ASTM E2805-18

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: E2805 − 11 E2805 − 18
Standard Practice for
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
ribosomes. Ricin is a member of the class 2 ribosome inactivating proteins (1). Other members of this
class of toxins include the proteins abrin and Shiga toxin.
Ricin Ricinus communis (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. Various research reports exist
describing different means of detecting ricin activity.
1. 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 and
Prevention (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.
This practice is under the jurisdiction of ASTM Committee E54 on Homeland Security Applications and is the direct responsibility of Subcommittee E54.01 on CBRNE
Sensors and Detectors.
Current edition approved Jan. 1, 2011Dec. 1, 2018. Published March 2011December 2018. Originally approved in 2011. Last previous edition approved in 2011 as
E2805 – 11. DOI: 10.1520/E2805-11.10.1520/E2805-18.
The boldface numbers in parenthesis refer to the list of references at the end of this standard.
Available at http://www.bt.cdc.gov/Agent/agentlist.asp.https://emergency.cdc.gov/agent/agentlist.asp.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2805 − 18
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 safety, health, and healthenvironmental practices and determine the
applicability of regulatory limitations prior to use. Ricin is an extremely dangerous toxin. See Section 9 for specific hazards
information.
1.9 This international standard was developed in accordance with internationally recognized principles on standardization
established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued
by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
2. Referenced Documents
2.1 ASTM Standards:
E2458 Practices for Bulk Sample Collection and Swab Sample Collection of Visible Powders Suspected of Being Biological
Agents and Toxins from Nonporous Surfaces
E3131 Specification for Nucleic Acid-Based Systems for Bacterial Pathogen Screening of Suspicious Visible Powders
F2149 Test Method for Automated Analyses of Cells—the Electrical Sensing Zone Method of Enumerating and Sizing Single
Cell Suspensions
2.2 Code of Federal Regulations:
21 CFR Part 58 Good laboratory practice for nonclinical laboratory studies
42 CFR Part 72 Interstate shipment of etiologic agents
2.3 ANSI/ATCC Standard:
ASN-0001-2009 Standardization of In-Vitro Assays to Determine Anthrax Toxin Activities
2.4 ISO Standard:
ISO Guide 34 General requirements for the competence of reference material producers
ISO 20391–1 Biotechnology – Cell Counting – Part 1: General Guidance on Cell Counting Methods
2.5 Federal Standard:
18 USC 178 Definitions
3. Terminology
3.1 Definitions:
3.1.1 aseptic technique, n—operation or performance of a procedure or method under carefully controlled conditions to reduce
the risk of exposure and prevent the introduction of unwanted material/matter (contamination) into a sample. E2458
3.1.2 calibration, n—set of operations that establish, under specified conditions, the relationship between the values of
quantities indicated by a measurement instrument or measuring system or values represented by a material measure or a reference
material and the corresponding values realized by standards. Eurochem Selection (6)
3.1.3 hazard, n—something that is potentially dangerous or harmful, often the root cause of an unwanted outcome; a danger or
peril. NIMS
3.1.4 inhibition, n—undesirable effect that can result in a false negative result and is typically caused by the presence of
compounds that interfere with the assay or detection process. E3131
3.1.5 reference material, n—material, sufficiently homogenous and stable with respect to one or more specified properties that
has been established to be fit for its intended use in the measurement process; properties can be quantitative or qualitative. ISO
Guide 34
3.1.6 risk, n—the probability of suffering a loss or harm or injury; peril. E2458
3.1.7 toxin, n—the toxic material or product of plants, animals, microorganisms (including but not limited to, bacteria, viruses,
fungi, rickettsiae, or protozoa), or infectious substances, or a recombinant or synthesized molecule, whatever their origin and
method of product, and includes: (1) any poisonous substance or biological product that may be engineered as a result of
biotechnology produced by a living organism; or (2) any poisonous isomer or biological product, homolog, or derivative of such
a substance. ISO USC 178
3.2 Abbreviations:Abreviations and Acronyms:
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
Available from Food and Drug Administration (FDA), 5600 Fishers Ln., Rockville, MD 20857, http://www.fda.gov.
Available from U.S. Government Printing Office Superintendent of Documents, 732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http://
www.access.gpo.gov.
Available from American National Standards Institute (ANSI), 25 W. 43rd St., 4th Floor, New York, NY 10036, http://www.ansi.org.
The sole source of supply of the apparatus (MicroLumi XS) known to the committee at this time is Harta Instruments, Inc., 8 Russell Ave Unit 106, Gaithersburg, MD
20877, www.hartainstruments.com. If you are aware of alternative suppliers, please provide this information to ASTM International Headquarters. Your comments will receive
careful consideration at a meeting of the responsible technical committee,Available from International Organization for Standardization (ISO), ISO Central Secretariat, BIBC
II, Chemin de Blandonnet 8, CP 401, which you may attend.1214 Vernier, Geneva, Switzerland, http://www.iso.org.
