1、Designation: D8238 18Standard Test Method forImmunological Assay to Quantify Extractable GuayuleNatural Rubber (GNR) Proteins1This standard is issued under the fixed designation D8238; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision
2、, the year 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. Scope1.1 This test method covers an immunological method todetermine the amount of protein in Guayule Natural Rub
3、ber(GNR) and its products using rabbit antisera specific forGuayule Natural Rubber latex proteins. This immunoassayprocedure quantitatively measures the level of GNR proteins insolution using an inhibition format. The samples may includebut are not restricted to, gloves or other GNR product extracts
4、,and liquid GNR samples which have been collected in order tomeasure the protein levels.1.2 For the purpose of this test method, the range of proteinwill be measured in terms of microgram to milligram quanti-ties.1.3 This standard does not purport to address all of thesafety concerns, if any, associ
5、ated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety, health, and environmental practices and deter-mine the applicability of regulatory limitations prior to use.1.4 This international standard was developed in accor-dance with internationally reco
6、gnized principles on standard-ization established in the Decision on Principles for theDevelopment of International Standards, Guides and Recom-mendations issued by the World Trade Organization TechnicalBarriers to Trade (TBT) Committee.2. Referenced Documents2.1 ASTM Standards:2D5712 Test Method fo
7、r Analysis of Aqueous ExtractableProtein in Latex, Natural Rubber, and Elastomeric Prod-ucts Using the Modified Lowry MethodD6499 Test Method for Immunological Measurement ofAntigenic Protein in Hevea Natural Rubber (HNR) and itsProductsD7427 Test Method for Immunological Measurement ofFour Principa
8、lAllergenic Proteins (Hev b 1, 3, 5 and 6.02)in Hevea Natural Rubber and Its Products Derived fromLatexE691 Practice for Conducting an Interlaboratory Study toDetermine the Precision of a Test MethodE177 Practice for Use of the Terms Precision and Bias inASTM Test Methods3. Terminology3.1 Definition
9、s:3.1.1 allergens, nprotein antigens which induce allergicimmune reactions typically mediated through IgE antibodies.3.1.2 antibody, nan immunoglobulin, a protein that isproduced as a part of the immune response which is capable ofspecifically combining with the antigen.3.1.3 antigen, nany substance
10、 that provokes an immuneresponse when introduced into the body.3.1.4 background absorbance, nthe absorbance reading inthe solution resulting from the presence of chemicals, ions etc.other than the substrate being determined.3.1.5 blocking solution, na non-reactive protein solutionused to prevent non
11、specific antibody adsorption.3.1.6 calibration, nthe standardization of an instrumentsetting or an assay configuration.3.1.7 concentration range, nthe recommended analyteconcentration range in g/mL that produces an absorbancereading of 0.1 to 2.0 OD units.3.1.8 enzyme linked immunosorbent assay (ELI
12、SA), nanimmunological test method to quantify antigen or antibodylevels using an enzyme as the detection mechanism.3.1.9 primary antibody, nthe antibody used first in asequence that is specific for the antigen.3.1.10 reference solution, nthe solution to which the testsample is being compared against
13、.3.1.11 repeatability, nthe variability or test error betweenindependent test results obtained within a single laboratory.3.1.12 reproducibility, nthe variability or error betweentest results obtained in different laboratories.1This test method is under the jurisdiction of ASTM Committee D11 on Rubb
14、erand Rubber-like Materials and is the direct responsibility of Subcommittee D11.40on Consumer Rubber Products.Current edition approved Dec. 1, 2018. Published January 2019. DOI: 10.1520/D8238-18.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at
15、 serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United StatesThis international standard was developed in accorda
16、nce with internationally recognized principles on standardization established in the Decision on Principles for theDevelopment of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.13.1.13 secondary antibody, nthe en
17、zyme conjugated anti-body used second in the sequence that is specific for the heavychain of the primary antibody.3.1.14 standard solution, nthe preparation of standardanalyte used as a reference to which the unknown sample beingmeasured is compared.3.1.15 substrate, nthe material or substance upon
18、whichan enzyme reacts.3.1.16 titer, nthe strength of the antibody solution (forexample, concentration and affinity of antibody).4. Summary of Test Method4.1 The test sample is extracted in an aqueous buffer. Theextract is recovered, and the antigen levels are determinedusing inhibition Enzyme Linked
