FAQs – Polyclonal antibodies

Polyclonal antibodies are valued for their enhanced sensitivity and broad reactivity. These properties make them useful and irreplaceable reagents for many immunoassays which are recurrently used to detect and/or capture low abundance or complex markers in research and diagnostic applications. Moreover, polyclonal antiserum is still one of the most effective ways to counter the adverse effects of envenoming (bites and stings from venomous animals). On this page, we provide answers to the most frequently asked question about the process of polyclonal antibody production and describe ProteoGenix’s platform and unique guarantees.

ProteoGenix’s polyclonal antibody production platform

What types of antigens can you use for polyclonal antibody production?

We have the capacity to produce all types of antigens at our facilities including peptides, proteins, DNA, small molecules, and cells overexpressing the target antigen. We can also produce polyclonal antibodies using customer-provided antigens.

What host species can you use for polyclonal antibody production?

We can produce polyclonal antibodies in many different species including rabbits, mice, goats, sheep, camelids, and chickens, among others. Although we recommend our clients to use rabbit or camelids for their polyclonal antibody production projects given the robustness of rabbit’s immune response and the advantageous properties of camelid heavy-chain antibodies (HCAbs).

How do you measure antibody titers?

To monitor the response to immunization, we recurrently take samples from hyperimmunized hosts to measure their antibody titers. Conventionally, this is done using ELISA, but alternatively, our clients may request antibody titer verification by Western Blot as well.

Do you comply with ethical standards and animal safety guidelines?

At ProteoGenix, we are committed to maintaining the highest standards of the ethical use of animals in research. As a result of our efforts to minimize animal use and ensure the highest quality standards (housing and management conditions, veterinary care, and facilities), we have earned AAALAC accreditation, one of the most reputable independent organisms that regularly evaluates our facilities.

What are the unique advantages of your polyclonal antibody production service?

The possibility to generate polyclonal antibodies in any species (except human) and using any antigen are the strongest advantages of our service. Additionally, all our service packages come with an antibody titer guarantee.

What types of guarantees do you offer?

Our polyclonal antibody production service packages come with a unique antibody titer guarantee (1/64000 in ELISA). Moreover, when using protein antigens, we offer our clients the possibility to confirm antibody titers by Western Blot. All these guarantees allied to the most competitive price on the market make our services the best choice for your polyclonal antibody generation process.

Polyclonal antibodies – definition and applications

What are polyclonal antibodies?

Polyclonal antibodies are a mixture of monoclonal antibodies that originated in different B cell clones. Consequently, these antibodies display different epitope-specificity and binding affinity. This property makes them highly sensitive to low abundance markers and highly effective at tackling complex targets.

What are the main applications of polyclonal antibodies?

Polyclonal antibodies are widely used for a multitude of assays including Western Blot, ELISA, Immunoprecipitation, Immunocyto- and Immunohistochemistry, among others. They can be used for the direct detection or capture of specific targets or indirect detection in tandem with monoclonal antibodies.

When polyclonal and monoclonal antibody pairs are used, they need to be generated in phylogenetically distant host species to avoid cross-reactivity. The monoclonal antibody is designed to target a specific antigen/epitope (primary antibody), while the polyclonal antibodies are designed to target the primary antibody (secondary antibody). The coupling of the two types of antibodies maximizes both the specificity and sensitivity of an assay, ideal for diagnostics, research, and food monitoring applications.

From a therapeutic point of view, polyclonal antibodies are still one of the best ways to treat acute reactions resulting from venomous stings and bites.

To learn more about the applications of polyclonal antibodies, read the complete article: What are polyclonal antibodies used for?

How are polyclonal antibodies classified?

Given their diverse nature, polyclonal antibodies are often classified according to the host species and antigen used for their generation. These antibodies are generally produced in rabbits, goats, and sheep. Rabbit polyclonal antibodies are particularly useful because the rabbit’s immune system can generate high-affinity antibodies in higher abundance than other host species.

Polyclonal antibodies are also named according to this classification, in this way, anti-mouse rabbit polyclonal antibodies are pAbs designed to bind to murine IgG and produced in a rabbit host.

What are the advantages of using polyclonal antibodies?

The benefits of using polyclonal antibodies are numerous. Due to their enhanced sensitivity in comparison to their monoclonal counterparts, polyclonal antibodies can serve to detect low abundance targets, tackle multiple epitopes or antigens in a single sample, or capture targets in complex samples.

