Figure 5

Luminex Sandwich assay. To measure the utility of sdAbs as tracer antibody, luminex sandwich assays were conducted. Purified antibodies for each tested target along with Ni-SA-PE were mixed with toxins captured by microspheres coated with the capture antibodies. Mab-Ric 03, llama sdAb Ric E7, and llama sdAb CT-C11 (as negative control) were used to capture ricin, which was at concentrations of 0.1–10 μg/mL. Ricin binder, P4RA7-1, along with Ni-SA-PE were used as a tracer and were able to detect ricin at 1 μg/mL captured by Mab-Ric 03 and llama sdAb Ric E7, but not negative control (A). Llama anti-BoNT, Lx-Rab-anti-BoNT/A and Lx-Rab anti-SEB (negative control) were used to capture BoNT/A complex toxoid, which was at concentrations between 0.1 to 200 ng/mL. P4BF7-3 and Ni-SA-PE were used as tracers to detect BoNT/A complex at a minimum concentration of 40 ng/mL captured by Llama anti-BoNT, Lx-Rab-anti-BoNT/A, but not Lx-Rab anti-SEB (B). The same tracers were used to detect BoNT/B complex toxoid at a concentration of 40 ng/mL captured by only Llama anti-BoNT, but not by Lx-Rab-anti-BoNT/A or Lx-Rab anti-SEB (C). Mouse monoclonal antibody against SEB, Mab2b3a, and Rab anti-Ricin (negative control) were used to capture SEB, at concentrations range from 0.01 to 100 ng/mL. P1SD3-3 and Ni-SA-PE were used as tracer to detect the SEB at a minimum concentration of 10 ng/mL, captured by Mab2b3a, but not by Rab anti-Ricin (D).