ASTM E2805-11
(Practice)Standard Practice for Measurement of the Biological Activity of Ricin
Standard Practice for Measurement of the Biological Activity of Ricin
SIGNIFICANCE AND USE
The CFT assay provides a sensitive and reliable method to detect ricin biological activity and results can be generated within 3 h. The assay measures the amount of ricin biological activity when compared to a known ricin standard and provides a quantitative measurement for active ricin.
The lower limit of quantitation and the upper limit of quantitation for ricin using the CFT assay was measured at 10 ng/mL and 170 ng/mL, respectively (5).
This practice is focused on the measurement of reference materials and not environmental samples. Additional control runs may be needed for measurements of environmental samples to ensure that the presence of additional materials in the samples (also referred to as the matrix) will interfere with the measurements.
The CFT assay may be used to determine the presence of active ricin in forensic or bioterrorist samples if the appropriate controls are utilized to ensure valid results (5).
The methods described in this document measure the biological activity of ricin and do not detect the presence of inactivated ricin in a given sample.
Ricin reference materials have a number of applications, such as testing detection devices, laboratory instruments, environmental sampling methods, disinfection studies, and basic research.
SCOPE
1.1 This guide is intended for the manufacturers and users of ricin reference material. Ricin reference materials are well-characterized materials that can be used to test detection devices and calibrate laboratory measurements. It is anticipated that ricin reference materials will be characterized by biochemical methods in addition to the measurement of biological activity.
1.2 This practice details the measurement of ricin biological activity using a cell-free translation (CFT) assay (4).
1.3 The CFT assay has been developed for use in any biotechnology laboratory where determination or confirmation of ricin biological activity is required.
1.4 The CFT assay has been validated by the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) VP-016 Validation of Cell-Free Translation Assay for the Detection of Ricin Toxin Biological Activities in compliance (5) with Good Laboratory Practices (GLP) Regulations of the Food and Drug Administration (21 CFR Part 58). Strict adherence to the protocol is necessary for validity of the test results.
1.5 Appendix X1 and Appendix X2 also provide guidance for the measurement of the biological activity of ricin using cell-based assays and the use of synthetic enzyme substrates.
1.6 Ricin is a category 2 select agent and acquisition of the ricin standard must adhere to the Center for Disease Control (CDC) regulations. Ricin is listed on the select agent list (42 CFR Part 72). The possession, transfer, and use of ricin are restricted under the Public Health Security Preparedness Act (CRS Report RL31263 Public Health Security and Bioterrorism Preparedness and Response Act (P.L. 107-188): Provision and Changes to Preexisting law). Access to stores of ricin is limited (USA Patriot Act, P.L. 107-56). Ricin is also a prohibited substance under the Biological Weapons Convention and the Chemical Weapons Convention (CRS Report RL31559 Proliferation Control Regimes: Background and Status).
1.7 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.8 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. Ricin is an extremely dangerous toxin. See Section 9 for specific hazards information.
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Designation: E2805 − 11
Standard Practice for
1
Measurement of the Biological Activity of Ricin
This standard is issued under the fixed designation E2805; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
INTRODUCTION
Ricin is a member of the protein toxins that cause their physiological effect by inactivation of
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ribosomes.Ricinisamemberoftheclass2ribosomeinactivatingproteins(1). Othermembersofthis
class of toxins include the proteins abrin and Shiga toxin.
Ricin consists of two chains, the A-chain that is responsible for the N-glycosidase enzymatic
activity and the B-chain that is needed for cell binding and intra-cellular processing. Ricin is a
heterogeneous protein with molecular weights ranging from approximately 62 to 64 kilodaltons (kDa)
(2). Both chains are glycosylated and of similar size (approximately 32 kDa). There are several genes
encoding putative ricin and ricin-like proteins in the genome of R. communis (3) resulting in
differences in the amino acid sequence of the subunits. The differences in amino acid sequence and
glycosylation both contribute to the heterogeneity of ricin.
1. Scope 1.5 Appendix X1 and Appendix X2 also provide guidance
for the measurement of the biological activity of ricin using
1.1 This guide is intended for the manufacturers and users
cell-based assays and the use of synthetic enzyme substrates.
of ricin reference material. Ricin reference materials are
well-characterized materials that can be used to test detection
1.6 Ricin is a category 2 select agent and acquisition of the
devicesandcalibratelaboratorymeasurements.Itisanticipated
ricin standard must adhere to the Center for Disease Control
that ricin reference materials will be characterized by bio- (CDC) regulations. Ricin is listed on the select agent list (42
3
chemical methods in addition to the measurement of biological
CFR Part 72). The possession, transfer, and use of ricin are
activity.
restricted under the Public Health Security Preparedness Act
(CRS Report RL31263 Public Health Security and Bioterror-
1.2 This practice details the measurement of ricin biological
ism Preparedness and Response Act (P.L. 107-188): Provision
activity using a cell-free translation (CFT) assay (4).
and Changes to Preexisting law). Access to stores of ricin is
1.3 The CFT assay has been developed for use in any
limited (USA Patriot Act, P.L. 107-56). Ricin is also a
biotechnology laboratory where determination or confirmation
prohibited substance under the Biological Weapons Conven-
of ricin biological activity is required.
tion and the Chemical Weapons Convention (CRS Report
RL31559 Proliferation Control Regimes: Background and
1.4 The CFT assay has been validated by the U.S. Army
Status).
Medical Research Institute of Infectious Diseases (USAM-
RIID) VP-016 Validation of Cell-Free Translation Assay for
1.7 The values stated in SI units are to be regarded as
the Detection of Ricin Toxin Biological Activities in compli-
standard. No other units of measurement are included in this
ance (5) with Good Laboratory Practices (GLP) Regulations of
standard.
the Food and Drug Administration (21 CFR Part 58). Strict
1.8 This standard does not purport to address all of the
adherence to the protocol is necessary for validity of the test
safety concerns, if any, associated with its use. It is the
results.
responsibility of the user of this standard to establish appro-
priate safety and health practices and determine the applica-
bility of regulatory limitations prior to use. Ricin is an
1
This practice is under the jurisdiction of ASTM Committee E54 on Homeland
extremely dangerous toxin. See Section 9 for specific hazards
Security Applications and is the direct responsibility of Subcommittee E54.01 on
information.
CBRNE Sensors and Detectors.
Current edition approved Jan. 1, 2011. Published March 2011. DOI: 10.1520/
E2805-11.
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The boldface numbers in parenthesis refer to the list of references at the end of
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this standard. Available at http://www.bt.cdc.gov/Agent/agentlist.asp.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
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E2805 − 11
2. Referenced Documents (cytotoxicity). Ricin is added to the cells and after an incuba-
4 tion period, the effect on cell cytotoxicity is measured. The
2.1 ASTM Standards:
ricin-treated cells are compared to control cells (without added
F2149 Test Method for Automated Analyses of Cells—the
ricin)maintainedunderthesameconditions.Guidanceisgiven
Electrical Sensing Zone Method
...
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