1、Designation: E1482 12Standard Practice forUse of Gel Filtration Columns for Cytotoxicity Reductionand Neutralization1This standard is issued under the fixed designation E1482; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the yea
2、r of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. ScopeNOTE 1The title was formerly Standard Test Method for Neutraliza-tion of Virucidal Agents in Virucidal Efficacy Evalu
3、ations.1.1 This practice is intended to be used to reduce thecytotoxic level of the virus-test product mixture prior toassaying for viral infectivity. It is used in conjunction withevaluations of the virucidal efficacy of disinfectant solutions,wipes, trigger sprays, or pressurized disinfectant spra
4、y prod-ucts intended for use on inanimate, nonporous environmentalsurfaces. This practice may also be used in the evaluation ofhygienic handwashes/handrubs, or for other special applica-tions. The practice may be employed with all viruses and hostsystems.1.2 This practice should be performed only by
5、 personstrained in virology techniques.1.3 This practice utilizes gel filtration technology. Theeffectiveness of the practice is dependent on the ratio of gel bedvolume to sample size and uniformity in the preparation ofcolumns as well as the conditions of entrifugation. Theeffectiveness of this pra
6、ctice is maximized by investigatorpractice and experience with gel filtration techniques.1.4 This practice will aid in the reduction, but not necessar-ily elimination, of test product toxicity while preserving thetiter of the input virus.1.5 The values stated in SI units are to be regarded asstandar
7、d. No other units of measurement are included in thisstandard.1.6 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 appro-priate safety and health practices and determine the applica
8、-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2E1052 Test Method to Assess the Activity of Microbicidesagainst Viruses in SuspensionE1053 Test Method to Assess Virucidal Activity of Chemi-cals Intended for Disinfection of Inanimate, NonporousEnvironmental S
9、urfaces3. Summary of Test Methods3.1 After the exposure of a virus to a test product (orhandwash/rub product), the virus-product suspension is addedto a column of Sephadex3LH-60, Sephadex3LH-20, orSephacryl3S-1000 Superfine. The column (encased within asterile centrifuge tube in order to capture the
10、 filtrate) is placedin a centrifuge and centrifuged to separate the virus from thetest product by gel filtration. Alternatively, samples may behand-plunged using a syringe plunger. The filtrate (the columnflow-through which contains the virus) is assayed in theappropriate host system. The untreated
11、virus control suspen-sion is gel-column filtered, using the same methods/techniques,and the virus titer of the filtrate is determined by assay ofinfectivity. The residual cytotoxicity of the disinfectant isdetermined by gel filtration of the test product control under thesame conditions as those whi
12、ch were used in the test. Resultsfor the virus inactivation and test product cytotoxicity ofgel-column filtrates are recorded in the same manner asdescribed in Test Methods E1052 and E1053. The gel-columnfiltration procedures described in this practice are a modifica-tion of the method of Blackwell
13、and Chen.4NOTE 2A limitation of utilizing columns in virological assays is thatthey are unable to effectively neutralize all actives. Prior to testing, ensure1This practice is under the jurisdiction of ASTM Committee E35 on Pesticides,Antimicrobials, and Alternative Control Agentsand is the direct r
14、esponsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved Oct. 1, 2012. Published November 2012. Originallyapproved in 1992. Last previous edition approved in 2004 as E1482 04). DOI:10.1520/E1482-12.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orco
15、ntact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3Sephadex is a registered trademark of Amersham Biosciences. The sole sourceof supply of the apparatus known to the committee at this t
16、ime is AmershamBiosciences. If you are aware of alternative suppliers, please provide this informa-tion to ASTM International Headquarters. Your comments will receive carefulconsideration at a meeting of the responsible technical committee,1which you mayattend.4Blackwell, H. H., and Chen, J. H. S.,
17、“Effects of Various Germicidal Chemicalson H.EP.2 Cell Culture and Herpes simplex Virus,” Journal of the AOAC, Vol 53,1970, pp. 12291236.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1the effectiveness of gel-filtration columns with
18、the intended productchemistry. In addition, chemical neutralization is recommended to ensure/aid neutralization of certain difficult to neutralize product active(s) inaddition to the use of Sephadex columns.4. Significance and Use4.1 This practice is to be used for the removal of virucidalagents fro
