyclonal or monoclonal, starts with a process known as immunization. Injecting the intended antigens into the host causes them to be flagged as foreign and targeted by antibodies.
If an immune response is not initially generated, adjuvants, which are immunogenic substances like Freund's adjuvant or keyhole limpet hemocyanin (KLH), may be used to further stimulate immune recruitment. These components are essential for producing the high-quality polyclonal antibodies acquired through immunization.
Major Processes Involved in Polyclonal Antibody Production
The steps involved in this type of antibody production:
1. Initial Immune Response-Inducing Exposure
"Primary" immunization refers to the first exposure of the host to an antigen in the presence of suitable adjuvants. Injections into the epidermis, peritoneum, subcutaneous layer, and muscle are typical routes of administration, and less common alternatives are administered by mouth or through the nose.
Another cutting-edge technique for toxic or otherwise challenging to-manufacture antigens is DNA immunization. Any additional antigen exposure is referred to as a "boost" immunization.
2. Processing of Antigens by Antigen-Presenting Cells
All antigen-presenting cells (APCs), including macrophages, dendritic cells (DCs), and, to some degree, B cells, take up antigens through phagocytosis or endocytosis. The antigen is broken down during antigen processing, and the smaller fragments are subsequently transferred to the APC's surface.
3. B-cell stimulation
Helper T-Cells release signaling molecules called cytokines when they recognize antigens on the surface of APCs. These cytokines stimulate B-cell activation and proliferation, which is responsible for antigen recognition thanks to their B-cell receptors (BCR).
Some B-cells don't need Helper T-Cell activation after BCR-antigen interaction. This generally causes a polyclonal antibody response to occur faster, with lower-affinity antibodies. Helper T-Cell participation is essential for a comprehensive immune response and the regulation of self-tolerance.
4. Increased B-Cell Growth and Antibody Synthesis
When stimulated, B-cells divide to produce plasma cells and memory B-cells. Both types of cells recognize the same epitope. Plasma cells release antibodies into the bloodstream, allowing the immune system to detect and neutralize antigens. At the same time, memory B-cells persist for longer periods and respond quickly when the same antigen is encountered again.
Immune Response Assessment
To gauge progress, antibody levels must be measured throughout the vaccination procedure. The level of the immunization-induced immune response can be determined by comparing serum samples taken before and after immunization using an ELISA against the target antigen. The amount of the antibody is found by serially diluting samples.
In most cases, a titer of 1:64,000 is believed to be an effective immune response and is adequate for moving on to the next step, either serum harvest or lymphocyte fusion. If your antibody titer is low, you may need a booster shot, a different delivery route, or additional adjuvants.
Harvesting Polyclonal Antibody Serum
To make polyclonal antibodies, you must take blood from the host animal and separate the cellular portion from the serum fraction, where antibody secretion products are found. The phrase "polyclonal antibody" refers to a preparation that contains numerous antigen-specific antibodies released by multiple activated B cells.
Antigen fragmentation and the subsequent generation of multiple clonal antibodies directed against a single antigen are essential steps in this process.
Certain BCRs can identify different antigenic epitopes.
Against the same antigen, distinct B-cell populations can produce a variety of antibodies, each of which is specific to the antigen. Additionally, the serum comprises all immunoglobulins aimed against various targets, including medicines, hormones, and so on, along with antibodies targeted toward the desired target antigen.
It is possible to employ serum in its natural state as a molecular biology probe; in most cases, additional purification is necessary to improve the sensitivity and specificity of a given test. Depending on the method, affinity purification can recover a significant fraction of the original antibody.
Given these mechanisms, let's dive further into rabbit polyclonal antibody production.
Polyclonal Antibodies in Rabbits
Below are the processes involved in rabbit antibody production.
Antigen preparation
The manufacturing procedure begins with the preparation of protein or peptide antigens. It's vital to ensure the chosen antigen is suitable, especially if you want it to present a flexible epitope. Polyclonal antibodies obtained are only as specific as the antigen used.
Impurities with concentrations of less than one percent have the potential to be immunodominant (for example, with many bacterial antigens), which can lead to polyclonal antibody generation that exerts a more significant amount of reactivity towards the impurity than towards the target antigen.
The process of antigen purification is arduous and time-consuming.
Fortunately, Biomak provides antigen preparation services based on our recombinant protein production platforms. We have extensive experience with recombinant protein and recombinant antibody production of different kinds.
Animal immunization
The type of animal used to produce polyclonal antibodies is determined by the quantity of antiserum required, the ancestral distance between the organism's species of origin and the species that will receive the immunization, and the animal's past exposure to the immunogens.
Most of the time, rabbits are used because their genes differ from those of humans and mice, and the origins of the proteins are frequently studied. In addition, rabbits can secrete up to 25 milliliters of serum without adverse outcomes.
Adjuvants stimulate a more robust immunological response in preparation for generating polyclonal antibodies (pAbs). Freund's Adjuvant is among the adjuvants employed the most frequently in the field of research. When an adjuvant is present, the protein antigen is introduced into the muscle, skin, or subcutaneously in a living organism of the chosen species.