E2805 − 18
3.2.1 CFT—cell free translation.
3.2.2 CPS—counts per second, units of luminescence instrument.
3.2.3 IC50—concentration of ricin that produces inhibition of 50 % of the activity in an assay.
3.2.4 kDa—molecular mass in kilo Dalton units.
3.2.5 NIMS—National Incident Management System
3.2.6 PBS—phosphate buffered saline.
4. Summary of Practice
4.1 The CFT assay for measuring biologically active ricin is based on its inhibitory effects on protein synthesis (67, 78). When
added to a rabbit reticulocyte translation mixture containing luciferase mRNA, messenger ribonucleic acid (mRNA), ricin inhibits
translation of the mRNA into the enzyme luciferase. Luciferase is then detected using a buffer containing the luciferin substrate.
The test is a bioluminescence assay that measures the amount of luminescence proportional to the amount of luciferase produced
from protein translation (RNA → protein). When active ricin is present, the amount of luminescence decreases corresponding to
a decrease in the production of the luciferase enzyme. The amount of protein (luciferase) produced is directly proportional to the
amount of luminescence generated. The decrease in luminescence is directly proportional to the amount of active ricin in the
sample. Confirmation that translation inhibition is caused by the presence of active ricin is determined by mixing an aliquot of the
ricin samples with anti-ricin antibody before adding to the translation mixture. The neutralized ricin does not inhibit luciferase
translation, and therefore, luminescence does not decrease.
4.2 Cell-based assays use mammalian cells maintained in culture to measure the effect of ricin on cell death or damage
(cytotoxicity). Ricin is added to the cells and after an incubation period, the effect on cell cytotoxicity is measured. The
ricin-treated cells are compared to control cells (without added ricin) maintained under the same conditions. Guidance is given in
Appendix X1.
4.3 The N-glycosidase enzymatic activity of the A-chain of ricin can be measured using synthetic oligonucleotides. The enzyme
activity is measured either by the released adenine or the effect on the depurinated substrate using a number of methods. Guidance
is given in Appendix X2.
5. Significance and Use
5.1 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.
5.2 The lower limit of quantitation and the upper limit of quantitation for ricin using the CFT assay waswere measured at 10
ng/mL and 170 ng/mL, respectively (5).
5.3 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 not interfere with the measurements.
5.4 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).
5.5 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.
5.6 Ricin reference materials have a number of applications, such as testing detection devices, laboratory instruments,
environmental sampling methods, disinfection studies, and basic research.
6. Apparatus
6.1 List of Equipment—The make and model are provided as examples, however equivalent apparatus may also be used.
6.1.1 Mixer, Mixer—vortex mixing motionmotion.
6.1.2 Display timers.
6.1.3 Incubator, Incubator—capable of maintaining temperature of (37 6 1°C).
6.1.4 96 Well Microplate Luminometer and Luminescence Test Plate.
6.1.5 Microplate Data Analysis Software,Software. KC4, with PowerReports,™ v3.0.
6.1.6 Plateshake.Plateshaker.
6.1.7 Laboratory Refrigerators (4°C), Freezer (-20°C), and Ultralow Freezer (-70°C or lower).
6.1.8 Water Bath, Bath—37 6 1°C.
6.1.9 Fixed Volume Pipettes, 1000 μL (200 to 1000 μL), 200 μL (20 to 200 μL), 20 μL (5 to 20 μL), 10 μL (1 to 10 μL), and
2 μL (0.5 to 2 μL), or adjustable pipettes of this range—Pipettes should be regularly calibrated to ensure accurate dispensing of
the set volumes.
E2805 − 18
6.1.10 Multi-Channel Pipettes including 12-Channel (20 to 200 μL), 8-Channel Pipettor (2 to 20 μL), and 8-Channel pipettor
(5 to 50 μL)—Pipettes should be regularly calibrated to ensure accurate dispensing of the set volumes.
7. Reagents
7.1 Reagents for the CFT Assay—The validation of the assay was performed with reagents purchased from the specific vendors.
The reproducibility and precision of the assay is dependent upon the quality of the reagents. The specific reagents have been tested
to work in the validated assay. Substitution of reagents will require testing to ensure the same performance.
7.2 Rabbit Reticulocyte Lysate,Lysate (nuclease treated)—nuclease treated.The rabbit reticulocyte lysate is prepared from New
Zealand white rabbits using a standard protocol under quality-controlled conditions (89). After the reticulocytes are lysed, the
lysate is treated with micrococcal nuclease in order to destroy endogenous mRNA. The lysate is further optimized for mRNA
translation by addition of an energy generating system, a mixture of tRNAs, transfer RNAs (tRNAs), hemin (to prevent inhibition
of initiation), potassium acetate, and magnesium acetate. The rabbit reticulocyte lysate contains no endogenous mRNA and
therefore translates only the mRNA added to the lysate (910). The ability to translate only one protein that can be detected permits
a more accurate analysis of the biological effect of ricin’s enzymatic reaction.
