A battery of assays for chasing ricin and its activity

Ricin, a type-2 Ribosome-Inactivating Protein (RIP), is a dangerous biotoxin derived from castor plant seeds. It is classified as a Schedule 1 agent by the Chemical Weapon Convention (CWC) and a Category B agent by the Biological and Toxin Weapon Convention (BTWC). Despite their high toxicity, castor seed plants are widely used for the production of castor oil and in folk medicine systems for the treatment of various diseases. Due to the lack of a Food and Drug Administration (FDA) approved medication, early detection of biologically active ricin is critical for implementing suitable countermeasures in time to avert casualties. It is required to employ an integrated approach to distinguish between pure and crude ricin as well as active/inactive ricin. In the present study, a series of bioassays were performed to identify biologically active ricin and its different forms. This assessment included both field and lab-based assays to detect and differentiate different isoforms of ricin. The assays used are the lateral flow assay (LFA), hemagglutination assay (HA), In-vitro translational system (IVTS), cytotoxicity assay (CA), and mouse protection assay (MPA). Considering the ricin-contaminated scenario, the first step was to qualitatively determine the presence of ricin using LFA. Following that, a HA was optimized to differentiate between crude and pure ricin. In addition to this, the assay was also able to differentiate various cultivars and isoforms. IVTS assay was used to identify the enzymatic activity of the ricin A chain that inhibited translational machinery with IC₅₀ (50 % inhibitory concentration) of 11.2 ng/ml. Further neutralization with anti-ricin rabbit polyclonal antibodies (RPAb) confirmed the ricin-mediated translation inhibition and excluded the use of other RIPs (abrin, saproin, and viscumin). Cytotoxicity in HeLa cells was used as a cellular model with an estimated CC₅₀ value (50 % cytotoxic concentration) of 36 ng/ml. The neutralization experiment with RPAb specifically reversed the ricin-induced cytotoxicity. A mouse protection assay was done using 5X LD₅₀ of ricin, which caused mortality within 48 h. RPAb increased the survival, verdict the presence of ricin, and eliminated the presence of related RIPs. All the proposed assays suffice the requirement during ricin exposure scenario from the field to the laboratory. These assays are also capable of distinguishing crude/pure/cultivars, and isoforms of ricin. During an emergency, a combination of these assays will help us to make faster decisions and increase the therapeutic time window for treatment.