Single domain antibodies for the detection of ricin using silicon photonic microring resonator arrays

Abstract

Ricin is a lethal protein toxin derived from the castor bean plant. Given its notorious history as a biowarfare agent and homicidal weapon, ricin has been classified as a category B bioterrorism agent. Current ricin detection methods based on immunoassays lack the required sensitivity and specificity for many homeland security surveillance applications. Importantly, many conventional antibody-based methodologies are unable to distinguish ricin from RCA 120, a nontoxic protein also found in the castor bean plant. Single domain antibodies (sdAbs), which are recombinantly derived from immunized llamas, are known to have high affinities for ricin A or B chains and low cross-reactivity with RCA 120. Herein, we demonstrate the use of silicon photonic microring resonators for antibody affinity profiling and one-step ricin detection at concentrations down to 300 pM using a 15 min, label-free assay format. These sdAbs were also simultaneously compared with a commercial anti-RCA IgG antibody in a multicapture agent, single target immunoassay using arrays of microrings, which allowed direct comparison of sensitivity and specificity. A selected sdAb was also found to exhibit outstanding specificity against another biotoxin, saporin, which has mechanism of action similar to ricin. Given the rapidity, scalability, and multiplexing capability of this silicon-based technology, this work represents a step toward using microring resonator arrays for the sensitive and specific detection of biowarfare agents.

Figure 1

Responses of a 3-capture agent sensor array exposed to 10 nM of a) RCA 120 and b) ricin. sdAb clones C8 and B4 both show greater selectivity for ricin compared to the goat anti-RCA IgG, which shows the largest response to RCA 120. Both sdAb clones show a significantly reduced response to RCA 120 while displaying good binding responses to ricin. In both sensing experiments, blank control rings show insignificant levels of non-specific binding.

Figure 2

Response of sdAb C8-modified microrings upon addition of 30 nM saporin, followed by addition of 30 nM ricin (red lines). Sensors were initially in PBST-BSA buffer and arrows indicate the times when analyte solutions were introduced. Dark gray lines indicate responses of thermal control rings.

Figure 3

Concentration-dependent binding response of ricin as a function of target concentration. Each measurement was made eight times redundantly on the same sensor chip, functionalized identically with sdAb C8. For the sake of clarity, data from four rings is presented. Following the establishment of an initial baseline by equilibrating with PBST-BSA running buffer, ricin-containing solutions were flowed across the array (staring at t = 5 min) and persisting for a total of 10 minutes.

Figure 4

Calibration curve illustrating the concentration-dependent response of sdAb C8 functionalized microrings to solutions of various concentrations. Real-time binding curves were obtained (as in Figure 3) for samples prior to the analysis of a prepared solution containing an unknown amount of ricin. The sensor response for the unknown solution was then compared against the standard calibration curve, allowing for quantitative detection. Error bars represent the 95% confidence interval from n=8 measurements.