7.3 Amino Acid Mixture, Complete—The amino acid mixture, complete, has been prepared for use in the rabbit reticulocyte
lysate systems and is an aqueous solution containing 1 mM each of the 20 essential amino acids. The mixture is sterile and
RNA-free.
7.4 Luciferase Control RNARNA——Luciferase control RNA used in these studies is commercially made using SP6 RNA
polymerase transcription of a plasmid bearing the coding region for the luciferase gene with an additional 30 adenine residues that
creates an uncapped, polyadenylated mRNA. The product of this luciferase control RNA is a monomeric protein (61 kDa) that does
not require post-translational processing or modification for enzymatic activity. Only full-length luciferase is active. In most
laboratories, contamination with extraneous luciferase or luciferase mRNA does not occur because luciferase is not found in
laboratory environments.
7.5 Luciferase Reporter BufferBuffer——The luciferin reaction system is purchased as two components, lyophilized assay
reagent and assay buffer. When mixed, the luciferase assay buffer provides high quantum efficiency and no background
luminescence in the reticulocyte system or in the assay chemistry. Light is produced by converting the chemical energy of luciferin
oxidation through an electron transition, forming the product molecule of oxyluciferin. The luciferase reaction buffer contains
coenzyme A that improves kinetics and allows for greater enzymatic turnover resulting in increased light intensity. Unlike many
chemiluminescent reactions, this reaction remains constant for nearly two minutes, thereby permitting accurate measurements
using a microtiter format (4). As noted, the amount of luminescence emitted with this reaction buffer is proportional to the amount
of luciferase present and therefore provided a comparative measurement of the luciferase amount in samples treated with toxins.
7.6 RNase InhibitorInhibitor—, aA 50 kDa protein that noncovalently binds to RNases in a 1:1 ratio, is a broad-spectrum RNase
inhibitor. The product is purified using a combination of ion exchange and affinity chromatography.
7.7 Sterile Nuclease-Free De-ionized Water, (sterile and RNase free).sterile and RNase free.
7.8 Ricin Standard and Antibody—Reagents should be comparable to the following products:
7.8.1 Ricin Toxin (Ricin), Ricinus Communis Agglutinin II, 5 mg/mLmg/mL——This product, purified from castor beans, is the
approximately 60 kDa molecular weight protein that is highly toxic with little agglutinin activity. The toxin is purchased as a liquid
(5 mg/mL) and contains 0.08 % sodium azide. At this concentration, the sodium azide does not affect the assay.
7.8.2 Antibody to Ricinus Communis Agglutinin I and II, Affınity Purified from Goat, 2 mg/mLmg/mL—, This product is a
polyclonal anti-ricin IgG and provides excellent neutralization of ricin toxin.
7.9 Phosphate Buffered Saline (PBS) , (pH of 7.2)—with a pH of 7.2. Sterile PBS solutions were tested in the CFT assay and
this PBS did not interfere with the assay.
7.10 Polyoxethylenesorbitan MonolaurateMonolaurate—, molecularMolecular biology grade, ensured to be endonuclease,
exonuclease, and RNAse free.
7.11 Disinfectant—Bleach, 5 % sodium hypochlorite. Ensure that the expiration date on the bleach has not passed and that the
bleach is still active.
8. Materials
8.1 96 V Shaped Bottom Plates, sterile, tissue culture grade.
8.2 96 Well Flat Bottom Plate, black with clear bottom.
8.3 Sterile Pipette Tip.
8.4 15 mL and 50-mL Conical Tubes, sterile.
8.5 1.5 mL Microtubes, sterile.
E2805 − 18
8.6 100 mL Disposable Reagent Reservoir, sterile.
8.7 Low-Density Polyethylene Bag, zip-lip, 5 by 8 in.
8.8 250 mL Glass Bottle, sterile, for preparing buffer.
8.9 Ice Bucket (Ice Pan).
8.10 Waste Container.
8.11 Paper Towels, disposable.
8.12 Nitrile Gloves, nonsterile.
9. Hazards
9.1 Ricin is extremely toxic in very small quantities (1011, 1112). The user of this practice is responsible for ensuring that the
procedures are carried in full compliance with the institute’s safety/biosurety regulations.
9.2 Ricin should be managed as a hazardous chemical and included in the laboratory specific chemical hygiene plan. Ricin in
powder form is more hazardous, therefore work with ricin powder should be avoided if possible. The institute’s biosurety/biosafety
office should be contacted prior to any work with powder samples containing (or thought to contain) ricin.
9.3 The use of ricin requires biosafety level 2 (BSL2) or higher and specialized procedures including training for working with
toxins of biological origin (1213).
9.4 Any materials containing ricin waste should be inactivated by autoclaving or treating with active solutions of bleach (10 %
vol/vol) for 30 min before disposal.
10. Calibration and Standardization
10.1 Calibration of instruments should be performed in accordance with the institute’s standard operating procedures or
manufacturer’s instructions.
10.2 The assay is standardized using the reagents listed. If different reagents are used, standardization of the materials is
required.
