1、Designation: E2805 11Standard Practice forMeasurement of the Biological Activity of Ricin1This standard is issued under the fixed designation E2805; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision. A numbe
2、r in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.INTRODUCTIONRicin is a member of the protein toxins that cause their physiological effect by inactivation ofribosomes. Ricin is a member of the class 2
3、ribosome inactivating proteins (1).2Other members of thisclass of toxins include the proteins abrin and Shiga toxin.Ricin consists of two chains, the A-chain that is responsible for the N-glycosidase enzymaticactivity and the B-chain that is needed for cell binding and intra-cellular processing. Ric
4、in is aheterogeneous 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 genesencoding putative ricin and ricin-like proteins in the genome of R. communis (3) resulting ind
5、ifferences in the amino acid sequence of the subunits. The differences in amino acid sequence andglycosylation both contribute to the heterogeneity of ricin.1. Scope1.1 This guide is intended for the manufacturers and usersof ricin reference material. Ricin reference materials arewell-characterized
6、materials that can be used to test detectiondevices and calibrate laboratory measurements. It is anticipatedthat ricin reference materials will be characterized by bio-chemical methods in addition to the measurement of biologicalactivity.1.2 This practice details the measurement of ricin biologicala
7、ctivity using a cell-free translation (CFT) assay (4).1.3 The CFT assay has been developed for use in anybiotechnology laboratory where determination or confirmationof ricin biological activity is required.1.4 The CFT assay has been validated by the U.S. ArmyMedical Research Institute of Infectious
8、Diseases (USAM-RIID) VP-016 Validation of Cell-Free Translation Assay forthe Detection of Ricin Toxin Biological Activities in compli-ance (5) with Good Laboratory Practices (GLP) Regulations ofthe Food and Drug Administration (21 CFR Part 58). Strictadherence to the protocol is necessary for validi
9、ty of the testresults.1.5 Appendix X1 and Appendix X2 also provide guidancefor the measurement of the biological activity of ricin usingcell-based assays and the use of synthetic enzyme substrates.1.6 Ricin is a category 2 select agent and acquisition of thericin standard must adhere to the Center f
10、or Disease Control(CDC) regulations. Ricin is listed on the select agent list (42CFR Part 72).3The possession, transfer, and use of ricin arerestricted under the Public Health Security Preparedness Act(CRS Report RL31263 Public Health Security and Bioterror-ism Preparedness and Response Act (P.L. 10
11、7-188): Provisionand Changes to Preexisting law). Access to stores of ricin islimited (USA Patriot Act, P.L. 107-56). Ricin is also aprohibited substance under the Biological Weapons Conven-tion and the Chemical Weapons Convention (CRS ReportRL31559 Proliferation Control Regimes: Background andStatu
12、s).1.7 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.8 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish a
13、ppro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use. Ricin is anextremely dangerous toxin. See Section 9 for specific hazardsinformation.2. Referenced Documents2.1 ASTM Standards:41This practice is under the jurisdiction of ASTM Committee E
14、54 on HomelandSecurity Applications and is the direct responsibility of Subcommittee E54.01 onCBRNE Sensors and Detectors.Current edition approved Jan. 1, 2011. Published March 2011. DOI: 10.1520/E2805-11.2The boldface numbers in parenthesis refer to the list of references at the end ofthis standard
15、.3Available at http:/www.bt.cdc.gov/Agent/agentlist.asp.4For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.1Copy
16、right ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.F2149 Test Method for Automated Analyses of CellstheElectrical Sensing Zone Method of Enumerating and Siz-ing Single Cell Suspensions2.2 Code of Federal Regulations:21 CFR Part 58 Good labor
17、atory practice for nonclinicallaboratory studies542 CFR Part 72 Interstate shipment of etiologic agents62.3 ANSI/ATCC Standard:7ASN-0001-2009 Standardization of In-Vitro Assays to De-termine Anthrax Toxin Activities3. Terminology3.1 Abbreviations:3.1.1 CFTcell free translation.3.1.2 CPScounts per se
18、cond, units of luminescence in-strument.3.1.3 IC50concentration of ricin that produces inhibitionof 50 % of the activity in an assay.3.1.4 kDamolecular mass in kilo Dalton units.3.1.5 PBSphosphate buffered saline.4. Summary of Practice4.1 The CFT assay for measuring biologically active ricin isbased