19、m test product-virus mixtures, or from test product-neutralizer-virus mixtures, at or after the contact period andbefore the inoculation of these mixtures into host systems forassay of viral infectivity.4.2 The purpose of the practice is to reduce the concentra-tion of the cytotoxic properties of th
20、e test product andneutralizers in order to permit the evaluation of viral infectivityat dilutions that would otherwise be toxic to the host cells.4.3 The practice is applicable to the testing of liquid,pre-saturated towelettes, and pressurized disinfectant products,as well as handwash/rub products.N
21、OTE 3When testing handwash/rub products, the ability of thesolution to pass through the column must be verified prior to testing.Certain products with high viscosities are unable to pass through columns.If the product is determined to be too viscous, alternative neutralizationmethods should be emplo
22、yed.4.4 This practice is compatible with organic soil loads, hardwater, disinfectants containing organic solvents, and chemicalneutralizers.5. Reagents and Materials5.1 Reagents:5.1.1 Purity of ReagentsReagent grade chemicals shall beused in all tests. Unless otherwise indicated, it is intended that
23、all reagents shall conform to the specifications of the Commit-tee on Analytical Reagents of the American Chemical Society,where such specifications are available.5Other grades may beused, provided it is first ascertained that the reagent is ofsufficiently high purity to permit its use without lesse
24、ning theaccuracy of the determination.5.1.2 Phosphate Buffered Saline (PBS).65.1.3 Sterile Distilled or Deionized Water.5.1.4 1% Albumin Solution (in PBS).5.2 Sephadex Gel Filtration Column Assembly:5.2.1 Sephadex LH-60 or LH-20, compatible with organicsolvents. (Sephacryl S-1000 Superfine may be su
25、bstituted.)5.2.2 Syringe, 5-cc or 10-cc, disposable.5.2.3 Glass wool, sterilized.5.2.4 Centrifuge tube, 15- and/or 50-mL, conical, sterile,and disposable.5.3 Labware:5.3.1 Pipettes, serological, 10-, 5-, and 2-mL.5.3.2 Erlenmeyer Flask, sterile, 250-mL or other suitablesterilizable container.5.3.3 T
26、est Tube Rack or Holder, for 15- and 50-mL tubes.5.3.4 Test Tubes, 18 by 150 mm.5.3.5 Laboratory Film, or other sealing film. (Aluminumfoil may also be used to cover the syringe/glass-wool/tubeassembly and then autoclaved).5.4 Equipment:5.4.1 Centrifuge, clinical, with rotor and shields capable ofho
27、lding 15- and/or 50-mL centrifuge tubes, and running at ar/min that generates 550 to 650 g.5.4.2 Refrigerator, 2to8C5.4.3 Autoclave.6. Procedure6.1 Suspend the Sephadex in a large excess of steriledistilled or deionized water in an Erlenmeyer flask or othersuitable sterilizable container. Use an amo
28、unt of Sephadexsufficient for the number of columns to be prepared (approxi-mately 0.5 g of Sephadex per column) or prepare a largervolume slurry to give a final suggested concentration of 5 to22 % Sephadex g/v. Sterilize slurry by autoclaving. (Exampleparameters for autoclaving are 121C at 15 psi (
29、pounds ofpressure per square inch) for at least 15 min. Autoclaveparameters vary depending on autoclave model, altitude, andso forth.) Allow slurry to cool to room temperature. Store at 2to 8C for longer term storage if desired.6.1.1 Alternatively, Sephadex may be prepared by firstpreparing a 1% alb
30、umin, antibiotics (optional), and PBSsolution. This solution is filter sterilized. Sephadex is thenadded to this filter sterilized solution at the desired concentra-tion of 5 to 22%.6.2 Select the syringe size to be used depending on the sizeof the column desired.A5-cc (mL) syringe is used for 3 to
31、5-cc(mL) columns (1.0 mL of sample to be added); a 10-cc (mL)syringe is used for 6 to 8-cc (mL) columns (1.0 to 5.0 mL ofsample to be added).NOTE 4If sample added is at the higher volume range, the bed sizemay need to be adjusted so that it is at the maximum allowable height inorder to ensure remova
32、l of cytotoxic properties.6.3 Remove the cap from the syringe tip, remove theplunger from the syringe, and place the syringe in an 18 by150-mm test tube or in another suitable tube holder which cancapture the column flow-through during column preparationprocedures.6.4 Place a small wad of glass wool
33、 in the syringe to coverthe internal tip opening. The wad should have a diameterapproximately the same size as the internal syringe diameter,and it should be sufficiently thick to hold the swollen Sephadexbeads while allowing water to pass readily. Cover assemblywith aluminum foil and autoclave to s
34、terilize.NOTE 5Sterilized column assembly without Sephadex slurry can beprepared and stored prior to testing. Additionally, sterile, individually-wrapped syringes and sterile glass wool may be utilized and handled underaseptic techniques to eliminate the need for autoclaving column assem-blies.5Reag