10.3 The performance and concentration of the anti-ricin antibody used should be determined.
10.4 The purity and concentration of the ricin standard used should be verified by analytical measurements.
10.4.1 Denaturing gel electrophoresis using sodium dodecyl sulfate (SDS) and conditions that reduce the disulfide bond
between the A and B subunits, results in two bands of approximately 32 kDa (1314).
10.4.2 Techniques such as size exclusion chromatography can also be used to determine the heterogeneity of the ricin sample,
pure samples should give a single peak with an approximate molecular weight of 60 kDa (1415).
10.4.3 Other methods can be used for characterizing ricin preparations such as ion-exchange chromatography and isoelectric
focusing (1516).
10.5 The ricin protein concentration of pure ricin samples should be determined for reference samples. The molar absorption
-1 -1
coefficient of ricin in phosphate-buffered saline (PBS) at 279 nm was measured as (93 900 6 3300) L mol cm , amino acid
analysis is used to determine the protein concentration (1617).
11. Procedure
11.1 Preparation of CFT Luciferase Reaction Buffer:
11.1.1 Thaw the luciferase assay buffer using a 37°C water bath.
11.1.2 Add the thawed assay buffer to the luciferase assay substrate powder that is contained in a brown glass bottle. Dispense
10 mL to each tube of the reaction buffer into conical tubes. Each tube (10 mL) is enough for 2 plates.
11.1.3 Assign a lot number and label the tube with the name (luciferin assay buffer), the date made, and the expiration date (1
month if stored at -20 6 10°C, or 1 year if stored at –70 6 10°C after the preparation date). Wrap each tube with aluminum foil
and store the buffer at a -20°C or -70°C.
11.2 Preparation of the Assay Buffer (PBS Containing 0.02 % Tween 20):
11.2.1 Pipette 200 mL of PBS into a sterile glass bottle and add 40 μlμL Tween 20. Mix gently to insure that Tween 20 is
dispersed. Larger volumes can be prepared as long as sterility of the container is maintained.
11.2.1.1 Prepare assay buffer fresh each day.
11.3 Preparation of Ricin Standard Working Solution (0.5 μg/mL):
11.3.1 Add 5 μL of ricin (5 mg/mL) to 2495 μL of assay buffer to make ricin stock solution (10 μg/mL). Label the stock solution
and store at 4°C. The stock solution is stable for 2 weeks when stored at 4°C (5), but caution should be exercised in storage of
dilute concentrations of ricin to confirm the stability or if losses due to adsorption to storage containers have occurred. When in
doubt prepare a fresh solution from the concentrated stock.
E2805 − 18
11.3.2 Then add 50 μL of the stock solution to 950 μL assay buffer to prepare a 0.5 μg/mL ricin working solution.
11.3.3 Place the ricin standard working solution (0.5 μg/mL) in ice pan. on ice.
11.4 Preparation of Anti-Ricin Antibody Working (Neutralization) Solution (10 μg/mL):
11.4.1 Add 10 μL goat anti-ricin antibody stock solution (2 mg/mL) to 1090 μL assay buffer for every set of 3 samples.
11.4.2 Keep anti-ricin antibody stock solution at 4°C.
11.5 Equipment Check:
11.5.1 Check to ensure that the incubator, vortex mixer, and shaker platform are working properly.
11.5.2 Turn on and test the luminometer following the manufacturer’s users manual, using the luminescence filter and settings.
Run the luminescence test plate to ensure that the instrument is functioning correctly.
11.6 Prepare the Necessary Amount of Luciferase Reporter, Assay Buffer, and Reagents on Ice:
11.6.1 Place the assay buffer, ricin working solution, anti-ricin antibody working solution, test samples, water, amino acid
solution, and luciferase mRNA in the ice bath. on ice. Except for incubating ricin with antibody, all reagents shall be at a
temperature between 0 to 4 0–4°C in order to minimize translation before incubating the lysate.
11.7 Serial Dilution of Ricin Standards and Unknown Test Samples:
11.7.1 Seven concentrations of ricin have been characterized as providing a suitable sigmoid standard curve for this assay. The
standards are prepared by serially diluting ricin working solution with assay buffer. The final concentrations of the standards are:
S1 = 250 ng/mL, S2 = 125.0 ng/mL, S3 = 62.5 ng/mL, S4 = 31.3 ng/mL, S5 = 15.6 ng/mL, S6 = 7.8 ng/mL, and S7 = 3.9 ng/mL.
The concentrations for the seven standards may need to be adjusted when using a different lot of ricin or ricin purchased from a
different vendor. The ricin standards are prepared in separate tubes and then added to the plates, so that the same standards can
be added to multiple plates if needed.
11.7.2 The starting dilution of test samples should be determined by the assay supervisor based on sample information or
pre-test results before starting the assay. The amount used for serial dilution of unknown test samples varies, depending on
quantity/amount of the sample.