19、 ImmunoSorbent Assay(ELISA) technology.3The ELISAassay is based on polyclonalantiserum which recognizes GNR proteins. ELISA technologytakes advantage of the specificity and sensitivity of theantibody-antigen reaction.As a variation of the ELISAmethodthis inhibition ELISAhas been developed for the de
20、tection andquantification of GNR protein antigens. In the inhibitionELISA, the GNR antigen is immobilized by absorption to thewells of a 96-well test plate. The sample extract is mixed withantibody specific for GNR protein in a dilution plate. Follow-ing a brief incubation to allow for antibody reco
21、gnition of therelevant GNR antigens, the mixture is added to the immobi-lized antigen in the assay plate. Anti-GNR antibody which isnot bound to the soluble GNR protein in the sample will bindto the immobilized antigen. The plate is washed to remove thesoluble antigen antibody complexes and a second
22、ary antibody(enzyme-labeled anti-immunoglobulin) is added which at-taches to the immobilized antigen-bound specific antibody.Next, the enzyme substrate is added and the reaction of theenzyme on the substrate results in a color change. A reductionin the amount of color in comparison to an uninhibited
23、 controlis an indicator of the amount of antigen present in the sample.Comparison to a standard curve generated using knownamounts of GNR protein permits quantification. The assay ishighly sensitive and can quantitate GNR proteins in thenanogram per millilitre range.5. Significance and Use5.1 NRL de
24、rived from Hevea contains over 200 proteins anumber of which are allergens capable of eliciting Type 1anaphylactic reactions. Guayule Natural Rubber derived fromthe Parthenium argentatum plant contains many fewer proteinsand at much lower levels than Hevea NRL. While there havebeen no reports of all
25、ergic reactions to GNR proteins atpresent, they have been shown to induce antibody productionin exposed individuals.4An immunological assay that tests forthe presence of GNR has the advantage of providing thespecificity and sensitivity that other ASTM standards such asTest Methods D5712 and D6499 do
26、 not offer.5.2 This test method describes an immunological methodfor the quantitation of Guayule natural rubber proteins usingrabbit anti-GNR serum. Rabbits immunized with GNR proteinsreact to the majority of the proteins present, and their sera havethe capability to detect most if not all of the pr
27、oteins in GNR.This test method may be used to determine if a test itemcontains material derived from a natural source.6. Interferences6.1 Substances such as detergents or surfactants have thepotential to prevent antibody binding to antigen and couldinterfere in an ELISA assay. However, due to the se
28、nsitivity ofthe ELISAassay, these interferences often can be controlled byserially diluting the sample.7. Apparatus7.1 96-Well Microtiter Assay Plate, (recommended NuncMaxiSorb, #442-404,).7.2 Dilution Plate, a low protein binding 96 well plate forsample dilution and antibody reaction (recommend Cor
29、ning#9017, or equivalent).7.3 Multichannel Pipettors.7.4 Analytical Balance.7.5 Centrifuge, (capable of 1000 g) and tubes.7.6 Incubator, capable of regulating the temperature at 37 63C.7.7 Microtiter Plate Reader, and optional computer for dataanalysis.7.8 ELISA Plate Sealing Tape, or plastic lids f
30、or coveringplates.8. Reagents and Materials8.1 BuffersBuffers and solutions should be prepared be-fore beginning the protocol. Make sure that all solutionscontaining protein are made in polypropylene tubes throughoutthe assay.8.1.1 Carbonate Buffer, pH 9.6 6 0.1:Na2CO30.795gNaHCO31.465gNaN30.1 g8.1.
31、1.1 Dissolve above in distilled H2O and dilute to a finalvolume of 500 mL. Check pH and adjust if necessary.NOTE 1Carbonate buffer can be stored for at least one month at 2 to8C. Alternatively, carbonate buffer capsules can be purchased from acommercial source.8.1.2 Phosphate-Buffered Saline (PBS),
32、pH 7.4 6 0.1; 10Xstock:NaH2PO4.H2O 5.125 gNa2HPO4.7H2O48.1.2.1 Dissolve above in 1.5 L distilled water and adjust topH 7.4, if necessary. Add 175.3 g NaCl and distilled water upto a total of 2 L. Prior to use, dilute an appropriate volume of10X stock 1:10 v/v with distilled water to obtain 1X PBS.3C
33、urrent Protocols in Molecular Biology, eds.Ausbel, F., Brent, R.M., KingstonR.E. et al. Unit 11, J. Wiley and Sons, Inc., 1997.4Robert G. Hamilton and Katrina Cornish, “Immunogenicity studies of guayuleand guayule latex in occupationally exposed workers,” Industrial Crops andProducts, Volume 31, Iss
34、ue 1, January 2010, pages 197-201.D8238 182NOTE 2Alternatively, PBS buffer solution can be purchased from acommercial source.8.1.3 T-PBS Wash BufferTo prepare T-PBS washingsolution, add 0.5 mLTween 20 to 1 Lof 1X PBS (0.05 %), mixwell.8.2 Dry Milk Solutions:8.2.1 Blocking SolutionPrepare 100 mLof 3
