Polyclonal Antibody Production – What’s Involved and When to Outsource?
Polyclonal antibodies are among the most widely used tools for scientific research. This article explains how polyclonal antibodies are made and suggests factors to consider when selecting a partner for custom polyclonal antibody production.
What is a polyclonal antibody?
When you purchase a vial of polyclonal antibody, what you receive is a heterogeneous mixture of antibodies that recognize different epitopes on a protein antigen. In contrast, a vial of monoclonal antibody contains many copies of a single antibody, all targeting the same epitope. Multi-epitope recognition gives polyclonal antibodies several advantages, not least high sensitivity to the target protein. However, it can also result in increased background noise for applications such as ELISA and western blot due to off-target binding. The advantages and disadvantages of polyclonal and monoclonal antibodies are summarized in Table 1.
Table 1: Advantages and disadvantages of polyclonal and monoclonal antibodies.
| Advantages | Disadvantages | |
|---|---|---|
| Polyclonal antibodies | ✔️ Multi-epitope recognition is useful for detecting proteins in low abundance, proteins whose conformational state may change, and proteins whose epitopes may be masked by crosslinking ✔️ May work in a broader range of applications than monoclonal antibodies ✔️ Relatively quick and inexpensive to produce | ✖️ Potential for cross-reactivity and increased background noise ✖️ Lot-to-lot variability ✖️ Limited supply, corresponding to the lifetime of the production host |
| Monoclonal antibodies | ✔️ High specificity ✔️ Lot-to-lot consistency offers more reproducible experimental results ✔️ Unlimited supply | ✖️ Longer, more costly production process than polyclonal antibodies ✖️ Risk of genetic drift over time |
How are polyclonal antibodies produced?
Polyclonal antibodies are produced in rabbits, as well as in larger mammals such as goats, donkeys, and sheep. The production process involves a series of steps, as follows:
- Antigen preparation
Antigens for polyclonal antibody production are typically purified endogenous proteins, recombinant proteins, or haptens. The term hapten describes a peptide or other small molecule that can only stimulate antibody production when coupled to a larger carrier protein, such as keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA). Often, the antigen will be mixed with an adjuvant to enhance the immune response. Common examples include Complete Freund’s Adjuvant and various aluminum salts, collectively referred to as alums.
- Immunization
Immunization schedules for polyclonal antibody production vary with the antigen and host species, but generally take place over a period of several weeks or months. Following initial immunization, a series of boosters is administered, with test bleeds being taken in between doses. For example, after initial immunization on day 0, a booster might be administered on day 14 and a blood sample taken on day 21, followed by a further booster on day 28, if necessary. When Complete Freund’s Adjuvant is used for the initial immunization, it is common for boosters to be prepared in Incomplete Freund’s Adjuvant, which is less likely to elicit a potentially harmful inflammatory response.
- Serum testing
The purpose of collecting test bleeds is to determine the antibody titer, which reflects the strength of the immune response. After allowing the blood to clot, usually by leaving it undisturbed at room temperature or 37oC for around 30 minutes, the samples are centrifuged and the serum supernatant is collected. This is typically tested with an ELISA, whereby the target antigen is captured on the surface of the microplate wells and a serial dilution of the serum is added. A labeled secondary antibody is then introduced to provide a measurable readout.
- Antibody purification
Once an acceptable antibody titer is reached, a larger blood sample is collected for antibody purification. This is generally achieved with antigen-specific affinity chromatography, which serves to remove any antibodies that might otherwise cause unwanted background signal in downstream applications. It is perhaps worth noting that monoclonal antibodies do not routinely require antigen-specific purification, but are instead purified using resins functionalized with Protein A, G or A/G, depending on the antibody species and isotype/subtype.
- Antibody characterization
Before a polyclonal antibody can be used for scientific research, it must be carefully characterized to confirm its specificity and sensitivity for the target antigen. The types of tests that might be performed include ELISA, western blot, and immunohistochemistry, as well as tests for evaluating potential cross-reactivities. In all cases, the use of relevant controls is essential to verify results, and it is recommended to compare the data to that generated with another antibody for the same target, if possible.
Factors to consider for custom polyclonal antibody production
Although a vast array of polyclonal antibodies is available commercially, there may be times when you need to consider a custom production. For example, you may be working with a newly discovered protein or be using an off-the-shelf product that simply doesn’t deliver on what is required. When selecting a partner for custom antibody production, factors to consider include the likelihood for success, the predicted timescale, and the validation strategies used to ensure optimal results. It is also important to understand the prospective partner’s commitment to animal welfare, as well as whether they can offer long-term support should your needs change.
How can we help?
LubioScience represents some of the most trusted brands in life science research and works closely with our partners to offer a comprehensive selection of custom antibody production services. Contact us today to discuss how we can help with generating high-quality antibodies for your project.
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