Following the priming immunization, booster shots are given between 4 and 8 weeks later at 2- to 3-week intervals. After the initial immunization and at regular intervals, the animal is bled, and whole blood serum is collected for antibody analysis.
The synthesis of polyclonal antibodies should be stopped as soon as the antibody titer reaches a level that is considered satisfactory.
Purifying Antibodies
When it comes to purifying polyclonal antibodies, affinity purification is a method that works well.
Immunoglobulin G (IgG) can be enriched from the raw antiserum using protein A/G affinity purification, effectively removing most other proteins.
However, these solutions still include a considerable amount of background IgGs. As a result, during the use of the antibody in assays like ELISA, immunohistochemistry, Western blot, etc., the noise level will be significantly increased.
Frequently, antigen-specific affinity purification is utilized to separate polyclonal antibodies from antiserum. Most non-specific IgG fractions are removed through antigen affinity purification, enhancing the immunoglobulin fraction that exclusively reacts with the desired antigen.
Quality Assurance
After purification, polyclonal antibodies undergo several quality control checks to ensure they are acceptable. Absorption at 280 nm, often known as A280, is used to determine the concentration of antibodies. SDS-PAGE is used to determine the purity of polyclonal antibodies.
The ELISA test presents the most appropriate method to determine the antibody titer of polyclonal antibodies.
Lastly, labeling polyclonal antibodies with HRP or biotin allows for accurate enzymatic detection in IHC and will enable you to use different types of conjugated avidin.
The following are guidelines and starting principles for other organisms.
Rats
Animals should be injected with 100–200 μg of antigen intraperitoneally or subcutaneously when they are about six weeks old.
Repeat the initial dose every two to three weeks until a satisfactory antibody response develops.
Under general anesthesia, withdraw 400–500 μl from the tail vein 10–12 days following every boost for testing and a maximum of 2 ml each month after observing an appropriate antibody response.
Mice
Animals should be injected with 50–100 μg of antigen intraperitoneally or subcutaneously when they are about six weeks old.
Repeat the initial dose every two to three weeks until a satisfactory antibody response is seen.
Under general anesthesia, withdraw 200-300 μl through the tail vein 6-12 days from the boost for testing and as much as 300 μl on the same schedule after establishing an adequate antibody response.
Goats
500 μg of antigen is injected intradermally at up to 20 locations in a 6-7-month-old goat.
Repeat the dose every two weeks until a satisfactory immune response is achieved.
If an adequate antibody response is seen within two weeks of each boost, 5 ml of blood should be drawn from the jugular or cephalic vein every two weeks and up to 400 ml monthly.
Chickens
Give an 18–20-week-old hen 80 μg of antigen through the skin or muscle.
Boost with the same quantity every two weeks until a satisfactory antibody response is seen.
Start collecting eggs the day before your planned inoculation.
Custom Antibody Service vs. In-house Polyclonal Production
Based on your prior knowledge, the solution might seem straightforward, but it's a little more complicated and less cut-and-dry than it looks at first glance.
For instance, if you have in-house chromatography expertise, you could outsource injection and blood collection while handling both the front- and back-end purification of antigens and antibodies. This allows you to avoid the high expenditures associated with using an animal facility or the provider's core facilities while giving you complete control over the processes involved.
Small-scale enterprises that want to get into biotech can offer a great value proposition. Still, you may need to give them specific protocols and directions on handling samples, or you may need to send a professional to oversee the initial inoculations and bleeds.
Even though you employ the services of a full-service custom antibody and gene synthesis company, your provider may offer you a price reduction if you can do these procedures on their behalf.
Employing a custom antibody service can help guarantee the availability of materials for some laboratories in the short, medium, and long term.
Deciding Factors for the Use of Polyclonal Antibodies
Compared to mAbs, pAbs can be produced in a far more comprehensive range of host species, including common hosts like rabbits, goats, rats, and more specialized hosts like chickens, ducks, and sheep.
As a result, it's important to weigh many variables before settling on whether to create polyclonal, monoclonal, or recombinant antibodies (pAbs, mAbs, or rAbs) and which host species to employ.
If the pAb is going to be deployed for a long-term application that requires a large quantity of antibodies, a host with a greater body weight, like a donkey, goat, or sheep, and consequently a larger total blood volume, may be more suitable.
Although mouse polyclonal antibodies (pAbs) can be made quickly and affordably, they are not a good choice because they are challenging to maintain and reproduce consistently. When an animal is immunized against a disease that generally affects a heterologous species, such as a hamster, a more pronounced immune response may be observed.
Conclusion
Antibodies, particularly custom polyclonal antibody samples, are powerful tools for diagnosing and treating disease. They continue to be widely used as critical reagents in life science research. Given the complexity of the processes involved, it seems logical that most scientists use commercial peptide synthesis and antibody production services instead of developing their own.