Validation of Immunodetection (ELISA) of Ricin Using a Biological Activity Assay

Abstract

The suitability of enzyme-linked immunosorbent assay (ELISA) for residual ricin toxicity determination was investigated in this study. Ricin was thermally treated at 80 to 90 °C for up to 9 min, and its residual concentration was determined by means of a commercial ELISA kit, and its bioactivity (amount of adenine released from DNA) was determined by means of a biological activity assay (BAA). Results showed that inactivation of ricin followed 1st-order kinetics. The half-life values for loss of bioactivity at 80, 85, and 90 °C were 1.93, 0.65, and 0.41 min, respectively. Similarly, the half-life values for reduction in ricin concentration determined by ELISA were 3.06, 0.79, and 0.43 min, respectively. The half-lives determined by both assays were only significantly different at 80 °C. The Z, Q(10), and Arrhenius activation energy values determined by both assays were dissimilar: 11.74 ˚C, 7.12 and 50.1 kcal/mol, respectively, by ELISA; and 14.87 °C, 4.71 and 39.5 kcal/mol, respectively, by BAA. Nevertheless, our findings indicate that the 2 assays were highly correlated (R(2) = 1), and it can be concluded that ELISA would be a reliable method for detecting residual toxicity of heat-treated ricin based on fraction lost. Practical Application: The results of this study indicate that immunodetection, even though not a direct measurement of the biological activity of ricin, is suitable for determining the residual bioactivity of ricin since immunodetection and the biological activity assay used in this investigation were highly correlated. Therefore, ELISA can be used for routine assessment of residual activity or toxicity of ricin in thermally treated foods.

Comparison of the Presence of Shiga Toxin 1 in Food Matrices as Determined by an Enzyme-Linked Immunosorbent Assay and a Biological Activity Assay

“…This observation suggests that the BAA was more sensitive than the ELISA for residual Stxl determination. This finding is in agreement with that of a previous study (14) in which the thermal inactivation rate of the active site of ricin was compared with that of the epitope(s) responsible for immunodetection. These results also suggest that a negative Stxl determination with an ELISA should be considered reliable because a negative outcome also would be obtained with a BAA.…”

“…The release of adenine from polynucleotides by RIPs has been reported (l, 2,5,14). In the present study, the adenine released by Stxl was reacted with chloroacetaldehyde to form the fluorescent sA (8)(9)(10), which was quantified and used as a measure of Stxl biological activity.…”

“…The BAA measured the ability of an RIP (Stxl or ricin) to remove adenine residues from a 2,551-bp DNA substrate as previously described (14). The release of adenine residues from polynucleotides has been used by previous investiga tors as an indicator of RIP biological activity (1,2,5).…”

Antibody Interactions withRicinus communisAgglutinins Studied by Biolayer Interferometry

“…Food samples and their extracts contain a complex mixture of protein-binding compounds, enzymes, and enzyme inhibitors and may produce matrix effects in immunoassay and affect ricin activity assays (He et al 2008). Immunoassays for ricin have nonetheless been validated by activity assays (Lumor et al 2011).…”

Detection of Ricin Contamination in Liquid Egg by Electrochemiluminescence Immunosorbent Assay

“…Because ricin has been used maliciously in the past and has been found at a number of locations as a result of apparent criminal activity (CDC 2003; Schieltz and others 2011), it is important to have sensitive methods for detecting ricin and marker compounds associated with crude ricin preparations. As an alternative to animal models for quantifying toxins, in vitro tests, including immunochemical methods, can be used to quantify relevant structural determinants or enzymatic activities and provide essential analytical data to assure food safety (He and others 2008, 2010; Lumor and others 2011). The reported methodologies include various immunoassay formats and devices (for example, Poli and others 1994; Shyu and others 2002; Brandon and Hernlem 2009), activity assays (Hale 2001; He and others 2008), and array and sensor technologies (Feltis and others 2008; Garber and others 2010) for ricin.…”