19、 on its inhibitory effects on protein synthesis (6, 7). Whenadded to a rabbit reticulocyte translation mixture containingluciferase mRNA, ricin inhibits translation of the mRNA intothe enzyme luciferase. Luciferase is then detected using abuffer containing the luciferin substrate. The test is a biol
20、umi-nescence assay that measures the amount of luminescenceproportional to the amount of luciferase produced from proteintranslation (RNAprotein). When active ricin is present, theamount of luminescence decreases corresponding to a decreasein the production of the luciferase enzyme. The amount ofpro
21、tein (luciferase) produced is directly proportional to theamount of luminescence generated. The decrease in lumines-cence is directly proportional to the amount of active ricin inthe sample. Confirmation that translation inhibition is causedby the presence of active ricin is determined by mixing ana
22、liquot of the ricin samples with anti-ricin antibody beforeadding to the translation mixture. The neutralized ricin doesnot inhibit luciferase translation, and therefore, luminescencedoes not decrease.4.2 Cell-based assays use mammalian cells maintained inculture to measure the effect of ricin on ce
23、ll death or damage(cytotoxicity). Ricin is added to the cells and after an incuba-tion period, the effect on cell cytotoxicity is measured. Thericin-treated cells are compared to control cells (without addedricin) maintained under the same conditions. Guidance is givenin Appendix X1.4.3 The N-glycos
24、idase enzymatic activity of the A-chain ofricin can be measured using synthetic oligonucleotides. Theenzyme activity is measured either by the released adenine orthe effect on the depurinated substrate using a number ofmethods. Guidance is given in Appendix X2.5. Significance and Use5.1 The CFT assa
25、y provides a sensitive and reliable methodto detect ricin biological activity and results can be generatedwithin 3 h. The assay measures the amount of ricin biologicalactivity when compared to a known ricin standard and providesa quantitative measurement for active ricin.5.2 The lower limit of quant
26、itation and the upper limit ofquantitation for ricin using the CFT assay was measured at 10ng/mL and 170 ng/mL, respectively (5).5.3 This practice is focused on the measurement of refer-ence materials and not environmental samples. Additionalcontrol runs may be needed for measurements of environmen-
27、tal samples to ensure that the presence of additional materialsin the samples (also referred to as the matrix) will interferewith the measurements.5.4 The CFT assay may be used to determine the presenceof active ricin in forensic or bioterrorist samples if theappropriate controls are utilized to ens
28、ure valid results (5).5.5 The methods described in this document measure thebiological activity of ricin and do not detect the presence ofinactivated ricin in a given sample.5.6 Ricin reference materials have a number of applications,such as testing detection devices, laboratory instruments,environm
29、ental sampling methods, disinfection studies, andbasic research.6. Apparatus6.1 List of EquipmentThe make and model are providedas examples, however equivalent apparatus may also be used.6.1.1 Mixer, vortex mixing motion6.1.2 Display timers.6.1.3 Incubator, capable of maintaining temperature of (376
30、 1C).6.1.4 96 Well Microplate Luminometer and LuminescenceTest Plate8.6.1.5 Microplate Data Analysis Software, KC4, with Pow-erReports,y v3.0.96.1.6 Plateshake.105Available from Food and Drug Administration (FDA), 5600 Fishers Ln.,Rockville, MD 20857, http:/www.fda.gov.6Available from U.S. Governmen
31、t Printing Office Superintendent of Documents,732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http:/www.access.gpo.gov.7Available from American National Standards Institute (ANSI), 25 W. 43rd St.,4th Floor, New York, NY 10036, http:/www.ansi.org.8The sole source of supply of the appara
32、tus (MicroLumi XS) known to thecommittee at this time is Harta Instruments, Inc., 8 Russell Ave Unit 106,Gaithersburg, MD 20877, . If you are aware of alterna-tive suppliers, please provide this information to ASTM International Headquarters.Your comments will receive careful consideration at a meet
33、ing of the responsibletechnical committee,1which you may attend.9The sole source of supply of the apparatus known to the committee at this timeis BioTek, P.O. Box 998, Highland Park, Winooski, VT 05404, http:/ If you are aware of alternative suppliers, please provide thisinformation to ASTM Internat
34、ional Headquarters. Your comments will receivecareful consideration at a meeting of the responsible technical committee,1whichyou may attend.10The sole source of supply of the apparatus (DELFIA (Dissociation-EnhancedLanthanide Fluorescent Immunoassay), product # 1296-003) known to the commit-tee at
