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technical BRIEF
INNOVATIVE RESEARCH FOR A SUSTAINABLE FUTURE
Sample Processing Method for Ricin Analysis
Background
Ricin, the Ricinus communis (castor bean) toxin, is known as one of the most lethal natural
poisons. It is a Category B bioterrorism agent, Schedule 1 chemical warfare agent, and is
periodically used in the U.S. and abroad for nefarious intentions and homicide attempts [1],
The toxin is manufactured in a powder form by partial purification or refinement of the
castor bean pulp. Ricin toxicity can occur from inhalation, ingestion, or injection, and, if
ingested, as few as 8 castor beans can contain a lethal dose. For inhalation, symptoms
including respiratory distress, fever, cough, nausea, and chest tightness can appear as
early as 4-8 hours post-exposure, whereas symptoms from ingestion (nausea, vomiting,
and diarrhea) typically develop in less than ten hours. Death usually occurs after 36-72
hours depending on the exposure route and the dose received.
The U.S. Environmental Protection Agency (EPA) is the lead federal agency to support
states and local authorities in the cleanup of facilities/sites contaminated with ricin. In the
last five years, EPA responded to four separate ricin incidents. EPA supported the
sampling, analysis and decontamination work, with final clearance for re-entry and reuse of
the sites performed by local or state health departments. In June 2013, ricin-containing
letters were sent to then President Barack Obama from Tupelo, Mississippi [2], EPA was
called upon to decontaminate and clear for reuse the two sites where the ricin used in the
letters was prepared. During 2014, EPA responded to ricin-contaminated sites in Oklahoma
City, Oklahoma and Oshkosh, Wisconsin. During a 2017 ricin incident in Boulder, Colorado,
EPA was called upon to decontaminate the affected site. During these ricin incidents,
U.S. Environmental Protection Agency
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samples were collected to determine the extent of contamination and, after
decontamination, to determine if the sites could be released to the public for reuse.
The time-resolved fluorescence (TRF) immunoassay is one of the primary screening
methods for ricin in environmental samples. It is also a method used by the Laboratory
Response Network of the Centers for Disease Control and Prevention (CDC). However,
during the EPA response to the Tupelo, Mississippi ricin incident, unsatisfactory results
were obtained due to high fluorescence backgrounds. As a result, the TRF immunoassay
could not be used for samples collected from surfaces to which chlorine bleach had been
applied for decontamination [3], The assay interferences were attributed to various potential
factors including impurities in reagents used for the TRF assay, residuals resulting from
bleach application, sampling material, and the buffer used for wetting the sampling devices.
Without appropriate sample processing prior to analysis, bleaching residuals and/or other
matrix effects from environmental samples could cause assay interferences that could
ultimately lead to false negative or false positive results. False negative results could occur
if high background fluorescence masked actual ricin presence, possibly leading to human
exposure if facilities were re-opened prematurely. False positives could occur if controls
were within range, but samples showed elevated fluorescence due to the assay interfering
materials present in the sample, thereby triggering additional, unwarranted decontamination
activities. Furthermore, without sample concentration, ricin could be present below the
analytical method's ability to detect it, while still being present at hazardous levels.
Decision-makers with local, state, federal, and tribal governments require rapid and high-
confidence results that are not unduly impacted by false positives or false negatives to
safely clear areas for re-entry and reuse and to reopen facilities. Therefore, ricin analytical
methods must be reproducible, sensitive, and specific, even in complex environmental
backgrounds. To mitigate the TRF immunoassay interference issue for post-
decontamination ricin analysis, the Homeland Security Research Program of the EPA's
Office of Research and Development, in partnership with the Lawrence Livermore National
Laboratory, developed a sample processing approach to enable accurate ricin analysis.
Development of a Ricin Sample Processing Method
A ricin sample processing method (Appendix B & C) including sample cleanup and ricin
concentration was developed for preparing ricin in environmental samples for analysis [4],
The method is performed in two steps. Step 1 is a sample extract pre-filtration using a 0.22
micrometer syringe filter to remove potential assay interferences by particulate matter that
could be present in the sample. Step 2 is a further cleanup of the sample extract and ricin
concentration using a centrifugal ultrafiltration (UF) device (Amicon® Ultra centrifugal filter
devices, Millipore, Inc., Billerica, MA). Such devices are used for sample cleanup for many
substances. The UF devices are selected based on their nominal molecular weight limit
cutoff to retain the target analyte, such as ricin, while allowing other soluble materials with
lower molecular weight to pass through. Basically, the UF devices allow washing out or
removal of soluble materials present in the sample extract that could interfere with analysis
while also enabling several-fold concentration of the target analyte due to volume reduction
by centrifugation. The processed sample is then analyzed by analytical methods such as
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the TRF immunoassay. Both the 0.5-mL and 2-mL UF devices were evaluated. The results
indicated that there was no loss of ricin from the sample while using the UF device-based
sample processing method. Using this sample processing method, a 1-mL sample could be
concentrated to 100 pL (10-fold concentration) with a 0.5-mL UF device (by using multiple
loadings on the same device). Relatedly, a 2-mL sample could be concentrated to 100 pL
(20-fold concentration) with a 2-mL UF device. Such a volume-reduction-based
concentration also concentrated ricin in the sample. Further, the sample processing method
was also tested for sponge-sticks sample extracts containing bleach residues and
reference test dust [Arizona Test Dust (ATD), selected to be representative of dust found
on sampled surfaces]. The results indicated that this sample processing method is effective
even for dirty samples
Conclusions
The ricin sample processing method based on the application of a pre-filter and centrifugal
UF devices allows sample extract cleanup and ~10-fold to 20-fold concentration of ricin,
and thereby, enhances the performance and sensitivity of the ricin TRF immunoassay.
