Antibody Characterisation: Ranking, Binning and Mapping

Three questions, in the order you should ask them#

Given a panel of forty antibodies against one antigen, there are three things you want to know, and they get harder and more expensive in a fixed order: which bind most tightly (ranking), which compete with each other (binning), and where exactly each one binds (mapping).

1. Affinity ranking orders the panel. Cheap, fast, and — importantly — more reliable than the absolute constants it is derived from.
2. Epitope binning groups the panel into competition classes. Moderately expensive; scales as n², which is why imaging instruments dominate here.
3. Epitope mapping localises the binding site on the antigen. Expensive, and requires reagents — peptide arrays or mutant panels — that are a project in themselves.

Affinity ranking, and why it is trustworthy#

Ranking is more robust than absolute affinity measurement because systematic errors act in the same direction on every member of a panel measured under identical conditions. Surface activity, small concentration inaccuracies and partial transport limitation all shift the whole set, so the ordering survives even where the numbers do not. The cross-laboratory benchmark studies show exactly this pattern (Katsamba et al., 2006; Papalia et al., 2006).

The format matters, though, and here is where valency bites.

Epitope binning#

Binning asks a simple question pairwise: with antibody A already saturating the antigen, can antibody B still bind? If not, they compete — overlapping epitopes, or steric or allosteric interference — and belong in the same bin.

Three formats are in common use, and each has a characteristic weakness.

• Classical sandwich. Antibody 1 on the chip captures antigen; antibody 2 is injected (Abdiche et al., 2009). Straightforward, but requires that antibody 1 captures antigen well, which not all of a panel will.
• Premix / in-solution. Antigen is pre-incubated with a saturating excess of antibody 1, then the complex is injected over antibody 2 on the chip. Avoids the capture requirement and is generally the most robust (Abdiche et al., 2009).
• Tandem. Antigen is immobilised; antibody 1 then antibody 2 are injected sequentially without regeneration. Fast and sample-efficient, but antibody 1 must genuinely saturate, which must be demonstrated rather than assumed.

Abdiche and colleagues compared blocking assays across three biosensor platforms and documented how format choice and platform interact (Abdiche et al., 2009) — worth reading before committing a panel to any one approach.

Epitope mapping#

Two approaches, with quite different reach.

Overlapping peptide arrays

Synthesise peptides tiling the antigen sequence with overlaps, immobilise them, and test antibody binding (Löfås et al., 1995). Fast and systematic, and works beautifully for linear epitopes.

The limitation is fundamental rather than technical: most antibodies against folded proteins recognise conformational epitopes assembled from residues distant in sequence, which no linear peptide reproduces. A negative peptide-array result is therefore close to uninformative, while a positive one is strong evidence. Interpreting the absence of binding as absence of an epitope is the standard error here.

Mutational scanning

Substitute surface residues — alanine scanning is the classic form — and look for substitutions that abolish binding. This reaches conformational epitopes, which is its whole point.

Two controls are non-negotiable, and their absence invalidates the result:

• Fold verification. A mutation that destabilises the protein abolishes binding for every antibody, which looks exactly like a shared epitope (Abdiche et al., 2014). Confirm each mutant is folded — a second antibody with a known distant epitope is the cheapest way.
• Expression and concentration. A mutant expressed at a tenth the level gives a tenth the response, which reads as reduced binding. Normalise to a measured active concentration (Sigmundsson et al., 2002).

Sources cited on this page

Listed alphabetically. Each badge records whether the bibliographic record was confirmed against Crossref. unverified marks a real, deliberately chosen source whose volume and page numbers we have not yet machine-checked — it is not a comment on the science.

• Abdiche et al., 2009Y. N. Abdiche, D. S. Malashock, A. Pinkerton, J. Pons (2009). Exploring blocking assays using Octet, ProteOn, and Biacore biosensors. Analytical Biochemistry 386, 172–180. unverified
  Epitope binning across three platforms.
• Abdiche et al., 2014Y. N. Abdiche, K. C. Lindquist, A. Pinkerton, J. Pons, A. Rajpal (2014). Expanding the ProteOn XPR36 biosensor into a 36 × 36 array format provides a fast and economical means of determining antibody affinities and epitope bins. PLoS ONE 9, e92451. doi:10.1371/journal.pone.0092451 unverified
  High-throughput binning of a large antibody panel; the source for the n² scaling argument.
• Canziani et al., 2004G. A. Canziani, S. Klakamp, D. G. Myszka (2004). Kinetic screening of antibodies from crude hybridoma samples using Biacore. Analytical Biochemistry 325, 301–307. doi:10.1016/j.ab.2003.11.004 unverified
  Ranking antibodies directly from crude supernatant, without purification.
• Katsamba et al., 2006P. S. Katsamba, I. Navratilova, M. Calderon-Cacia, et al. (2006). Kinetic analysis of a high-affinity antibody/antigen interaction performed by multiple Biacore users. Analytical Biochemistry 352, 208–221. unverified
• Löfås et al., 1995S. Löfås, M. Malmqvist, I. Rönnberg, E. Stenberg, B. Liedberg, I. Lundström (1995). Methods for site controlled coupling to carboxymethyldextran surfaces in surface plasmon resonance sensors. Biosensors and Bioelectronics 10, 813–822. doi:10.1016/0956-5663(95)99220-F verified
  Thiol, aldehyde and other orientation-controlling couplings beyond plain amine chemistry.
• Nieba et al., 1996L. Nieba, A. Krebber, A. Plückthun (1996). Competition BIAcore for measuring true affinities: large differences from values determined from binding kinetics. Analytical Biochemistry 234, 155–165. doi:10.1006/abio.1996.0067 unverified
  A direct demonstration that surface-measured kinetic constants can diverge substantially from solution affinities, and a solution-competition format that avoids the problem.
• Papalia et al., 2006G. A. Papalia, S. Leavitt, M. A. Bynum, et al. (2006). Comparative analysis of 10 small molecules binding to carbonic anhydrase II by different investigators using Biacore technology. Analytical Biochemistry 359, 94–105. unverified
  A benchmark study in which many labs measured the same interactions; the spread between labs is the honest measure of SPR reproducibility.
• Sigmundsson et al., 2002K. Sigmundsson, G. Másson, R. Rice, N. Beauchemin, B. Öbrink (2002). Determination of active concentrations and association and dissociation rate constants of interacting biomolecules: an analytical solution to the theory for kinetic and mass transport limitations in biosensor technology and its experimental verification. Biochemistry 41, 8263–8276. doi:10.1021/bi020099h unverified
  Analytical treatment of combined kinetic and transport limitation, with experimental validation.