35、this time is PerkinElmer, 940 Winter St., Waltham MA 02451, http:/ If you are aware of alternative suppliers, please provide thisinformation to ASTM International Headquarters. Your comments will receivecareful consideration at a meeting of the responsible technical committee,1whichyou may attend.E2
36、805 1126.1.7 Laboratory Refrigerators (4C), Freezer (-20C), andUltralow Freezer (-70C or lower).6.1.8 Water Bath,376 1C.6.1.9 Fixed Volume Pipettes, 1000 L (200 to 1000 L), 200L (20 to 200 L), 20 L (5 to 20 L), 10 L (1 to 10 L), and2 L (0.5 to 2 L), or adjustable pipettes of this rangePipettes shoul
37、d be regularly calibrated to ensure accuratedispensing of the set volumes.6.1.10 Multi-Channel Pipettes including 12-Channel (20 to200 L), 8-Channel Pipettor (2 to 20 L), and 8-Channelpipettor (5 to 50 L)Pipettes should be regularly calibratedto ensure accurate dispensing of the set volumes.7. Reage
38、nts7.1 Reagents for the CFT AssayThe validation of theassay was performed with reagents purchased from the specificvendors. The reproducibility and precision of the assay isdependent upon the quality of the reagents. The specificreagents have been tested to work in the validated assay.Substitution o
39、f reagents will require testing to ensure the sameperformance.7.2 Rabbit Reticulocyte Lysate, nuclease treated.11The rab-bit reticulocyte lysate is prepared from New Zealand whiterabbits using a standard protocol under quality-controlledconditions (11). After the reticulocytes are lysed, the lysate
40、istreated with micrococcal nuclease in order to destroy endog-enous mRNA. The lysate is further optimized for mRNAtranslation by addition of an energy generating system, amixture of tRNAs, hemin (to prevent inhibition of initiation),potassium acetate, and magnesium acetate. The rabbit reticu-locyte
41、lysate contains no endogenous mRNA and thereforetranslates only the mRNA added to the lysate (12). The abilityto translate only one protein that can be detected permits amore accurate analysis of the biological effect of ricinsenzymatic reaction.7.3 Amino Acid Mixture, CompleteThe amino acid mix-tur
42、e, complete, has been prepared for use in the rabbitreticulocyte lysate systems and is an aqueous solution contain-ing1mM each of the 20 essential amino acids. The mixture issterile and RNA-free.7.4 Luciferase Control RNA11Luciferase control RNAused in these studies is commercially made using SP6 RN
43、Apolymerase transcription of a plasmid bearing the codingregion for the luciferase gene with an additional 30 adenineresidues that creates an uncapped, polyadenylated mRNA. Theproduct of this luciferase control RNA is a monomeric protein(61 kDa) that does not require post-translational processing or
44、modification for enzymatic activity. Only full-length luciferaseis active. In most laboratories, contamination with extraneousluciferase or luciferase mRNA does not occur because lu-ciferase is not found in laboratory environments.7.5 Luciferase Reporter Buffer11The luciferin reactionsystem is purch
45、ased as two components, lyophilized assayreagent and assay buffer. When mixed, the luciferase assaybuffer provides high quantum efficiency and no backgroundluminescence in the reticulocyte system or in the assaychemistry. Light is produced by converting the chemicalenergy of luciferin oxidation thro
46、ugh an electron transition,forming the product molecule of oxyluciferin. The luciferasereaction buffer contains coenzymeAthat improves kinetics andallows for greater enzymatic turnover resulting in increasedlight intensity. Unlike many chemiluminescent reactions, thisreaction remains constant for ne
47、arly two minutes, therebypermitting accurate measurements using a microtiter format(4). As noted, the amount of luminescence emitted with thisreaction buffer is proportional to the amount of luciferasepresent and therefore provided a comparative measurement ofthe luciferase amount in samples treated
48、 with toxins.7.6 RNase Inhibitor11, a 50 kDa protein that noncovalentlybinds to RNases in a 1:1 ratio, is a broad-spectrum RNaseinhibitor. The product is purified using a combination of ionexchange and affinity chromatography.7.7 Sterile Nuclease-Free De-ionized Water11, sterile andRNase free.7.8 Ri
49、cin Standard and AntibodyReagents should be com-parable to the following products:7.8.1 Ricin Toxin (Ricin), Ricinus Communis Agglutinin II,5 mg/mL12This product, purified from castor beans, is theapproximately 60 kDa molecular weight protein that is highlytoxic with little agglutinin activity. The toxin is purchased as aliquid (5 mg/mL) and contains 0.08 % sodium azide. At thisconcentration, the sodium azide does not affect the assay.7.8.2 Antibody to Ricinus Communis Agglutinin I an
