Reporting, Reviewing and Reading Critically

The uncomfortable background#

Long-running surveys of the published biosensor literature have documented, year after year, that a large fraction of papers omit the information a reader would need to judge them: no raw sensorgrams shown, no fit overlaid on data, no replicates, no statement of surface density or referencing scheme (Rich & Myszka, 2010).

This is worth stating not to be discouraging but because it changes how you should read. When you encounter a K_D in a paper with no sensorgram, the correct posture is not scepticism about the authors but recognition that you have been given a conclusion without the evidence for it. Whether it is right is unknowable from what is printed.

The reporting checklist#

Everything here is cheap to report and expensive to reconstruct afterwards.

Materials and surface

• Instrument model and analysis software, including version.
• Sensor chip type and immobilisation chemistry.
• Immobilised ligand level, in RU — not "the ligand was immobilised".
• Reference surface preparation, described specifically.
• Running buffer composition in full, including surfactant and any DMSO.
• Temperature.

The experiment

• Analyte concentration series, with actual concentrations and how they were determined — A₂₈₀, BCA, or active concentration by CFCA (Sigmundsson et al., 2002).
• Association and dissociation times.
• Flow rate.
• Regeneration conditions, and evidence that they did not degrade the surface.
• Number of independent repeats, distinguished clearly from replicate injections within one run.

The analysis

• Referencing scheme — state explicitly whether double referencing was used.
• Interaction model, and why that model rather than 1:1.
• Whether R_max was fitted globally or locally, and why.
• Which regions of the curve were excluded, and on what grounds.
• Reported constants with uncertainties from independent repeats, not from the fit’s standard errors (Katsamba et al., 2006).

The figures

• Raw sensorgrams, with the fit overlaid. This is the single most important item on the list.
• Residual plots, at least in supplementary material.
• A replicate overlay demonstrating stability across the run.
• For steady-state analysis, the R_eq-versus-concentration plot with the fitted isotherm and the individual points visible.

Reading and refereeing: what to look for first#

1. Is there a sensorgram at all? If not, the kinetic claims are unsupported. This is the fastest triage available.
2. Is the fit overlaid on the data? A fit shown alone is a picture of the model, not evidence about the sample.
3. Do the shapes look right? Linear association or a dragging dissociation means transport; a biphasic decay means avidity or heterogeneity. You can see both by eye (reading sensorgrams).
4. Is k_a physically plausible? Above ~10⁷ M⁻¹ s⁻¹ for proteins, ask what went wrong (Squires et al., 2008).
5. Was the analyte multivalent? An IgG over an antigen surface reported as an intrinsic affinity is the most common single error in the antibody literature (Nieba et al., 1996).
6. Does the concentration range bracket the K_D? A steady-state K_D derived entirely from concentrations below K_D is not measured, it is extrapolated.
7. Was the surface stable? Any evidence at all — a replicate overlay, a cycle-number plot — or none?
8. Is a complex model used, and is it justified experimentally? A two-state model with no supporting evidence is a fitted curve, not a mechanism.

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.

• Johnsson et al., 1995B. Johnsson, S. Löfås, G. Lindquist, Å. Edström, R.-M. Müller Hillgren, A. Hansson (1995). Comparison of methods for immobilization to carboxymethyl dextran sensor surfaces by analysis of the specific activity of monoclonal antibodies. Journal of Molecular Recognition 8, 125–131. doi:10.1002/jmr.300080122 unverified
  Measures surface activity across coupling chemistries on the same antibody — the primary source for the claim that amine coupling inactivates a substantial fraction of ligand.
• 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
• 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.
• Rich & Myszka, 2010R. L. Rich, D. G. Myszka (2010). Grading the commercial optical biosensor literature — Class of 2008: ‘The Mighty Binders’. Journal of Molecular Recognition 23, 1–64. unverifiedreview
  Cited only where the claim is about the state of the published literature. The reporting checklist used on this site derives from this series.
• 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.
• Squires et al., 2008T. M. Squires, R. J. Messinger, S. R. Manalis (2008). Making it stick: convection, reaction and diffusion in surface-based biosensors. Nature Biotechnology 26, 417–426. doi:10.1038/nbt1388 unverified
  The general transport analysis for any surface-based sensor, in dimensionless form. The clearest statement of when a measured rate is chemistry and when it is delivery.