11.7.3 Prepare two sets of serial dilutions for ricin standards (S), unknown test samples (T), and buffer control (BC) on a
v-shaped 96-well microplate. One set will be used for CFT assay and the second set will be neutralized with ricin antibody and
then analyzed by the assay to confirm ricin toxicity. The plate should be set up in accordance with Table 1.
11.7.4 Add 50 μL assay buffer to the wells for all of the BC samples, and the T samples in rows C, D, E, F, G, and H (but not
row B, according to the configuration shown in Table 1).
11.7.5 Add 50 μL of the corresponding ricin standard (S1 to S9) to the wells of columns 1 and 9.
11.7.6 Add 100 μL of unknown sample starting dilution to each corresponding well of row B (columns 2, 3, 4, 10, 11, and 12).
11.7.7 Perform 2-fold serial dilution of unknown samples by transferring 50 μL/well from row B to row C, and so on continuing
to row H. Remove and discard 50 μL from row H. After each transfer, mix dilutions by pipetting up and down 7 times. Clean pipette
tips should be used after each serial dilution stepstep.
11.8 Prepare Anti-Ricin Antibody Neutralization Reaction:
11.8.1 Add 50 μL/well of the anti-ricin antibody neutralization working solution (10 μg/mL) to one set of standards, samples,
and buffer control (columns 9-12). After each addition, mix solutions by pipetting up and down 5 times.
11.8.2 Add 50 μL/well of the assay buffer to the other set of standards, samples, and control (Columns 1-4). After each addition,
mix solutions by pipetting up and down at least 5 times.
11.8.3 Cover and gently shake the plate on a shaker at room temperature (25 6 3) for 20 6 5 min.
11.9 Transferring Solutions:
11.9.1 Place a clean V-shaped bottom plate on ice in an ice bucket. ice.
11.9.2 Remove the lysate plate from the shaker and place the plate on ice.
11.9.3 Transfer 5 μL/well from each set of standards, sample dilutions, and control to corresponding wells in a V-shaped bottom
plate.
TABLE 1 Configuration for 96-well Microplate for Serial Dilution of Ricin Standards and Unknown Samples
1 2 3 4 5 6 7 8 9 10 11 12
A A A A A A A A
A BC BC BC BC BC BC BC BC
B C C C B C C C
B S1 T1–1 T2–1 T3–1 S1 T1–1 T2–1 T3–1
B C C C B C C C
C S2 T1–2 T2–2 T3–2 S2 T1–2 T2–2 T3–2
B C C C B C C C
D S3 T1–3 T2–3 T3–3 S3 T1–3 T2–3 T3–3
B C C C B C C C
E S4 T1–4 T2–4 T3–4 S4 T1–4 T2–4 T3–4
B C C C B
F S5 T1–5 T2–5 T3–5 S5 T1–5 T2–5 T3–5
B C C C B C C C
G S6 T1–6 T2–6 T3–6 S6 T1–6 T2–6 T3–6
B C C C B C C C
H S7 T1–7 T2–7 T3–7 S7 T1–7 T2–7 T3–7
A
BC = buffer control.
B
Ricin standard samples: S1 = 250 ng/mL, S2 = 125.0 ng/mL, S3 = 62.5 ng/mL, S4 = 31.3 ng/mL, S5 = 15.6 ng/mL, S6 = 7.8 ng/mL, and S7 = 3.9 ng/mL.
C
Sample dilutions: T1, T2, and T3 = serial dilutions of unknown samples prepared in triplicate.
E2805 − 18
11.10 Preparation of Translation Reagent:
11.10.1 Place the reagent reservoir on ice in an ice bucket. ice.
11.10.2 Prepare the translation reagent by adding the following reagents in the reagent reservoir.
11.10.3 All work should be done on ice (4 6 2) in order to prevent the onset of translation.
11.10.4 Add reagents in the same order as listed in Table 2.
11.10.5 Mix reagents by pipetting the mixture gently up and down 5 times using a 1000 μL pipettor.
11.11 Incubation of Translation/Ricin Mixture:
11.11.1 Immediately add 25 μL of above mixed translation reagent solution into each well of the V-shaped plate containing 5
μL of the samples.
11.11.2 Gently tap the sides of the plate. Avoid creating air bubbles.
11.11.3 Place a damp paper towel around the plate and place the plate in a sealable plastic bag.
11.11.4 Incubate the plate at 37 6 1°C for 90 6 5 min.
11.11.5 After the 90 6 5 min incubation, remove the plate from the plastic bag.
11.11.6 Immediately place the plate on ice in the ice pan. ice. Leave on ice for 5 min.
11.11.7 Transferring Reaction Mixture and Adding Luciferase Reaction Buffer:
11.11.7.1 Transfer 5 μL per well of the reaction mixtures from the translation incubation plate to a new 96 well, clear bottom
black plate.
11.11.7.2 All dilutions should be run in triplicate. Each set of samples is run on one black plate. One plate shall be used for the
normal translation set and one plate for the anti-ricin antibody (neutralization) translation reaction set.
