Limits of Detection (and Credibility)

Methods of Effective Conjugation of Antigens to Nanoparticles as Non-Inflammatory Vaccine Carriers

Nanoparticles are promising carriers for recombinant vaccine antigens, although the physical properties that make some particles good vaccines and others not are still unknown. In this work[1], the authors conjugate model ovalbumin (OVA) antigen to polystyrene nanoparticles for use as a vaccine delivery system. They immunize mice and examine the in vivo responses in order to elucidate an immunological mechanism, however their poor statistical analysis of the resulting data leaves little in the way of conclusions.

Before I get to that, another issue I had with their data was an odd regression curve used to determine concentration from absorbance (Figure 3). In the adjacent figure, the authors used what appear to be serial dilutions of nanoparticles to generate a regression between optical density (OD) at 248 nm and the number of nanoparticles per mL. While their R² value is especially high, as would be expected for a serial dilution, there are no error bars on the points or indications of the number of replicates performed. More seriously, however, is the regression equation they use. In drawing a correlation between the number of nanoparticles in units of 10¹³/mL and the optical density, both numbers with one significant figure, it seemed odd that their regression equation would have an intercept with 4 significant figures, leading me to question the value of their equation and its corresponding R² value. Additionally, the fact that serial dilutions of a solution would have linearly decreasing absorbance values is not necessarily novel or informative, and this figure could have been put in the supplemental information.

More statistically egregious, however, is their table of multiplexed, cytokine-bead array results, expressed as pg/mL (Tables 3 and 4):

Looking only at the first column, one wonders what the difference between 1.4 ± 1.9, or 0 ± 0.21 and “Not detectable” is. Given the absurd concentrations seen in naïve serum and serum from mice injected with NPs, it’s clear that the data is very widely skewed, in which case actually seeing the data points in a graph would be much more helpful. It is also suspicious that these values were the only data in the paper displayed in tabular form, whereas all others were either histograms or bar graphs of some sort. No mention of the number of mice used for this analysis was found in the figure caption, or the results or methods sections either. Not only are their conclusions on immunological mechanism weak, but their poor statistical analysis calls the significance of other data they present into question as well.

[1] Xiang SD, Wilson K, Day S, Fuchsberger M, Plebanski M. Methods of effective conjugation of antigens to nanoparticles as non-inflammatory vaccine carriers. Methods 2013;60:232-41.