1、Designation: D6499 12Standard 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 t
5、his 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 hea
6、lth 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 Extractabl
7、eProtein in Natural Rubber and Its Products Using theModified Lowry MethodE691 Practice for Conducting an Interlaboratory Study toDetermine the Precision of a Test Method3. Terminology3.1 Definitions:3.1.1 allergens, nprotein antigens which induce allergicimmune reactions typically mediated through
8、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 provokes an immuneresponse when introduced into the body.3.1.4 background absorbance, nthe absor
9、bance 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 antibody adsorption.3.1.6 calibration, nthe standardization of an instrumentsetting or an ass
10、ay 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), nanimmunological test method to quantify antigen or antibodylevels using an enzyme as the detection
11、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 repeatability, nthe variability or test error betweenindependent test results obtained within a s
12、ingle laboratory.3.1.12 reproducibility, nthe variability or error betweentest results obtained in different laboratories.3.1.13 secondary antibody, nthe enzyme conjugated an-tibody used second in the sequence that is specific for theheavy chain of the primary antibody.3.1.14 standard solution, nthe
13、 preparation of standardanalyte used as a reference to which the unknown sample beingmeasured is compared.1This test method is under the jurisdiction of ASTM Committee D11 on Rubber, and is the direct responsibility of Subcommittee D11.40 on Consumer RubberProducts.Current edition approved May 1, 20
14、12. Published July 2012. Originally approvedin 2000. Last previous edition approved in 2007 as D6499 07. DOI: 10.1520/D6499-12.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information
15、, refer to the standards Document Summary page onthe ASTM website.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.3.1.15 substrate, nthe material or substance upon whichan enzyme reacts.3.1.16 titer, nthe strength of the antibody sol
16、ution (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 inhibition Enzyme Linked ImmunoSorbentAssay (ELISA) technology (1).3The ELISA assay is bas
17、ed 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) has been developed for thedetection and quantification of latex protein antigens. In thein
18、hibition 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 incubation to allow for antibodyrecognition of the relevant NRL antigens, the mixture is added
19、to 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 antibody complexes anda secondary antibody (enzyme-labeled anti-immunoglobulin)is added wh
20、ich 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 comparison to anuninhibited control is an indicator of the amount of antigenpresent in the
21、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. Significance and Use5.1 Type 1 latex allergy most commonly manifests aslocalized urticari
22、a 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 the rubber tree, whichremain on the finished natural rubber products. The quantifi-cation o
23、f 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 amount of protein found inNRL products are often below the detection limits of thestandard
24、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 the majority of the proteins present, and their sera have thecapability to detect most if n
25、ot 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 as detergents or surfactants have thepotential to prevent antibody binding to antigen and
26、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, (recommended NuncMaxiSorb, #442-404, round robin testing found this plate toprovide more cons
27、istent 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 Centrifuge, (capable of 1000 3 g) and tubes.7.6 An Incubator, capable of regulating the t
28、emperature 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 (GLP) and ensure that all reagents used arewithin their shelf life and that all equipment
29、 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 in polypropylene tubes throughoutthe assay.8.1.1 Carbonate Buffer pH 9.6:Na2CO30.795gNa
30、HCO31.465gNaN30.1 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, carbonate buffer capsules can be purchased from acommercial source.8.1.2 Phosphate-Buffered
31、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, dilute an appropriate volume of 10X stock1:10 v/v with distilled water to obtain 1X PBS.N
32、OTE 2Alternatively, PBS buffer solution can be purchased from acommercial source.8.1.3 T-PBS Wash BufferTo prepare T-PBS washing solu-tion, add 0.5 mL Tween 20 to 1 L of 1X PBS (0.05 %), mixwell.8.2 Dry Milk Solutions:3The boldface numbers given in parentheses refer to a list of references at theend
33、 of the text.D6499 1228.2.1 Blocking SolutionPrepare 100 mL of 3 % w/v non-fat dry milk in T-PBS (for blocking of assay plate and dilutionplate).8.2.2 Dilution buffer: Prepare 100 mL 0.2 % w/v nonfat drymilk in T-PBS (for dilution of antibodies and blocking in thecompetitive inhibition step.8.3 Refe
34、rence 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 thestandard antigen and the protocol for rabbit immunization aredescribed in an
35、 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. To reduce possible protein loss, all procedures thatinvolve protein containin
36、g 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 withdistilled H2O to a concentration of 1 mg/mL.Aliquot this stocksolution into s
37、mall 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 the assay plate,as described in 12.2.1.8.3.1.2 Reference StandardPrepare a 2 g/
38、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 rabbits using the same NRLprotein asthe antigen. This reference sera must be used f
39、or 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 anti-rabbit IgG (recommend Sigma #A-0545) is to be used to detect the primary
40、 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 SolutionA yellow colored re-action product is produced using o-phenylenediamin
41、e (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 H2O and 30 L of30 % H2O2is added just prior to use.9. Hazards9.1 Working perso
42、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 Method D5712 to allow total protein and antigenic proteinto
43、 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 should be25 6 5C.10.4 The entire natural rubber product or device should bewe
44、ighed 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 extraction medium.If the product is too large for all surfaces of the material to beeve
45、nly 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 the extractionmedium. Alternatively, the extraction vessel should be shakenthre
46、e 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 ratio ofextraction medium volume to the weight of natural rubber shallnot excee
47、d 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 reduce thepossible loss of proteins by adsorption to the inner surface ofthe
48、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 than500 3 g to remove particulate matter. Alternatively, filter theextract through a low protein binding
49、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.11.2 Zero the instrument as required in the manufacturersmanual.4The sole source of supply of the reference reagents known to the co
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