11.11.8 Set up black plates as shown in Table 3.
11.11.9 Without interruption, add 45 μL of luciferase reaction buffer to each well (in black plates) using the multichannel
pipettes to the wells containing the 5 μL samples. Once the luciferase reaction buffer is added the plate shall be read by the
luminometer within 2 min.
11.11.10 Gently tap the sides of the plate to mix.
11.12 Reading the Plates:
11.12.1 Immediately following the addition of the luciferase reporter buffer, place the plate in the luminometer and read within
2 min.
11.12.2 Save raw data, counts per second (CPS) to a disk.
11.13 Calculations:
11.13.1 Calculate the mean CPS, standard deviation (SD), and %CV of the triplicate wells, including standards, controls, and
samples, on each individual test black plate with the Bio-Tek microplate data analysis software with PowerReports™ (KC4, v3.0),
or a suitable statistical analysis software program.
11.13.2 The four parameters and R of ricin standard curves and mean ricin concentration of sample dilutions are calculated with
KC4 software, using the following four-parameter logistic (4PL) equation:
y 5 a 2 d / 11 x/c b 1d (1)
~ ! ~ ~ ! !
where:
y = the expected response,
x = concentration,
a = response at zero concentration,
d = response at infinite concentration,
c = concentration resulting in a response halfway between a and d (the IC50), and
b = slope parameter
11.13.3 Specific active ricin in the test samples is confirmed by comparing CPS of each sample dilution without and with the
addition of anti-ricin IgG, which neutralized the ricin toxic activity.
11.13.4 Raw data (CPS) should be stored in a computer spreadsheet program.
11.13.5 Calculate percent recovery (% recovery), percent of relative error (% RE), and all mean concentrations, standard
deviation (SD), and % CV for dilution-to-dilution, plate-to-plate, and day-to-day comparison, using the following equations:
% CPS 5 CPS /CPS x 100 (2)
sample buffer control
TABLE 2 Translation Reagent Preparation
Reagent Name Volume (per 3 unknown samples)
Nuclease-Free Water 1000 μL
Rabbit Reticulocyte Lysate, Nuclease Treated 1000 μL
RNasin RNase inhibitor 5 μL
Amino Acid Mixture, Complete 35 μL
Luciferase Control RNA 30 μL
E2805 − 18
TABLE 3 Configuration for 96-well Black Micro Plates for the Luciferase Reaction
1 2 3 4 5 6 7 8 9 10 11 12
A BC BC BC BC BC BC BC BC BC BC BC BC
B S1 S1 S1 T1–1 T1–1 T1–1 T2–1 T2–1 T2–1 T3–1 T3–1 T3–1
C S2 S2 S2 T1–2 T1–2 T1–2 T2–2 T2–2 T2–2 T3–2 T3–2 T3–2
D S3 S3 S3 T1–3 T1–3 T1–3 T2–3 T2–3 T2–3 T3–3 T3–3 T3–3
E S4 S4 S4 T1–4 T1–4 T1–4 T2–4 T2–4 T2–4 T3–4 T3–4 T3–4
F S5 S5 S5 T1–5 T1–5 T1–5 T2–5 T2–5 T2–5 T3–5 T3–5 T3–5
G S6 S6 S6 T1–6 T1–6 T1–6 T2–6 T2–6 T2–6 T3–6 T3–6 T3–6
H S7 S7 S7 T1–7 T1–7 T1–7 T2–7 T2–7 T2–7 T3–7 T3–7 T3–7
% recovery 5 observed/nominal x 100 (3)
% RE 5 Observed/Nominal 2 1 x 100 (4)
~ !
% CV 5 SD/mean x 100 (5)
Nominal concentration refers to the initial concentration of the ricin used as the standard.
11.14 The P value is calculated by paired T-test for neutralization data and un-paired T-test for stability data, using a suitable
statistical analysis software program (see Fig. 1).
12. Disposal of All Ricin Solutions
12.1 All liquid waste shall be decontaminated using a final concentration of 10 % bleach solution for a 30-min 30 minute contact
time.
12.2 Dispose decontaminated materials in accordance with the institution’s policies.
TABLE 4 Acceptance Criteria for Plate Data Report
Criteria Plate No. 1 Plate No. 2 Plate No. 3
Standards CV # 25 %
Control CV # 20 %
Slope B
R > 0.99
Plate Acceptable (if criteria listed above are
met)
TABLE 5 Acceptance Criteria Report for Unknown Samples
Test Sample ID CV # 20 % Toxin Concentration, μg/mL Ricin Confirmed Other Information
FIG. 1 Atypical A Typical Standard Curve of Ricin Toxin for Cell-Free Translation Assay (R = 0.9970)
S
E2805 − 18
13. Report
13.1 Acceptance Criteria for Plates—Determine whether the test black plate is acceptable by recording Y or N in the spaces
of Table 4.
