1、Designation: D6499 16D6499 18Standard Test Method forThe Immunological Measurement of Antigenic Protein inHevea Natural Rubber (HNR) and its Products1This standard is issued under the fixed designation D6499; the number immediately following the designation indicates the year oforiginal adoption or,
2、 in the case of revision, 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 to determine the amount of antig
3、enic protein in natural rubber HeveaNatural Rubber and its products using rabbit antisera specific for natural rubber latex (NRL) HNRL proteins. This immunoassayprocedure quantitatively measures the level of antigenic latex proteins in solution using an inhibition format. The samples mayinclude glov
4、e or other rubber product extracts which have been collected in order to measure the latex protein HNR levels.Although this method detects antigenic proteins, it should not be considered as a measure of allergenic proteins. Correlation ofprotein/antigen levels with the level of allergenic proteins h
5、as not been fully established.1.2 For the purpose of this test method, the range of protein will be measured in terms of microgram to milligram quantities.1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibilityof the user of
6、 this standard to establish appropriate safety safety, health, and healthenvironmental practices and determine theapplicability of regulatory limitations prior to use.1.4 This international standard was developed in accordance with internationally recognized principles on standardizationestablished
7、in the Decision on Principles for the Development of International Standards, Guides and Recommendations issuedby the World Trade Organization Technical Barriers to Trade (TBT) Committee.2. Referenced Documents2.1 ASTM Standards:2D4483 Practice for Evaluating Precision for Test Method Standards in t
8、he Rubber and Carbon Black Manufacturing IndustriesD5712 Test Method for Analysis of Aqueous Extractable Protein in Latex, Natural Rubber, and Elastomeric Products Using theModified Lowry MethodE177 Practice for Use of the Terms Precision and Bias in ASTM Test MethodsE691 Practice for Conducting an
9、Interlaboratory Study to Determine the Precision of a Test Method3. Terminology3.1 Definitions:3.1.1 allergens, nprotein antigens which induce allergic immune reactions typically mediated through IgE antibodies.3.1.2 antibody, nan immunoglobulin, a protein that is produced as a part of the immune re
10、sponse which is capable ofspecifically combining with the antigen.3.1.3 antigen, nany substance that provokes an immune response when introduced into the body.3.1.4 background absorbance, nthe absorbance reading in the solution resulting from the presence of chemicals, ions etc.other than the substr
11、ate being determined.3.1.5 blocking solution, na non-reactive protein solution used to prevent nonspecific antibody adsorption.3.1.6 calibration, nthe standardization of an instrument setting or an assay configuration.3.1.7 concentration range, nthe recommended analyte concentration range in g/mL th
12、at produces an absorbance reading of0.1 to 2.0 units.1 This test method is under the jurisdiction of ASTM Committee D11 on Rubber and Rubber-like Materials, and is the direct responsibility of Subcommittee D11.40 onConsumer Rubber Products.Current edition approved July 1, 2016Aug. 1, 2018. Published
13、 August 2016August 2018. Originally approved in 2000. Last previous edition approved in 20122016 asD6499 12.D6499 16. DOI: 10.1520/D6499-16.10.1520/D6499-18.2 For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer Service at serviceastm.org. For Annual Book of AST
14、M Standardsvolume information, refer to the standards Document Summary page on the ASTM website.This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Becauseit may not be technically po
15、ssible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current versionof the standard as published by ASTM is to be considered the official document.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700,
16、West Conshohocken, PA 19428-2959. United States13.1.8 enzyme linked immunosorbent assay (ELISA), nan immunological test method to quantify antigen or antibody levelsusing an enzyme as the detection mechanism.3.1.9 primary antibody, nthe antibody used first in a sequence that is specific for the anti
17、gen.3.1.10 reference solution, nthe solution to which the test sample is being compared against.3.1.11 repeatability, nthe variability or test error between independent test results obtained within a single laboratory.3.1.12 reproducibility, nthe variability or error between test results obtained in
18、 different laboratories.3.1.13 secondary antibody, nthe enzyme conjugated antibody used second in the sequence that is specific for the heavy chainof the primary antibody.3.1.14 standard solution, nthe preparation of standard analyte used as a reference to which the unknown sample beingmeasured is c
