1、Designation: D6499 16Standard Test Method forThe Immunological Measurement of Antigenic Protein inNatural Rubber and its Products1This standard is issued under the fixed designation D6499; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revi
2、sion, 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 antigenic protein in natur
3、al rubber andits products using rabbit antisera specific for natural rubberlatex (NRL) proteins. This immunoassay procedure quantita-tively measures the level of antigenic latex proteins in solutionusing an inhibition format. The samples may include glove orother rubber product extracts which have b
4、een collected inorder to measure the latex protein levels.Although this methoddetects antigenic proteins, it should not be considered as ameasure of allergenic proteins. Correlation of protein/antigenlevels with the level of allergenic proteins has not been fullyestablished.1.2 For the purpose of th
5、is 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, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and heal
6、th practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D4483 Practice for Evaluating Precision for Test MethodStandards in the Rubber and Carbon Black ManufacturingIndustriesD5712 Test Method for Analysis of Aqueous Extractable
7、Protein in Latex, Natural Rubber, and Elastomeric Prod-ucts Using the Modified Lowry MethodE177 Practice for Use of the Terms Precision and Bias inASTM Test MethodsE691 Practice for Conducting an Interlaboratory Study toDetermine the Precision of a Test Method3. Terminology3.1 Definitions:3.1.1 alle
8、rgens, 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 that provok
9、es 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 nonspecific ant
10、ibody 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 units.3.1.8 enzyme linked immunosorbent assay (ELISA), nanimmunol
11、ogical 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.3.1.11 repeata
12、bility, 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.3.1.13 secondary antibody, nthe enzyme conjugated anti-body used second in the sequence
13、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.1This test method is under the jurisdiction ofASTM Committee D11 on Rubber,and is the direct responsibil
14、ity of Subcommittee D11.40 on Consumer RubberProducts.Current edition approved July 1, 2016. Published August 2016. Originallyapproved in 2000. Last previous edition approved in 2012 as D6499 12. DOI:10.1520/D6499-16.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact AST
15、M Customer Service at 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 States13.1.15 substrate, nthe materia
16、l or substance upon 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 latex device is extracted for2hinanaqueousbuffer. The extract is recovered and the antigen levels aredetermined using i
17、nhibition Enzyme Linked ImmunoSorbentAssay (ELISA) technology (1).3The ELISA assay is based onpolyclonal antiserum which can detect NRL proteins. ELISAtechnology takes advantage of the specificity and sensitivity ofthe antibody-antigen reaction. A variation of the ELISAmethod (an inhibition ELISA) h
18、as been developed for thedetection and quantification of latex protein antigens. In theinhibition ELISA, the latex antigen is immobilized by absorp-tion to the wells of a 96-well test plate. The sample extract ismixed with antibody specific for NRL protein in a dilutionplate. Following a brief incub
19、ation to allow for antibodyrecognition of the relevant NRL antigens, the mixture is addedto the immobilized antigen in the assay plate. Anti-NRLantibody which is not bound to the soluble NRL protein in thesample will bind to the immobilized antigen. The plate iswashed to remove the soluble antigen a
20、ntibody complexes anda secondary antibody (enzyme-labeled anti-immunoglobulin)is added which attaches to the immobilized antigen-boundspecific antibody. Next, the enzyme substrate is added and thereaction of the enzyme on the substrate results in a colorchange.Areduction in the amount of color in co
21、mparison to anuninhibited control is an indicator of the amount of antigenpresent in the sample. Comparison to a standard curve gener-ated using known amounts of NRL protein permits quantifica-tion. The assay is highly sensitive and can quantitate NRLproteins in the nanogram per millilitre range.5.
22、Significance and Use5.1 Type 1 latex allergy most commonly manifests aslocalized urticaria after contact of skin with natural rubber butcan also include symptoms of allergic rhinoconjunctivitis,asthma and rarely anaphylaxis. This immediate (Type I) allergyis caused by natural proteins inherent to th
23、e rubber tree, whichremain on the finished natural rubber products. The quantifi-cation of protein levels in NRL products using the standardcolorimetric protein assays may give spurious results due tochemical additives in the latex formulations that interfere withthe assay (2,3). Furthermore, the am
24、ount of protein found inNRL products are often below the detection limits of thestandard colorimetric protein assay (4,5).5.2 This test method describes an immunological methodfor quantitation of natural rubber latex proteins using rabbitanti-NRL serum. Rabbits immunized with NRL proteins reactto th
25、e majority of the proteins present, and their sera have thecapability to detect most if not all of the proteins in NRL.Therefore, although rabbit antibody reacts with antigenicmaterial, this should not be considered as quantitative measureof total protein levels.6. Interferences6.1 Substances such a
26、s detergents or surfactants have thepotential to prevent antibody binding to antigen and couldinterfere in an ELISA assay. However, due to the sensitivity ofthe ELISAassay, these interferences often can be controlled byserially diluting the sample.7. Apparatus7.1 96-Well Microtiter Assay Plate, (rec
27、ommended NuncMaxiSorb, #442-404, round robin testing found this plate toprovide more consistent results).7.2 Dilution Plate, a low protein binding 96 well plate forsample dilution and antibody reaction (recommend Corning#25880-96, or equivalent).7.3 Multichannel Pipettors.7.4 Analytical Balance.7.5