Thus, this method could minimize false positive and false negative results, especially for
the samples collected for analysis during the post-decontamination phase of the response
to a ricin incident. Because the sample processing procedure developed here is intended
for use following the sample extraction steps, it could be used for both the pre- and post-
decontamination phase samples. Additionally, it could be used with any other fluorescence-
and electrochemiluminescence-based immunoassays, as well as other analytical methods
(e.g., PCR assays) used for ricin detection, although further verification and validation
would be required (i.e., for different surface types and potential interferences).
It is important to note how the generation of false positives and false negatives is related to
how specific assays function. For example, some assays, like TRF, are based on the
binding of an antibody to a specific part of the ricin molecule. However, this part of the ricin
molecule can be present even if the ricin cannot produce a toxic effect. On the other hand,
absence of the response is often considered to suggest that the ricin molecule has been
sufficiently degraded, especially by decontamination agent such as bleach, to preclude a
toxic effect. Thus, improvement in false positive and false negative rates, regardless of
what they mean in context of a particular assay, is an important goal, which this sample
processing method helped meet.
This sample processing method was successfully used to analyze post-bleach-
decontamination samples during the recent (2017) ricin incident in Boulder, Colorado. Data
quality objectives, including those related to false positives and false negatives, were met,
enabling the site — an apartment building where small children lived — to be returned to its
intended use in a timely manner.
References
[1] Centers for Disease Control and Prevention. CDC: Facts about ricin
https://emergency.cdc.gov/agent/ricin/
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[2]	Hughes, Brian. April 17, 2013. Feds arrest suspect in ricin-laced letters sent to Obama,
senator. Washington Examiner, Media DC, Washington DC.
https://www.washingtonexaminer.com/feds-arrest-suspect-in-ricin-laced-letters-sent-to-
obama-senator/article/2527471
[3]	U.S. Environmental Protection Agency (EPA). 2013. Pollution/situation report. Tupelo
ricin site - Removal Polrep. August 27, 2013.
http://www.epaosc.org/site/sitrep_profile.aspx?site_id=8630&counter=20255
[4]	Kane, S., Shah, S., Erler, A.M., and Alfaro, T. 2017. Sample processing approach for
detection of ricin in surface samples. J. Immunol. Methods 451: 54-60.
http://dx.doi.Org/10.1016/j.jim.2017.08.008
Additional Information
Shah, S. and Lawrence Livermore National Laboratory. 2016. Development of a sample
processing approach for post bleach-decontamination ricin sample analysis. U.S.
Environmental Protection Agency, Washington, DC, EPA/600/R-17/159.
https://cfpub.epa.gov/si/si public record report.cfm?dirEntrvld=339230
Disclaimer
The United States Environmental Protection Agency (EPA) through its Office of Research
and Development funded and managed the research described here and executed by the
Lawrence Livermore National Laboratory under an Interagency Agreement between EPA
and the Department of Energy (EPA IA DW-89-92328201-0). It has been subjected to the
Agency's review and has been approved for publication. Note that approval does not signify
that the contents necessarily reflect the views of the Agency. Mention of trade names,
products, or services does not convey official EPA approval, endorsement, or
recommendation.
For more information, visit the EPA Web site at http://www2.epa.gov/homeland-securitv-
research
Technical Contact: Dr. Sanjiv Shah (shah.saniiv@epa.gov)
General Feedback/Questions: Amelia McCall (mccall.amelia@epa.gov)
U.S. EPA's Homeland Security Research Program (HSRP) develops products based on scientific
research and technology evaluations. Our products and expertise are widely used in preventing,
preparing for, and recovering from public health and environmental emergencies that arise from
terrorist attacks or natural disasters. Our research and products address biological, radiological, or
chemical contaminants that could affect indoor areas, outdoor areas, or water infrastructure. The
HSRP provides these products, technical assistance, and expertise to support EPA's roles and
responsibilities under the National Response Framework, statutory requirements, and Homeland
Security Presidential Directives.
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