ASTM E2805-2018 Standard Practice for Measurement of the Biological Activity of Ricin.pdf

1、Designation: E2805 11E2805 18Standard 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.

2、 A number 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

3、class 2 ribosome inactivating proteins (1).2 Other members of thisclass of toxins include the proteins abrin and Shiga toxin.Ricin Ricinus communis (Ricin) consists of two chains, the A-chain that is responsible for theN-glycosidase enzymatic activity and the B-chain that is needed for cell binding

4、and intra-cellularprocessing. Ricin is a heterogeneous protein with molecular weights ranging from approximately 62to 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 genom

5、e of R. communis(3) resulting in differences in the amino acid sequence of the subunits. The differences in amino acidsequence and glycosylation both contribute to the heterogeneity of ricin. Various research reports existdescribing different means of detecting ricin activity.1. Scope1.1 This guide

6、is intended for the manufacturers and users of ricin reference material. Ricin reference materials arewell-characterized materials that can be used to test detection devices and calibrate laboratory measurements. It is anticipated thatricin reference materials will be characterized by biochemical me

7、thods 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 ricinbi

8、ological 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) withGood Laboratory Practices (GLP

9、) Regulations of the Food and Drug Administration (21 CFR Part 58). Strict adherence to theprotocol 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 usingcell-based assays and the use of synthe

10、tic 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 andPrevention (CDC) regulations. Ricin is listed on the select agent list (42 CFR Part 72).3 The possession, transfer, and use of ricinare restricted under

11、 the Public Health Security Preparedness Act (CRS Report RL31263 Public Health Security and BioterrorismPreparedness 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 un

12、der the Biological Weapons Convention and the ChemicalWeapons 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 This practice is under th

13、e jurisdiction ofASTM Committee E54 on Homeland SecurityApplications and is the direct responsibility of Subcommittee E54.01 on CBRNESensors and Detectors.Current edition approved Jan. 1, 2011Dec. 1, 2018. Published March 2011December 2018. Originally approved in 2011. Last previous edition approved

14、 in 2011 asE2805 11. DOI: 10.1520/E2805-11.10.1520/E2805-18.2 The boldface numbers in parenthesis refer to the list of references at the end of this standard.3 Available at http:/www.bt.cdc.gov/Agent/agentlist.asp.https:/emergency.cdc.gov/agent/agentlist.asp.This document is not an ASTM standard and

15、 is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Becauseit may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only t

16、he current versionof the standard as published by ASTM is to be considered the official document.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States11.8 This standard does not purport to address all of the safety concerns, if any, associa

17、ted with its use. It is the responsibilityof the user of this standard to establish appropriate safety safety, health, and healthenvironmental practices and determine theapplicability of regulatory limitations prior to use. Ricin is an extremely dangerous toxin. See Section 9 for specific hazardsinf

18、ormation.1.9 This international standard was developed in accordance with internationally recognized principles on standardizationestablished in the Decision on Principles for the Development of International Standards, Guides and Recommendations issuedby the World Trade Organization Technical Barri

19、ers to Trade (TBT) Committee.2. Referenced Documents2.1 ASTM Standards:4E2458 Practices for Bulk Sample Collection and Swab Sample Collection of Visible Powders Suspected of Being BiologicalAgents and Toxins from Nonporous SurfacesE3131 Specification for Nucleic Acid-Based Systems for Bacterial Path

20、ogen Screening of Suspicious Visible PowdersF2149 Test Method for Automated Analyses of Cellsthe Electrical Sensing Zone Method of Enumerating and Sizing SingleCell Suspensions2.2 Code of Federal Regulations:21 CFR Part 58 Good laboratory practice for nonclinical laboratory studies542 CFR Part 72 In

21、terstate shipment of etiologic agents62.3 ANSI/ATCC Standard:7ASN-0001-2009 Standardization of In-Vitro Assays to Determine Anthrax Toxin Activities2.4 ISO Standard:8ISO Guide 34 General requirements for the competence of reference material producersISO 203911 Biotechnology Cell Counting Part 1: Gen

22、eral Guidance on Cell Counting Methods2.5 Federal Standard:618 USC 178 Definitions3. Terminology3.1 Definitions:3.1.1 aseptic technique, noperation or performance of a procedure or method under carefully controlled conditions to reducethe risk of exposure and prevent the introduction of unwanted mat

23、erial/matter (contamination) into a sample. E24583.1.2 calibration, nset of operations that establish, under specified conditions, the relationship between the values ofquantities indicated by a measurement instrument or measuring system or values represented by a material measure or a referencemate

24、rial and the corresponding values realized by standards. Eurochem Selection (6)3.1.3 hazard, nsomething that is potentially dangerous or harmful, often the root cause of an unwanted outcome; a danger orperil. NIMS3.1.4 inhibition, nundesirable effect that can result in a false negative result and is

25、 typically caused by the presence ofcompounds that interfere with the assay or detection process. E31313.1.5 reference material, nmaterial, sufficiently homogenous and stable with respect to one or more specified properties thathas been established to be fit for its intended use in the measurement p

26、rocess; properties can be quantitative or qualitative. ISOGuide 343.1.6 risk, nthe probability of suffering a loss or harm or injury; peril. E24583.1.7 toxin, nthe toxic material or product of plants, animals, microorganisms (including but not limited to, bacteria, viruses,fungi, rickettsiae, or pro

27、tozoa), or infectious substances, or a recombinant or synthesized molecule, whatever their origin andmethod of product, and includes: (1) any poisonous substance or biological product that may be engineered as a result ofbiotechnology produced by a living organism; or (2) any poisonous isomer or bio