19、ompared.3.1.15 substrate, nthe material or substance upon which an enzyme reacts.3.1.16 titer, nthe strength of the antibody solution (for example, concentration and affinity of antibody).4. Summary of Test Method4.1 The latex devicetest sample is extracted for 2 h in an aqueous buffer. The extract
20、is recovered and the antigen levels aredetermined using inhibition Enzyme Linked ImmunoSorbent Assay (ELISA) technology (1).3 The ELISA assay is based onpolyclonal antiserum which can detect NRLHNRL proteins. ELISA technology takes advantage of the specificity and sensitivityof the antibody-antigen
21、reaction.Avariation of the ELISAmethod (an inhibition ELISA) has been developed for the detection andquantification of latexHNR protein antigens. In the inhibition ELISA, the latexHNR antigen is immobilized by absorption to thewells of a 96-well test plate. The sample extract is mixed with antibody
22、specific for NRLHNRL protein in a dilution plate.Following a brief incubation to allow for antibody recognition of the relevant NRLHNRL antigens, the mixture is added to theimmobilized antigen in the assay plate. Anti-NRLAnti-HNRL antibody which is not bound to the soluble NRLHNRL protein inthe samp
23、le will bind to the immobilized antigen. The plate is washed to remove the soluble antigen antibody complexes and asecondary antibody (enzyme-labeled anti-immunoglobulin) is added which attaches to the immobilized antigen-bound specificantibody. Next, the enzyme substrate is added and the reaction o
24、f the enzyme on the substrate results in a color change.Areductionin the amount of color in comparison to an uninhibited control is an indicator of the amount of antigen present in the sample.Comparison to a standard curve generated using known amounts of NRLHNRL protein permits quantification. The
25、assay is highlysensitive and can quantitate NRLHNRL proteins in the nanogram per millilitre range.5. Significance and Use5.1 Type 1 latex allergy most commonly manifests as localized urticaria after contact of skin with natural rubber but can alsoinclude symptoms of allergic rhinoconjunctivitis, ast
26、hma and rarely anaphylaxis. This immediate (Type I) allergy is caused bynaturalHNRL proteins inherent to the rubber tree, which remain on the finished natural rubber products. The quantification ofprotein levels in NRLHNRL products using the standard colorimetric protein assays may give spurious res
27、ults due to chemicaladditives in the latex formulations that interfere with the assay (2,3). Furthermore, the amount of protein found in NRLHNRLproducts are often below the detection limits of the standard colorimetric protein assay (4,5).5.2 This test method describes an immunological method for qu
28、antitation of natural rubber latex HNRL proteins using rabbitanti-NRLanti-HNRL serum. Rabbits immunized with NRLHNRL proteins react to the majority of the proteins present, and theirsera have the capability to detect most if not all of the proteins in NRL.HNRL. Therefore, although rabbit antibody re
29、acts withantigenic material, this should not be considered as quantitative measure of total protein levels.6. Interferences6.1 Substances such as detergents or surfactants have the potential to prevent antibody binding to antigen and could interferein an ELISA assay. However, due to the sensitivity
30、of the ELISA assay, these interferences often can be controlled by seriallydiluting the sample.7. Apparatus7.1 96-Well Microtiter Assay Plate, (recommended Nunc MaxiSorb, #442-404, round robin testing found this plate to providemore consistent results).7.2 Dilution Plate, a low protein binding 96 we
31、ll plate for sample dilution and antibody reaction (recommend Corning#25880-96,#9017, or equivalent).7.3 Multichannel Pipettors.3 The boldface numbers given in parentheses refer to a list of references at the end of the text.D6499 1827.4 Analytical Balance.7.5 Centrifuge, (capable of 1000 g) and tub
32、es.7.6 An Incubator, capable of regulating the temperature at 37C.7.7 Microtiter Plate Reader, and optional computer for data analysis.7.8 ELISA Plate Sealing Tape or Plastic Lids.7.9 It is expected that all laboratories will adhere to good laboratory practices (GLP) and ensure that all reagents use
33、d are withintheir shelf life and that all equipment used has been calibrated or verified before use.8. Reagents and Materials8.1 BuffersBuffers and solutions should be prepared before beginning the protocol. Make sure that all solutions containingprotein are made in polypropylene tubes throughout th
