1、Designation:D649912Standard 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 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 antigenic protein in natural rubber
3、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 been collected
4、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 this test method,t
5、he 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 health practices and det
6、ermine 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 ExtractableProtein in Natural Ru
7、bber 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 IgE antibodies.3.1.2 ant
8、ibody,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 absorbance reading inthe solution
9、 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 assay configuration.3.1.7 concentr
10、ation 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 mechanism.3.1.9 primary antibody,n
11、the 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 single laboratory.3.1.12 reproducibili
12、ty,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 that is specific for the heavychain of the primary antibody.3.1.14 standard solution,nthe preparation of standardanalyte used as
13、a reference to which the unknown sample beingmeasured is compared.3.1.15 substrate,nthe material or substance upon whichan enzyme reacts.1This test method is under the jurisdiction ofASTM Committee D11 on Rubber,and is the direct responsibility of Subcommittee D11.40 on Consumer RubberProducts.Curre
14、nt edition approved May 1,2012.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
15、 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 States1 3.1.16 titer,nthe strength of the antibody solution(forexample,concentration and affinity of antibody).4.S
16、ummary of Test Method4.1 The latex device is extracted for 2 h in an aqueousbuffer.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 antibody-antigen reaction.A variation of the ELISAmethod(an inhibition ELISA)has been developed for thedetection and quantification o
