1、Designation: D 6499 07Standard Test Method forThe Immunological Measurement of Antigenic Protein inNatural Rubber and its Products1This standard is issued under the fixed designation D 6499; the number immediately following the designation indicates the year oforiginal adoption or, in the case of re
2、vision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) 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 na
3、tural 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 hav
4、e been 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 o
5、f 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, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and
6、health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D 4483 Practice for Evaluating Precision for Test MethodStandards in the Rubber and Carbon Black ManufacturingIndustriesD 5712 Test Method for Analysis of Aqueous Extra
7、ctableProtein in Natural Rubber and Its Products Using theModified Lowry MethodE 691 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 th
8、rough 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
9、 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 antibody adsorption.3.1.6 calibration, nthe standardization of an instrumentsetting or
10、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), nanimmunological test method to quantify antigen or antibodylevels using an enzyme as the dete
11、ction 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 with
12、in 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 an-tibody used second in the sequence that is specific for theheavy chain of the primary antibody.3.1.14 standard solution
13、, nthe 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 Nov
14、. 1, 2007. Published December 2007. Originallyapproved in 2000. Last previous edition approved in 2003 as D 6499 03.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to
15、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.Copyright by ASTM Intl (all rights reserved); Mon Mar 17 01:56:07 EST 2008Downloaded/printed byGuo Dehua (CNIS) pursuant to Licens
16、e Agreement. No further reproductions authorized.3.1.15 substrate, nthe material 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 for2hinanaqueo
17、usbuffer. The extract is recovered and the antigen levels aredetermined using inhibition 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 an
18、tibody-antigen reaction. A variation of the ELISAmethod (an inhibition ELISA) has 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 wit
19、h antibody specific for NRL protein in a dilutionplate. Following a brief incubation 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
20、 to the immobilized antigen. The plate iswashed to remove the soluble antigen antibody 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
21、 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 sample. Comparison to a standard curve gener-ated using known amounts of NRL protein permits quantifica-tion. The assay is highly se
22、nsitive and can quantitate NRLproteins in the nanogram per millilitre range.5. 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 anaphylax
23、is. This immediate (Type I) allergyis caused by natural proteins inherent to the 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
24、 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 colorimetric protein assay (4,5).5.2 This test method describes an immunological methodfor quantitation of natural rubber latex prot
25、eins 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 not all of the proteins in NRL.Therefore, although rabbit antibody reacts with antigenicmaterial, this should not be considered as qu
26、antitative measureof total protein levels.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 sensitivity ofthe ELISAassay, these interferences often can be controlled byse
27、rially diluting the sample.7. Apparatus7.1 96-Well Microtiter Assay Plate, (recommended 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#
28、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 temperature at37C.7.7 Microtiter Plate Reader, and optional computer for dataanalysis.7.8 ELISA Plate Sealing Tape.8. Reagents and Ma
29、terials8.1 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:Na2CO30.795gNaHCO31.465gNaN30.1 gDissolve above in distilled H2O and dilute to a
30、 final volumeof 500 mL. Check pH and adjust if necessary.NOTE 1Carbonate buffer can be stored for at least one month at 4C.Alternatively, carbonate buffer capsules can be purchased from a com-mercial source.8.1.2 Phosphate-Buffered Saline (PBS), pH 7.4; 10X stock:NaH2PO4.H2O 5.125 gNa2HPO4.7H2O4Diss
31、olve 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.NOTE 2Alternatively, PBS buffer solution can be purchased from acomme
32、rcial 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:8.2.1 Blocking SolutionPrepare 100 mL of 3 % w/v non-fat dry milk in T-PBS (for blocking of assay plate and dilutionplate).3The boldface numbers given
33、 in parentheses refer to a list of references at theend of the text.D6499072Copyright by ASTM Intl (all rights reserved); Mon Mar 17 01:56:07 EST 2008Downloaded/printed byGuo Dehua (CNIS) pursuant to License Agreement. No further reproductions authorized.8.2.2 Dilution buffer: Prepare 100 mL 0.2 % w
34、/v nonfat drymilk in T-PBS (for 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
35、 the preparation procedure for thestandard 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
36、activity, orantibody reactivity. 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
37、NRL protein is reconstituted withdistilled H2O to a concentration of 1 mg/mL.Aliquot this stocksolution into small polypropylene tubes and store at 20C.Aliquots, once thawed for use in the assay should be stored at4C.8.3.1.1 Coating AntigenPrepare a 3 g/mL solution of thestandard antigen in carbonat
38、e buffer for coating the 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
39、 was produced in rabbits 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 at20C until use.8.3.2.2 Secondary AntibodyA horseradish peroxidas
40、e(HRP) conjugated 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 20C until use.8.4 Substrate
41、Development SolutionA yellow colored re-action product is produced using o-phenylenediamine (OPD)and hydrogen peroxide. A 10 mg tablet of OPD is dissolved in10 mL of distilled H2O and 30 L of 30 % H2O2is added justprior to use.9. Hazards9.1 Working personnel should adhere to standard GoodLaboratory
42、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 D 5712 to allow total protein and antigenicprotein to be determined for the same sample extract.10
43、.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 beweighed and the total weight per device recorde
44、d. 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 beevenly exposed to extraction medium, it should b
45、e 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 shakenthree separate times for 15 s intervals at the be
46、ginning, middleand end of the extraction period (see Test Method D 5712).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 exceed 10 mL per gram of material. The material m
47、ust beextracted in polypropylene vessels to reduce the possible lossof proteins by adsorption to the inner surface of the containerwalls.10.7 Remove the test specimen from the extraction solution.Transfer the solution containing the extractable protein into apolypropylene tube and centrifuge for 15
48、min at not less than500 3 g to remove particulate 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 2 to 4Cand for greater than two
49、 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.12. Inhibition ELISA Assay ProcedureDAY 112.1 Blocking the Dilution PlateBlock a Corning lowprotein binding plate by adding 300 l of blocking bufferovernight at 4C.12.2 Coating the Assay Plate:12.2.1 Prepare the coating antigen (StAg) at 3 g/m
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