28、logical product, homolog, or derivative of sucha substance. ISO USC 1783.2 Abbreviations:Abreviations and Acronyms:4 For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer Service at serviceastm.org. For Annual Book of ASTM Standardsvolume information, refer to th

29、e standards Document Summary page on the ASTM website.5 Available from Food and Drug Administration (FDA), 5600 Fishers Ln., Rockville, MD 20857, http:/www.fda.gov.6 Available from U.S. Government Printing Office Superintendent of Documents, 732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 204

30、01, http:/www.access.gpo.gov.7 Available from American National Standards Institute (ANSI), 25 W. 43rd St., 4th Floor, New York, NY 10036, http:/www.ansi.org.8 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

31、 106, Gaithersburg, MD20877, . If you are aware of alternative suppliers, please provide this information toASTM International Headquarters.Your comments will receivecareful consideration at a meeting of the responsible technical committee,Available from International Organization for Standardizatio

32、n (ISO), ISO Central Secretariat, BIBCII, Chemin de Blandonnet 8, CP 401,1 which you may attend.1214 Vernier, Geneva, Switzerland, http:/www.iso.org.E2805 1823.2.1 CFTcell free translation.3.2.2 CPScounts per second, units of luminescence instrument.3.2.3 IC50concentration of ricin that produces inh

33、ibition of 50 % of the activity in an assay.3.2.4 kDamolecular mass in kilo Dalton units.3.2.5 NIMSNational Incident Management System3.2.6 PBSphosphate buffered saline.4. Summary of Practice4.1 The CFT assay for measuring biologically active ricin is based on its inhibitory effects on protein synth

34、esis (67, 78). Whenadded to a rabbit reticulocyte translation mixture containing luciferase mRNA, messenger ribonucleic acid (mRNA), ricin inhibitstranslation of the mRNA into the enzyme luciferase. Luciferase is then detected using a buffer containing the luciferin substrate.The test is a biolumine

35、scence assay that measures the amount of luminescence proportional to the amount of luciferase producedfrom protein translation (RNA protein). When active ricin is present, the amount of luminescence decreases corresponding toa decrease in the production of the luciferase enzyme. The amount of prote

36、in (luciferase) produced is directly proportional to theamount of luminescence generated. The decrease in luminescence is directly proportional to the amount of active ricin in thesample. Confirmation that translation inhibition is caused by the presence of active ricin is determined by mixing an al

37、iquot of thericin samples with anti-ricin antibody before adding to the translation mixture. The neutralized ricin does not inhibit luciferasetranslation, and therefore, luminescence does not decrease.4.2 Cell-based assays use mammalian cells maintained in culture to measure the effect of ricin on c

38、ell death or damage(cytotoxicity). Ricin is added to the cells and after an incubation period, the effect on cell cytotoxicity is measured. Thericin-treated cells are compared to control cells (without added ricin) maintained under the same conditions. Guidance is given inAppendix X1.4.3 The N-glyco

39、sidase enzymatic activity of theA-chain of ricin can be measured using synthetic oligonucleotides. The enzymeactivity is measured either by the released adenine or the effect on the depurinated substrate using a number of methods. Guidanceis given in Appendix X2.5. Significance and Use5.1 The CFTass

40、ay provides a sensitive and reliable method to detect ricin biological activity and results can be generated within3 h. The assay measures the amount of ricin biological activity when compared to a known ricin standard and provides aquantitative measurement for active ricin.5.2 The lower limit of qu

41、antitation and the upper limit of quantitation for ricin using the CFT assay waswere measured at 10ng/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 runsmay be needed for measurements of envir

42、onmental samples to ensure that the presence of additional materials in the samples (alsoreferred 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 appropriatecontrols are util

43、ized 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 inactivatedricin in a given sample.5.6 Ricin reference materials have a number of applications, such as testing detection devices, laboratory instrum

44、ents,environmental sampling methods, disinfection studies, and basic research.6. Apparatus6.1 List of EquipmentThe make and model are provided as examples, however equivalent apparatus may also be used.6.1.1 Mixer, Mixervortex mixing motionmotion.6.1.2 Display timers.6.1.3 Incubator, Incubatorcapabl

45、e of maintaining temperature of (37 6 1C).6.1.4 96 Well Microplate Luminometer and Luminescence Test Plate.6.1.5 Microplate Data Analysis Software,Software. KC4, with PowerReports, v3.0.96.1.6 Plateshake.Plateshaker. 106.1.7 Laboratory Refrigerators (4C), Freezer (-20C), and Ultralow Freezer (-70C o

46、r lower).6.1.8 Water Bath, Bath37 6 1C.6.1.9 Fixed Volume Pipettes, 1000 L (200 to 1000 L), 200 L (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 should be regularly calibrated to ensure accurate dispensing ofthe set volumes.E2805

47、1836.1.10 Multi-Channel Pipettes including 12-Channel (20 to 200 L), 8-Channel Pipettor (2 to 20 L), and 8-Channel pipettor(5 to 50 L)Pipettes should be regularly calibrated to ensure accurate dispensing of the set volumes.7. Reagents7.1 Reagents for the CFT AssayThe validation of the assay was perf

48、ormed 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 testedto work in the validated assay. Substitution of reagents will require testing to ensure the same performance.7.2

49、 Rabbit Reticulocyte Lysate,Lysate (nuclease treated)nuclease treated.The rabbit reticulocyte lysate is prepared from NewZealand white rabbits using a standard protocol under quality-controlled conditions (89). After the reticulocytes are lysed, thelysate is treated with micrococcal nuclease in order to destroy endogenous mRNA. The lysate is further optimized for mRNAtranslation by addition of an energy generating system, a mixture of tRNAs, transfer RNAs (tRNAs), hemin (to prevent inhibitionof initiat