POLYCLONAL ANTIBODY GENERATION

Polyclonal antibody production at ProteoGenix: FAQ

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.

How to optimize the polyclonal antibody production process?

Antigen Design And Production

Achieving high titers of target-specific antibodies depends on the antigen’s capacity for eliciting a strong immune response. For this reason, choosing a suitable antigen for immunization remains one of the most important steps of the polyclonal antibody production process

Several antigens may be used for polyclonal antibody generation including:

• Proteins – proteins are the most conventional antigens for antibody generation. They ensure polyclonal antibodies recognize different relevant epitopes naturally exposed in the native conformation of the protein.
• Peptides – using peptides for immunization is useful when developing antibodies for linear epitopes (important in Western Blot applications) or when developing antibodies against a specific epitope (increases assay specificity). Since most peptides aren’t immunogenic, adjuvants are typically used to enhance the immune response.
• DNA – genetic immunization is reserved for special cases. For instance, when the target protein is hard to produce, unstable or contains complex transmembrane domains, genetic immunization may be a suitable alternative.

Other antigens may be used for immunization such as small molecules or even whole cells (native or recombinant); however, these projects require the development and testing of custom immunization solutions.

Animal Immunization

Our standard immunization protocol starts at:

• 51 days for anti-protein polyclonal antibody production
• 70 days for anti-peptide polyclonal antibody production

Both protocols can be extended if guaranteed antibody titers are not reached. Typically, it is better to immunize animals with a lower quantity of antigen and for longer periods, rather than using higher quantities of antigen and shorter immunization times.

Polyclonal Antibody Purification

Polyclonal antibodies are typically harvested by bleeding the hosts after desired antibody titers are reached. The cellular fraction and the antibody-enriched serum can be separated by centrifugation resulting in a crude polyclonal antibody solution.

Crude preparations are useful for many applications. However, for enhanced sensitivity and reduced off-target binding, these preparations should be purified. Polyclonal antibody purification can be carried out by:

• Protein A or G purification – these proteins are produced by Staphylococcus aureus and Streptococcus spp., respectively, and they can bind the Fc fragment of antibodies with high affinity. Protein A/G purification allows the straightforward separation of immunoglobulins from all other serum components. However, unspecific antibodies with a low affinity towards the target are not eliminated with this type of purification.
• Antigen-specific purification – using affinity chromatography to recover polyclonal antibodies with high affinity towards a specific antigen is a widely used process of polyclonal antibody purification. It ensures only the antibodies with the highest affinity are recovered, reducing off-target binding and, consequently, reducing background noise in immunoassays.

Need advice for your custom polyclonal antibody production? Please feel free to contact your dedicated account manager!

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.

Polyclonal antibodies

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.

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 main advantages and uses of polyclonal antibodies?

Polyclonal antibodies are a mixture of monoclonal antibodies with distinct epitope-specificity and binding affinity. They are produced by immunizing animal hosts with a specific target (protein, peptide, DNA, etc.) and harvested by separation and purification of their serum after the development of a strong immune response.

Unlike monoclonal antibodies, prized for their high specificity and selectivity, polyclonal antibodies are known for their enhanced sensitivity. This property makes them invaluable tools for multiple applications including research (basic, medical, etc.), therapy, and diagnostics. They are particularly advantageous reagents to detect low abundance markers, toxins, among other substances. The detection of these rare markers is extremely useful for the design of early diagnostic tools, food monitoring applications, and for capturing rare targets and purifying them before further analysis.

From a therapeutic perspective, polyclonal antisera are invaluable to treat complex acute conditions like snakebite envenoming. Venoms cause acute reactions and are composed of a complex mixture of different proteins with key roles in pathogenesis. In this way, successful antivenom therapies need to be able to tackle several antigens and epitopes present in a single venom to block the pathogenic pathways and avoid adverse reactions.

From a technical point of view, polyclonal antibodies are cheaper to produce than their monoclonal counterparts. Plus, the timelines for polyclonal antibody production are significantly shorter than the lead time of monoclonal antibody production. In contrast, their most notable limitations are the difficulty in scaling up the process and high batch-to-batch variability. The impact of the latter can be mitigated by including proper controls (positive and negative) and standards in every assay.

Overcoming scale-up hurdles is more challenging, but recent breakthroughs have provided interesting solutions. One of the most interesting solutions is the purification of IgY antibodies present in egg yolk using ionic liquids. Due to the non-invasive methods of antibody harvesting and the cost-effective new purification methods, these molecules may soon gain ground over other polyclonal antibodies for a multitude of applications.

What are the advantages of pairing monoclonal and polyclonal antibodies?

Polyclonal antibodies can be used by themselves or in tandem with their monoclonal counterparts in different assays. The most common application of polyclonal and monoclonal antibody pairs is the enzyme-linked immunosorbent assay (ELISA). In ELISA, monoclonal antibodies (also called primary antibodies) are typically used to bind a specific target, while polyclonal antibodies (secondary antibodies) are used to bind the primary antibodies.

In this configuration, the primary antibodies are “naked” (no tags) while the secondary antibodies carry the enzymatic tag. This format allows the amplification of the detection signal, lowering detection thresholds considerably. The signal amplification occurs because, while primary antibody binds to a single epitope on the target molecule, secondary antibodies bind to multiple regions of the primary antibodies. In this way, multiple secondary antibodies conjugated with an antibody can coat the surface of the primary antibody, dramatically enhancing the detection signal.

The enhanced sensitivity of these molecules comes with its own set of specific hurdles. For instance, polyclonal antibodies are notorious for their propensity for off-target binding, which may lead to false-positive results. To overcome this hurdle, polyclonal antibodies must be submitted to stricter purification processes (e.g. by performing antigen-specific antibody purification). Additionally, the inclusion of controls and standards can help researchers correctly identify thresholds of detection.