34、e assay.8.1.1 Carbonate Buffer pH 9.6:Na2CO3 0.795gNaHCO3 1.465gNaN3 0.1 gDissolve above in distilled H2O and dilute to a final volume of 500 mL. Check pH and adjust if necessary.NOTE 1Carbonate buffer can be stored for at least one month at 463C.Alternatively, carbonate buffer capsules can be purch
35、ased from a commercialsource.8.1.2 Phosphate-Buffered Saline (PBS), pH 7.4; 10X stock:NaH2PO4. H2O 5.125 gNa2HPO4. 7H2O 45 gDissolve 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
36、 of 10X stock 1:10 v/v with distilled water to obtain 1X PBS.NOTE 2Alternatively, PBS buffer solution can be purchased from a commercial source.8.1.3 T-PBS Wash BufferTo prepare T-PBS washing solution, add 0.5 mL Tween 20 to 1 L of 1X PBS (0.05 %), mix well.8.2 Dry Milk Solutions:8.2.1 Blocking Solu
37、tionPrepare 100 mL of 3 % w/v nonfat dry milk in T-PBS (for blocking of assay plate and dilution plate).8.2.2 Dilution buffer: Prepare 100 mL 0.2 % wv nonfat dry milk in T-PBS (for dilution of antibodies and blocking in thecompetitive inhibition step.8.3 Reference ReagentsThe lyophilized standard re
38、ference antigen (StAg) and the reference anti-NRLanti-HNRL serumevaluated during development of this protocol will be supplied to the test users.4 Details of the preparation procedure for thestandard antigen and the protocol for rabbit immunization are described in an ASTM Research Reports for the I
39、ndustry ReferenceMaterial (IRM).5NOTE 3Do not use frost free freezers which have temperatures that fluctuate and can result in degradation of proteins, enzyme activity, or antibodyreactivity. To reduce possible protein loss, all procedures that involve protein containing solutions must be performed
40、in polypropylene tubes or vessels.Polystyrene or glass vessels must be avoided.8.3.1 Standard Antigen (StAg) Solutions (IRM # 913)The lyophilized preparation of NRLHNRL protein is reconstituted withdistilled H2O to a concentration of 1 mg/mL. Aliquot this stock solution into small polypropylene tube
41、s and store at 20 6 10C.Aliquots, once thawed for use in the assay, should be stored at 4 6 3C.8.3.1.1 Coating AntigenPrepare a 3 g/mL solution of the standard antigen in carbonate buffer for coating the assay plate, asdescribed in 12.2.1.8.3.1.2 Reference StandardPrepare a 2 g/mL solution of StAg i
42、n dilution buffer for the reference standard to be used in thecompetitive inhibition 12.4.3.8.3.2 Antisera (IRM # 914):8.3.2.1 Primary AntiseraAn anti-NRLanti-HNRL protein reference antisera was produced in rabbits using the sameNRLHNRLprotein as the antigen. This reference sera must be used for thi
43、s standard protocol.Analyst should dilute 1:5 in dilutionbuffer, aliquot into convenient aliquots (for example, 50 l), and store at 20 6 10C until use.8.3.2.2 Secondary AntibodyAhorseradish peroxidase (HRP) conjugated anti-rabbit IgG (recommend Sigma #A-0545) is to beused to detect the primary antib
44、ody recognition of the NRL protein bound to the solid phase. Analyst should dilute 1:5 in dilutionbuffer, aliquot into convenient aliquots (for example, 50 L), and store at 20 6 10C until use.4 The sole source of supply of the reference reagents known to the committee at this time is Akron Rubber De
45、velopment Lab, 2887 Gilchrist Rd., Akron, OH 44305. Ifyou are aware of alternative suppliers, please provide this information to ASTM International Headquarters. Your comments will receive careful consideration at a meetingof the responsible technical committee,1 which you may attend.5 Supporting da
46、ta have been filed at ASTM International Headquarters and may be obtained by requesting Research Report RR:D11-1094.D6499 1838.4 Substrate Development SolutionA yellow colored reaction product is produced using o-phenylenediamine (OPD) andhydrogen peroxide. Dissolve the OPD tablet in dH2O and add th
47、e appropriate volume of H2O2 following the manufacturers-manufacturers instructions. For example: A 10 mg tablet of OPD from Sigma is dissolved in 10 mL of distilled H2O and 30 Lof 30 % H2O2 is added just prior to use.9. Hazards9.1 Working personnel should adhere to standard Good Laboratory Practice
48、s. Care should be taken when working with allchemical reagents including acids and bases.10. Sample Extraction and Preparation10.1 Sample extraction is designed to be compatible with Test Method D5712 to allow total protein and antigenic protein to bedetermined for the same sample extract.10.2 An aq
49、ueous buffer of pH 7.4 and a minimum of 25 mM must be used as the extraction medium. Phosphate buffered salineis recommended.10.3 The temperature of the extraction medium should be 25 6 5C.10.4 The entire natural rubber product or device should be weighed and the total weight per device recorded. When possible,the surface area of the device should be recorded.10.5 The length of the extraction period should be 1206 5 min with all surfaces evenly exposed to the extraction medium. Ifthe product is too large for all surfaces of the