13.2 Acceptance Criteria/Concentration/Toxin Type for Unknown Test Samples:
13.2.1 Record toxin concentration, %CV, and whether the toxin is confirmed to be ricin.
13.2.2 Record negative sample as “ – ” in the form shown in Table 5.
14. Keywords
14.1 biodefense; biological activity; cell culture; cell free translation (CFT) assay; enzymatic activity; forensic test; ricin; toxin
APPENDIXES
(Nonmandatory Information)
X1. CELL BASED AND SYNTHETIC SUBSTRATE ASSAYS
X1.1 Cells Used to Measure Ricin Activity
X1.1.1 Ricin is a galactose-specific toxin lectin that is taken into cells through the binding of the B chain to galactose-terminated
molecules on the cell surface and also through mannose receptors expressed on the surface of macrophagic cells (1718).
X1.1.2 A number of mammalian cell lines have been used to measure the cytotoxicity of ricin (1819).
X1.1.3 Cells should be screened for contamination by microorganisms, such as mycoplasma, to ensure the purity and quality of
the cell culture results
X1.1.4 Cells should be obtained from a reputable repository to ensure authenticity of the line.
X1.1.5 It is recommended that cells should be passaged no more than 30 times when used in measuring the activity of ricin, due
to potential changes in genetic and phenotypic characteristics that are associated with cells with high passage numbers.
X1.1.6 Vero cells (African green monkey epithelial cells ) are used here as an example to demonstrate one method for measuring
ricin activity in vitro.
The sole source of supply of the apparatus known to the committee at this time is BioTek, P.O. Box 998, Highland Park, Winooski, VT 05404, http://www.biotek.com/.
If you are aware of alternative suppliers, please provide this information to ASTM International Headquarters. Your comments will receive careful consideration at a meeting
of the responsible technical committee, which you may attend.
The sole source of supply of the apparatus (DELFIA (Dissociation-Enhanced Lanthanide Fluorescent Immunoassay), product # 1296-003) known to the committee at
this time is PerkinElmer, 940 Winter St., Waltham MA 02451, http://www.perkinelmer.com/. If you are aware of alternative suppliers, please provide this information to ASTM
International Headquarters. Your comments will receive careful consideration at a meeting of the responsible technical committee, which you may attend.
The sole source of supply of the reagent known to the committee at this time is Promega Corporation, 2800 Woods Hollow Rd., Madison, WI 53711, www.promega.com.
If you are aware of alternative suppliers, please provide this information to ASTM International Headquarters. Your comments will receive careful consideration at a meeting
of the responsible technical committee, which you may attend.
The sole source of supply of the reagent known to the committee at this time is Vector Laboratories, 30 Ingold Rd., Burlington, CA 94010, www.vectorlabs.com. If you
are aware of alternative suppliers, please provide this information to ASTM International Headquarters. Your comments will receive careful consideration at a meeting of
the responsible technical committee, which you may attend.
The sole source of supply of the reagent known to the committee at this time is Sigma-Aldrich Corp., St. Louis, MO, www.sigmaaldrich.com. If you are aware of
alternative suppliers, please provide this information to ASTM International Headquarters. Your comments will receive careful consideration at a meeting of the responsible
technical committee, which you may attend.
The sole source of supply of the cells (#CCL-81) known to the committee at this time is American Type Culture Collection (ATCC) , (ATCC), P.O. Box 1549, Manassas,
VA 20108. If you are aware of alternative suppliers, please provide this information to ASTM International Headquarters. Your comments will receive careful consideration
at a meeting of the responsible technical committee, which you may attend.
E2805 − 18
X1.2 Referenced Documents
X1.2.1 Polio Laboratory Manual, 4th edition, 2003, World Health Organization, Department of Immunization, Vaccines and
Biologicals, CH-1211, Geneva 27, Switzerland.
X1.3 Terminology
X1.3.1 IC50—the concentration of ricin that produces an inhibition of cell viability (cytotoxicity) or decrease in protein synthesis
by 50 %. This value is determined from a series of concentrations of ricin.
X1.3.2 Definitions—The following cell culture terms are from either the Health Protection Agency Culture Collections (NIH;
11 12
UK) or the National Institutes of Health (NIH) Chemical Genomics Center (NCGC).
X1.3.2.1 Cell Culture—Establishment and maintenance of cultures derived from dispersed cells taken from original tissues,
primary culture, or from a cell line or cell strain.
X1.3.2.2 Continuous Cell Culture—A culture that is apparently capable of an unlimited number of population doublings; often
referred to as an immortal cell culture.
X1.3.2.3 Immortalization—The attainment by a finite cell culture, whether by perturbation or intrinsically, of the attributes of a
continuous cell line. An immortalized cell is not necessarily one, which is neoplastically or malignantly transformed.
X1.3.2.4 Passage—The transfer or transplantation of cells, with or without dilution, from one culture vessel to another. It is
understood that any time cells are transferred from one vessel to another, a certain portion of the cells may be lost and therefore,
dilution of cells, whether deliberate or not, may occur. This term is synonymous with the term subculture.
X1.3.2.5 Passage Number—The number of times the cells in the culture have been subcultured or passed. In descriptions of this
process, the ratio or dilution of the cells should be stated so that the relative cultural age gap can be ascertained.
