Ultrasensitive Detection of Ricin Toxin in Multiple Sample Matrixes Using Single-Domain Antibodies

Abstract

Ricin is an extremely potent ribosomal inactivating protein listed as a Category B select agent. Although ricin intoxication is not transmittable from person to person, even a single ricin molecule can lead to cell necrosis because it inactivates 1500 ribosomes/min. Since there is currently no vaccine or therapeutic treatment for ricin intoxication, ultrasensitive analytical assays capable of detecting ricin in a variety of matrixes are urgently needed to limit exposure to individuals as well as communities. In this paper, we present the development and application of a single-molecule array (Simoa) for the detection of ricin toxin in human urine and serum. Single-domain antibodies (sdAbs), among the smallest engineered binding fragments, were chemically coupled to the surface of paramagnetic beads for the sensitive detection of ricin toxin. The Simoa was able to detect ricin at levels of 10 fg/mL, 100 fg/mL, and 1 pg/mL in buffer, urine and serum, respectively, in a fraction of the assay time need using immuno-polymerase chain reaction (IPCR). Using a fully automated state-of-the-art platform, the Simoa HD-1 analyzer, the assay time was reduced to 64 min.

Characterization of Ricin and R. communis Agglutinin Reference Materials

“…Various ELISAs have been developed by different groups [ 70 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 ]. Only a few of them are able to quantify ricin with detection limits down to a few pg/mL with limits of detection of 10 fg/mL [ 84 ], 2 pg/mL [ 70 ], 8 pg/mL [ 85 ], and 40 pg/mL [ 86 ]. Several ricin-specific ELISAs developed in different laboratories have been evaluated in a common ricin proficiency test panel organized in the framework of the EQuATox project, and results are presented in different manuscripts in this special issue of Toxins [ 42 , 87 ].…”

Single‐Molecule Sensors: Challenges and Opportunities for Quantitative Analysis

“…This is achieved by using antibodymodified magnetic beads that capture the analyte in solution, followed by binding of asecondary antibody with biotin labels to which streptavidin-b-galactosidase conjugate is attached. TheW alt research group has demonstrated the power of this technique for awide range of assays such as the cancer biomarker prostate-specific antigen, [43] the toxin ricin, [44] cytokines, [45] dengue fever, [46] and bacterial genomic DNA. We refer to this assay format as wide-field sampling/near-field detection measurements.T he detection limits achieved are exquisite:f or biotin-streptavidin it was 350 zm, which is equivalent to 10-20 enzyme labels in a100 mL sample.…”

Biological toxins of potential bioterrorism risk: Current status of detection and identification technology

“…For screening purposes, mostly sandwich enzymelinked immunosorbent assays (ELISAs) are being used, detecting the presence of the protein and reaching detection limits from ng/mL to fg/mL depending on the antibodies and the read-out system used [19], where ultrasensitive detection has been obtained by using the proprietary single molecule array technology [20]. A less sensitive, but much faster approach is on-site detection assays such as lateral flow assays (LFA) or biosensor approaches based on different principles and read-outs that usually reach detection limits in the ng/mL range within about 30 min [15].…”