Polyclonal antibodies are also much quicker, cheaper, and less labor-intensive to produce than monoclonal antibodies. Plus, they display enhanced stability at extreme pH values and temperatures.

To learn more about the advantages of these reagents, read the complete article: What are the advantages of using polyclonal antibodies?

What are the limitations of using polyclonal antibodies?

Despite the enhanced sensitivity of these reagents, current polyclonal antibody production methods are hard to scale-up. Moreover, these reagents suffer from batch-to-batch variability, making standardization difficult to achieve. This variability stems from the diversity of the host-to-host immune response (i.e. depends on the age of the host and its natural antibody repertoire). Even when the same host species, antigen, and immunization protocols are used, each host will produce a mixture of antibodies with different epitope-specificity and binding affinity.

Proper purification (i.e. antigen-based purification) of polyclonal antisera is one of the most widely used strategies to overcome this limitation. Despite considerably reducing the antibody production yield, antigen-based purification can greatly enhance the quality of the reagent by reducing off-target binding and background noise. Another way to reduce the impact of batch-to-batch variability is rigorous standardization. This can be done by including adequate controls and standards in every assay.

Polyclonal antibody production technologies

How are polyclonal antibodies produced?

The production of polyclonal antibodies involves the immunization of a host with the desired antigen.

Immunization protocols depend on the nature of the antigen, as some antigens can generate a robust immune response quicker than others. However, conventional immunization schedules involve a first injection followed by one or several boosters – until antibody titers measured by ELISA and/or Western Blot stabilize. At this point, hosts are bled and the serum is harvested by centrifugation.

How are polyclonal antibodies purified?

Crude antisera may be useful for certain applications, but, conventionally, polyclonal antibodies are submitted to an additional purification step before their use. Purification can be immunoglobulin- or antigen-specific, depending on the desired quality and yield of the reagent.

In the former, IgG immunoglobulins can be easily recovered by protein A or protein G purification. These proteins, produced by Staphylococcus aureus and Streptococcus spp., respectively, can interact with the Fc region with high affinity allowing the straightforward purification of these molecules. However, only 10% of this antiserum contains antibodies with a high affinity towards the desired target.

In this case, researchers may choose to enrich polyclonal preparations by using the target antigen to further purify the antisera. Antigen-specific purification is known to dramatically reduce production yields; however, it greatly enhances the specificity of these reagents which is useful for certain applications.

How to choose the best host species for polyclonal antibody production?

The most commonly used host species for polyclonal antibody production are rabbits, chickens, goats, and sheep. Rabbits are particularly useful hosts given the high-specificity of their immune response. In general, these hosts generate a polyclonal response that is richer in high-affinity and antigen-specific antibodies than found in other mammalian hosts. Thus, higher yields and quality are expected when using rabbits for polyclonal antibody production.

Similarly, chickens are valuable hosts for polyclonal antisera production. Chickens transfer IgY antibodies from their circulation into the egg yolk. These antibodies are then subsequently absorbed by the embryo. The fact that hens transfer their antibodies to eggs makes the harvesting procedure less invasive in comparison to other host species. Moreover, egg yolk is richer in antibodies than conventional antisera. The only challenge remains in the process of purification of IgY antibodies from egg yolk, a process that has been greatly improved by the introduction of ionic liquids for cost-effective purification.

What types of antigens can be used for polyclonal antibody production?

Antigen design and production are two of the most important steps of the polyclonal antibody production process. This process should take into account the desired application of the antigen, sample type, and sample preparation protocol. For instance, in Western Blot, antigens are denatured before the detection step; in this case, polyclonal antibodies for WB should be preferably generated using linear peptides. In contrast, if polyclonal antibodies are intended for Flow Cytometry applications, it is necessary to take into account the native structure of the target. In this case, polyclonal antibodies should be generated using the recombinant protein or by genetic immunization (for hard-to-express or transmembrane proteins).

How long does it take to produce polyclonal antibodies?

The timelines for polyclonal antibody generation depend on the nature of the antigen (peptides and DNA are quicker to produce than proteins with complex structures) and the immunogenicity of the target. In general, polyclonal antibody production takes up to 9-15 weeks. Exceptionally, some highly immunogenic proteins may generate a strong immune response in less than a month. Serum purification is a straightforward procedure that, depending on the quantity, may take less than a week.