ASTM D6499-2012 6250 Standard Test Method for The Immunological Measurement of Antigenic Protein in Natural Rubber and its Products《天然橡胶及其制品中抗原蛋白免疫测量的标准试验方法》.pdf
《ASTM D6499-2012 6250 Standard Test Method for The Immunological Measurement of Antigenic Protein in Natural Rubber and its Products《天然橡胶及其制品中抗原蛋白免疫测量的标准试验方法》.pdf》由会员分享,可在线阅读,更多相关《ASTM D6499-2012 6250 Standard Test Method for The Immunological Measurement of Antigenic Protein in Natural Rubber and its Products《天然橡胶及其制品中抗原蛋白免疫测量的标准试验方法》.pdf(9页珍藏版)》请在麦多课文档分享上搜索。
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
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