35、ent Chemicals, American Chemical Society Specifications, AmericanChemical Society, Washington, DC. For Suggestions on the testing of reagents notlisted by the American Chemical Society, see Annual Standards for LaboratoryChemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeiaand
36、 National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,MD.6Dulbecco, R., and Vogt, M., “Plaque Formation and Isolation of Pure Lineswith Poliomyelitis Virus,” Journal of Experimental Medicine, Vol 99, 1954, p. 167.E1482 1226.5 Just prior to testing, swirl the Sephadex slurry and
37、pipetSephadex into the syringe barrel. Allow the excess water todrain, and repeat until the desired bed size of Sephadex hasformed. If the column is not used immediately, cover withlaboratory film or aluminum foil and store at 2-8C for a shortperiod of time to prevent the column from drying out.6.6
38、To use the column, allow the water to flow through, andthen equilibrate (optional) with PBS or a 1 % albumin solutionby passing 10 to 20 mL through the column.6.7 Place the column in a sterile 15-or 50-mL conicalcentrifuge tube. Cover the column with a tube cap, laboratoryfilm or other suitable cove
39、r. Place the tube with the preparedcolumn in the centrifuge and centrifuge at approximately 550to 650 g for 3 to 4 min to clear the void volume.6.8 Remove the column, discard the void volume, and/orplace the column in a new tube.6.9 Gently pipet the appropriate volume of the virus-testproduct mixtur
40、e (depending on the column size) onto theSephadex, place the column in the centrifuge, and centrifugeagain for 3 to 4 min at exactly the same r/min as in the previousstep. Alternatively, samples may be hand plunged utilizing asyringe plunger to push the liquid through the column to collectthe filtra
41、te. Caution should be taken to avoid over plunging ofsamples, which will push the Sephadex out into the filtrate.6.10 Remove the column from the centrifuge; if necessary,collect the filtrate (column flow-through), and titrate forinfectivity.6.11 The virus and test product control samples are handled
42、in the same manner as previously described.6.12 Optional ControlTo determine the reduction of cy-totoxicity of a specific test substance when utilizing gel-filtration columns, a control whereas the chemically neutral-ized test product is run through a column is compared with thechemically neutralize
43、d test product which is not passed througha column. Compare the toxicity induced cytopathic effects onthe host cells to calculate the reduction in toxicity with the useof Sephadex columns.NOTE 6 It is up to the end user to decide whether the use ofgel-filtration columns is appropriate in order to ob
44、tain the assaysnecessary log reduction.7. Spray Products7.1 Prior to applying the virus-product mixture to a Sepha-dex column (when not using chemical neutralizers), the vol-ume of the mixture may be adjusted (that is, up to 2 mL) withan appropriate aqueous medium such as water, PBS, tissueculture m
45、edium, or neutralizer solution in order to obtainenough sample to allow for titration. For example, utilize thispractice when alcohol-based spray products which evaporateduring the contact period are being tested.8. Chemical NeutralizersNOTE 7Chemical neutralization is recommended for difficult tone
46、utralize actives.8.1 When utilized, the chemical neutralizer is added at theend of the contact time, and the virus-test product-neutralizermixture is then immediately added to the Sephadex column.9. Precision and Bias9.1 A precision and bias statement cannot be made for thispractice at this time.10.
47、 Keywords10.1 cytotoxicity; disinfectant; gel filtration; neutralization;tissue culture; virucidal; virucidal neutralization methodASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentionedin this standard. Users of this standard
48、are expressly advised that determination of the validity of any such patent rights, and the riskof infringement of such rights, are entirely their own responsibility.This standard is subject to revision at any time by the responsible technical committee and must be reviewed every five years andif no
49、t revised, either reapproved or withdrawn. Your comments are invited either for revision of this standard or for additional standardsand should be addressed to ASTM International Headquarters. Your comments will receive careful consideration at a meeting of theresponsible technical committee, which you may attend. If you feel that your comments have not received a fair hearing you shouldmake your views known to the ASTM Committee on Standards, at the address shown below.This standard is copyrighted by ASTM International, 100 Barr Harbor Drive, PO Box C700, West C
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