28、Centrifuge, (capable of 1000 g) and tubes.7.6 An Incubator, capable of regulating the temperature at37C.7.7 Microtiter Plate Reader, and optional computer for dataanalysis.7.8 ELISA Plate Sealing Tape or Plastic Lids.7.9 It is expected that all laboratories will adhere to goodlaboratory practices (G
29、LP) and ensure that all reagents used arewithin their shelf life and that all equipment used has beencalibrated or verified before use.8. Reagents and Materials8.1 BuffersBuffers and solutions should be prepared be-fore beginning the protocol. Make sure that all solutionscontaining protein are made
30、in polypropylene tubes throughoutthe assay.8.1.1 Carbonate Buffer pH 9.6:Na2CO30.795gNaHCO31.465gNaN30.1 gDissolve above in distilled H2O and dilute to a final volumeof 500 mL. Check pH and adjust if necessary.NOTE 1Carbonate buffer can be stored for at least one month at 4 63C. Alternatively, carbo
31、nate buffer capsules can be purchased from acommercial source.8.1.2 Phosphate-Buffered Saline (PBS), pH 7.4; 10X stock:NaH2PO4.H2O 5.125 gNa2HPO4.7H2O4Dissolve above in 1.5 L distilled water and adjust to pH 7.4,if necessary.Add 175.3 g NaCl and distilled water up to a totalof 2 L. Prior to use, dil
32、ute an appropriate volume of 10X stock1:10 v/v with distilled water to obtain 1X PBS.NOTE 2Alternatively, PBS buffer solution can be purchased from acommercial source.3The boldface numbers given in parentheses refer to a list of references at theend of the text.D6499 1628.1.3 T-PBS Wash BufferTo pre
33、pare 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 % w/v nonfatdry milk in T-PBS (for blocking of assay plate and dilutionplate).8.2.2 Dilution buffer: Prepare 100 mL 0.2 % wv nonfat drymilk in T-PBS (for
34、dilution of antibodies and blocking in thecompetitive inhibition step.8.3 Reference ReagentsThe lyophilized standard refer-ence antigen (StAg) and the reference anti-NRL serum evalu-ated during development of this protocol will be supplied to thetest users.4Details of the preparation procedure for t
35、hestandard antigen and the protocol for rabbit immunization aredescribed in an ASTM Research Reports for the IndustryReference Material (IRM).5NOTE 3Do not use frost free freezers which have temperatures thatfluctuate and can result in degradation of proteins, enzyme activity, orantibody reactivity.
36、 To reduce possible protein loss, all procedures thatinvolve protein containing solutions must be performed in polypropylenetubes or vessels. Polystyrene or glass vessels must be avoided.8.3.1 Standard Antigen (StAg) Solutions (IRM # 913)Thelyophilized preparation of NRL protein is reconstituted wit
37、hdistilled H2O to a concentration of 1 mg/mL.Aliquot this stocksolution into small polypropylene tubes and store at 20 610C. Aliquots, once thawed for use in the assay, should bestored at 4 6 3C.8.3.1.1 Coating AntigenPrepare a 3 g/mL solution of thestandard antigen in carbonate buffer for coating t
38、he assay plate,as described in 12.2.1.8.3.1.2 Reference StandardPrepare a 2 g/mL solution ofStAg in dilution buffer for the reference standard to be used inthe competitive inhibition 12.4.3.8.3.2 Antisera (IRM # 914):8.3.2.1 Primary AntiseraAn anti-NRL protein referenceantisera was produced in rabbi
39、ts using the same NRLprotein asthe antigen. This reference sera must be used for this standardprotocol. Analyst should dilute 1:5 in dilution buffer, aliquotinto convenient aliquots (for example, 50 l), and store at 206 10C until use.8.3.2.2 Secondary AntibodyA horseradish peroxidase(HRP) conjugated
40、 anti-rabbit IgG (recommend Sigma #A-0545) is to be used to detect the primary antibody recognitionof the NRL protein bound to the solid phase. Analyst shoulddilute 1:5 in dilution buffer, aliquot into convenient aliquots(for example, 50 L), and store at 20 6 10C until use.8.4 Substrate Development
41、SolutionA yellow colored re-action product is produced using o-phenylenediamine (OPD)and hydrogen peroxide. Dissolve the OPD tablet in dH2O andadd the appropriate volume of H2O2following the manufac-turers instructions. For example: A 10 mg tablet of OPD fromSigma is dissolved in 10 mL of distilled
42、H2O and 30 L of30 % H2O2is added just prior to use.9. Hazards9.1 Working personnel 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 c
43、ompatible withTest Method D5712 to allow total protein and antigenic proteinto be determined for the same sample extract.10.2 An aqueous buffer of pH 7.4 and a minimum of 25 mMmust be used as the extraction medium. Phosphate bufferedsaline is recommended.10.3 The temperature of the extraction medium
44、 should be25 6 5C.10.4 The entire natural rubber product or device should beweighed and the total weight per device recorded. Whenpossible, the surface area of the device should be recorded.10.5 The length of the extraction period should be 1206 5min with all surfaces evenly exposed to the extractio
45、n 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 extractionvessel. The extraction vessel should be continuously rotated bya mechanical device to ensure even exposure to t
46、he 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).10.6 A volume of 5 to 10 mL of extraction medium shouldbe used per gram of natural rubber material. The r
47、atio ofextraction medium volume to the weight of natural rubber shallnot exceed 10 mL per gram of material. Extraction ratios ofless than 5:1 can be used provided that the volume of extract issufficient to cover all surfaces of the test item. The materialmust be extracted in polypropylene vessels to
48、 reduce thepossible loss of proteins by adsorption to the inner surface ofthe container walls.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 particulat
49、e matter. Alternatively, filter theextract through a low protein binding 0.45 m filter into apolypropylene tube.10.8 The aqueous extracts of residual proteins should beused immediately but can be stored up to two days at 4 6 3Cand for greater than two days at or below 15C.11. Calibration and Standardization11.1 Microtiter Plate Spectrophotometer Warm-UpUndernormal operation, switch “on” the spectrophotometer andallow to warm up following the manufacturers recommenda-tions.4The sole source of supply of the reference reagents kno
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