X1.3.2.6 Population Doubling Level—The total number of population doublings of a cell line or strain since its initiation in vitro.
X1.3.2.7 Primary Culture—A culture started from cells, tissues, or organs taken directly from organisms. A primary culture may
be regarded as such until it is successfully subcultured for the first time, when it becomes a cell line.
X1.3.2.8 Tissue Culture—Traditionally, the maintenance of fragments of tissue in vitro, but commonly is used as a generic term
including tissue explant culture, organ culture, and dispersed cell cultures (for example, cell lines and cell strains).
X1.4 Apparatus
X1.4.1 Mixer, vortex mixing motion.
X1.4.2 Display Timers.
X1.4.3 Laboratory Refrigerators (4°C), Freezer (-20°C) and Ultralow Freezer (-70 to -80°C).
X1.4.4 Water Bath, 37 6 1 ºC.
X1.4.5 Fixed Volume Pipettes—1000 μL, 200 μL, 20 μL, 10 μL, and 2 μL or adjustable pipettes of similar range. Pipettes should
be calibrated to ensure accurate dispensing of the set volumes.
http://www.who.int/vaccines/en/poliolab/webhelp/
http://www.hpacultures.org.uk/glossary/index.jsp
http://www.ncgc.nih.gov/guidance/section7.html#cell-culture-glossary
E2805 − 18
X1.4.6 Multi-Channel Pipettes, including 12-channel (20 to 200 μL), 8-channel pipettor (2 to 20 μL) and 8-channel pipettor (5
to 50 μL). Pipettes should be calibrated to ensure accurate dispensing of the set volumes.
X1.4.7 Biological Safety Cabinet, Class II, type A2, that provides a sterile environment for cell culture that will provide sterile
air flow in the cabinet and inward flow of air to protect the user. The correct performance of the biological safety cabinet should
be checked every time it is used. The user is responsible for ensuring the proper maintenance and certification of performance of
the biological safety cabinet.
X1.4.8 Tissue Culture Incubator, capable of maintaining a temperature of 37 6 1 ºC),ºC, a high relative humidity (95 %), and a
CO level of 5 % in air.
X1.4.9 Cell Culture Flasks, with sterile 25 and 75 cm surface area for cell growth.
X1.4.10 Cell Counting Slides, such as a hemocytomer or a laboratory instrument to determine cell concentration.
X1.5 Reagents
X1.5.1 Cells used for Cytoxicity Measurement:
X1.5.1.1 The choice of cells is important, since the sensitivity of cells to ricin is dependent on the binding of ricin to the surface
of cells and intracellular processing.
X1.5.1.2 Permanent cells lines obtained from biological repositories provide a source of uniform characterized cells that can be
used by a number of investigators.
X1.5.1.3 Cell lines have been engineered to express a protein that is easily measured and can be used to detect the effect of ricin
on cellular protein synthesis. The enzyme luciferase expressed in Vero cells is used to measure ricin activity (1819). The amount
of luciferase produced in the cells is measured using an assay based on light production by the enzyme activity. The expression
of Vero cells that have been engineered to express green fluorescent protein (GFP) is also used to measure the effect of ricin on
protein synthesis (1920). The decrease in cellular fluorescence in response to ricin was measured using fluorescence microscopy
or with a flow cytometer.
X1.5.2 The purity, authenticity, and stability of the cells should be assessed prior to beginning the measurement of ricin biological
activity for ricin.
X1.5.3 The purity and concentration of ricin material used as a standard should be confirmed as described in Section 7.
X1.5.4 The performance and purity of the cell culture media used should be confirmed by testing with cells of known properties.
X1.5.5 Trypsin, 0.05 % in 0.53 mM EDTA, sterile cell culture grade.
X1.5.6 Fetal Bovine Serum, cell culture grade (mycoplasma, virus, and endotoxin negative).
X1.5.7 Eagle’s Minimum Essential Media, sterile, cell culture grade.
X1.6 Procedure
X1.6.1 Cell Culture of Vero Cells:
X1.6.2 All cell work and media preparation should be performed in a sterile environment using a Class II Biological Safety
Cabinet (BSL2 hood). Proper aseptic techniques should be used to avoid contamination events.
E2805 − 18
X1.6.3 The base medium for Vero cells is Eagle’s Minimum Essential media. To make the complete growth medium, add fetal
bovine serum to a final concentration of 10 % (vol/vol).
X1.6.4 Culture the cells in an incubator maintaining an atmosphere of 95 % air, 5 % CO , and 37°C.
X1.6.5 Subculturing and Passaging:
X1.6.5.1 This example is based on Vero cells cultured in T-75 flasks (75 cm ).
X1.6.5.2 Remove and discard the culture medium. Briefly rinse the cell layer with 0.25 % (w/v) Trypsin- 0.53 Trypsin-0.53 mM
EDTA solution to remove all traces of serum that contains trypsin inhibitor.
X1.6.5.3 Add 2.0 to 3.0 mL of Try
...