35、% w/v nonfatdry milk in T-PBS (for blocking of assay plate and dilutionplate).8.2.2 Dilution BufferPrepare 100 mL 0.2 % w/v nonfatdry milk in T-PBS (for dilution of antibodies and blocking inthe competitive inhibition step.8.3 Reference ReagentsThe lyophilized standard refer-ence antigen (StAg) and
36、the reference anti-GNR serum evalu-ated during development of this protocol will be supplied to thetest users. Details of the preparation procedure for the standardantigen and the protocol for rabbit immunization are describedin an ASTM Research Report for the Industry ReferenceMaterial (IRM).5NOTE
37、3Do not use frost free freezers which have temperatures thatfluctuate and can result in degradation of proteins, enzyme activity, orantibody reactivity. To reduce possible protein loss, all procedures thatinvolve protein containing solutions must be performed in polypropylenetubes or vessels. Polyst
38、yrene or glass vessels must be avoided.8.3.1 Standard Antigen (StAg) Solution (IRM #925)Thelyophilized preparation of GNR protein is reconstituted withdistilled H2O to a concentration of 1 mg/mL. Verify the proteinconcentration of the reconstituted protein by Test MethodD5712 with background subtrac
39、tion assay. Aliquot this stocksolution into small polypropylene tubes and store at 15C orlower. Aliquots, once thawed for use in the assay should bestored at 2 to 8C for up to one week.8.3.1.1 Coating AntigenPrepare a 3 g/mL solution of thestandard antigen in carbonate buffer for coating the assay p
40、late,as described in 12.2.8.3.1.2 Reference StandardPrepare a 0.5 g/mL solutionof StAg in dilution buffer for the reference standard to be usedin the competitive inhibition.8.3.2 Antisera (IRM #926):8.3.2.1 Primary AntiseraAn anti-GNR protein referenceantisera was produced in rabbits using the same
41、GNR proteinas the antigen. This reference sera must be used for thisstandard protocol. Analyst should dilute in dilution buffer,aliquot into convenient aliquots (for example, 50 l), and storeat 15C or lower until use. Once thawed the antisera shouldnot be refrozen.8.3.2.2 Secondary AntibodyA horsera
42、dish peroxidase(HRP) conjugated anti-rabbit IgG (recommend Sigma #A-0545) is to be used to detect the primary antibody recognitionof the GNR protein bound to the solid phase. Analyst shoulddilute the antibody 1:5 in dilution buffer, aliquot into conve-nient aliquots (for example, 50 L), and store at
43、 15C or loweruntil use.8.4 Substrate Development SolutionFollowing the manu-facturers directions, a yellow colored reaction product isproduced using o-phenylenediamine (OPD) and hydrogenperoxide. Sigma P-8412 and Sigma P-9187 are examples ofOPD substrates that can be used.9. Hazards9.1 Working perso
44、nnel should adhere to standard GoodLaboratory Practices. Care should be taken when working withall chemical reagents including acids and bases.10. Sample Extraction and Preparation10.1 Sample extraction is designed to be compatible withTest Methods D5712, D6499, and D7427 to allow total proteinand a
45、ntigenic protein to be determined for the same sampleextract.10.2 An aqueous buffer of pH 7.4 6 0.2 and a minimum of25 mM must be used as the extraction medium. Phosphatebuffered saline is recommended.10.3 The temperature of the extraction medium should be25 6 5C.10.4 The entire product or device sh
46、ould be weighed and thetotal weight per device recorded. When possible, the surfacearea of the device should be recorded.10.5 A volume of up to 5 mL of extraction medium shouldbe used per gram of sample. The ratio of extraction mediumvolume to the weight of the sample shall not exceed 5 mL pergram o
47、f material. The material must be extracted in polypro-pylene vessels to reduce the possible loss of proteins byadsorption to the inner surface of the container walls. The labshould take measures to ensure that the volume of buffer usedis adequate to contact all surfaces of the sample duringextractio
48、n.10.6 The length of the extraction period should be 120 6 5min with all surfaces evenly exposed to the extraction medium.If the product is too large for all surfaces of the material to beevenly exposed to extraction medium, it should be cut intopieces of appropriate size to accommodate the extracti
49、onvessel. The extraction vessel should be continuously rotated bya mechanical device to ensure even exposure to the extractionmedium. Alternatively, the extraction vessel should be shakenthree separate times for 15 s intervals at the beginning, middleand end of the extraction period (see Test Method D5712).10.7 Remove the test specimen from the extraction solution.Transfer the solution containing the extractable protein into apolypropylene tube and centrifuge for 15 min at not less than500gtoremove particulate matter. Alternatively, filt
