Monoclonal versus Polyclonal Antibody Sequencing
Antibody reagents used in biomedical research and diagnostics assays fall into two large categories: monoclonals and polyclonals. For high-quality reagents, an antibody needs to demonstrate specific reactivity in its intended assay, and maintain consistency across reagent batches for multiple experiments.
Monoclonals are generally preferred for their high specificity and consistent performance. Polyclonals are necessary for assays that require the reagent to bind to multiple parts of an antigen or where binding needs to tolerant variations in the antigen. For example, most secondary antibodies are polyclonal; they bind to multiple parts of the constant region of assay-specific primary antibodies in ELISA, ICC/IF or IHC applications.
In this post we discuss different sources for monoclonal and polyclonal antibodies, and methods for monoclonal versus polyclonal antibody sequencing.
Production of Antibody Reagents
The complexity of these reagents depends on their production method. Advances in synthetic biology allow for the recombinant production of high-purity monoclonal antibodies. This process involves synthesizing the known antibody sequence as genes, transfecting and transforming mammalian cells to express and secrete the antibody. Critically, the antibody sequence must be known for this production method.
Before efficient recombinant expression systems, monoclonal antibody reagents primarily originated from immunizing animals, isolating B cells, fusing them to immortal cell lines, and screening them for secretion of antibodies with the desired function (See our resource page on hybridoma technology). While generally labeled as monoclonal antibodies, hybridoma technologies may actually contain multiple antibody variants in the same reagent (see Bradbury et al for more on non-monoclonality). Additionally, culture stocks can become contaminated with other antibodies, accrue mutations over time, or innately express multiple antibody chains.
Monoclonal antibodies are often derived from immortalized, antibody-secreting cell lines called hybridomas. Hybridomas are created by fusing myeloma cells with B cells. This technology is most commonly used with rodents.
The most complex are polyclonal antibody reagents, which are derived from serum of immunized large animals. Most of the polyclonal reagents are affinity purified using Protein G or Protein A to only remove serum proteins, leaving a complex mix of antibody clones. Only a small proportion of antibody may be contributing to binding antigens in the assay. While 2%-5% of total mouse IgG is often reported as antigen-specific, this percentage can vary depending on the antigen, binding strength requirements, and immunized species. For example, rabbits generate high-affinity and specific antibodies, so only a small proportion of a rabbit polyclonal would need to be antigen-specific to drive activity in intended assays.
Polyclonal antibodies are derived from a variety of donor species. Serum or plasma is typically purified to enrich for total IgG (e.g. using Protein A or Protein G), but may also undergo antigen purification to enrich for clones that bind the target of interest.
The challenge with polyclonal antibody reagents lies in ensuring reproducibility between experiments, as every immunized animal generates a unique polyclonal response. Pooling sera from immunized animals can help create a large and consistent reagent stock, but quality will change when the stock needs to be replenished. Often, the polyclonal reagent relies on a single large animal with excellent polyclonal activity in its serum. If that animal dies, then production is lost.
Monoclonal versus Polyclonal Antibodies: Ensuring Reagent Consistency
So the big question: how do you ensure the consistency of your reagents? A surefire approach is to digitize your antibody sequences, and rely on synthetic biology and recombinant expression to future-proofing production.
Valens and Griffin digitizes antibody protein sample-to-sequence, providing means to consistent and scalable production of monoclonal antibody. Benefits include: high purity (>99%), flexible production yields (50ug to > 1g), and immortal with stop and restart production from preserved DNA plasmids or stable cell lines.
Digitizing Monoclonal versus Polyclonal Antibodies
Mass spectrometry-based de novo sequencing has become the dominant approach for analyzing antibody proteins to determine their amino acid sequence(s).
Valens (monoclonal antibody protein sequencing) is best suited for determining a single heavy and light chain from an antibody protein sample. This is useful, for example, when a monoclonal antibody reagent’s producing hybridoma cells have been lost. Valens can digitize functional antibody protein by recovering the amino acid sequence and reviving the reagent through recombinant expression. Alternatively, in rare cases, recombinant production may go awry and an antibody sequence expected to have certain activity does not express as a well-performing antibody. Valens can help diagnose issues in the production system by independently assuring the expressed protein sequence matches intended active sequence.
Analyzing a monoclonal antibody sample with Valens requires enough material for chain-separated, four-enzyme digests, nano-liquid chromatography mass spectrometry (nano LC-MS/MS) data generation, and highly accurate, automated de novo sequence analysis. Time to results is 1-3 weeks, and depends on mass spectrometer bandwith. For more information, check out our blog post on methods for monoclonal antibody sequencing.
For polyclonals, Griffin addresses the more complicated task of assembling multiple and sometimes very similar, heavy and light chains. This is appropriate for discovering functional monoclonal antibodies from polyclonal reagents derived from a single or small cohort of immunized animals. In some cases, antibody material is handed off between labs or researchers, and information about antibody clonality and complexity is lost, even though the activity and validated applications (WB, ELISA, IHC/ICC, or IF) are known. When a sample’s clonality is unknown, Griffin is appropriate for profiling the its complexity and then recovering activity as monoclonal antibodies.
Analyzing a polyclonal antibody sample with Griffin requires enough material for extensive mass spectrometry analysis. First, the polyclonal antibody reagent needs to be purified to enrich for antigen-specific antibodies. Griffin discovers the abundant clones in sample; low abundance antigen-specific antibodies will be out-competed as candidates by more high abundance sequences. In the first analysis stage, the polyclonal sample is profiled for clonal complexity. The sample is analyzed by a shallow pass LC-MS/MS data generation and de novo sequence analysis to identify clones, specifically detecting CDR-H3 sequences. Assessment of clonality is typically achieved in two weeks. A sample with more than 20 dominant clones carries a risk of being too complex to assemble full-length antibody chains; at this point, a stopping point or a revisit of the antigen-specific purification strategy is recommended. Provided the purified sample has a small number of dominant clones, additional LC-MS/MS data generation is performed to more deeply recover de novo peptides sequences spanning the full-length of antibody sequences. Candidate assembled sequences are expressed and validated for activity, extending the timeline for deliverables to eight weeks. Check out a our talk “Griffin: Accessing functional antibodies from serum without B cells“.