1、Designation: D 6499 03Standard 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:D 4483 Practice For Determining Precision For TestMethod Standards in the Rubber and Carbon Black Indus-tries2D 5712 Test Method for Analysis of Aqueous ExtractableProte
7、in in Natural Rubber and its Products Using theModified Lowry Method33. Terminology3.1 Definitions:3.1.1 allergensprotein antigens which induce allergic im-mune reactions typically mediated through IgE antibodies.3.1.2 antibodyan immunoglobulin, a protein that is pro-duced as a part of the immune re
8、sponse which is capable ofspecifically combining with the antigen.3.1.3 antigenany substance that provokes an immuneresponse when introduced into the body.3.1.4 background absorbancethe absorbance reading inthe solution resulting from the presence of chemicals, ions etc.other than the substrate bein
9、g determined.3.1.5 blocking solutiona non-reactive protein solutionused to prevent nonspecific antibody adsorption.3.1.6 calibrationthe standardization of an instrument set-ting or an assay configuration.3.1.7 concentration rangethe recommended analyte con-centration range in g/mL that produces an a
10、bsorbance readingof 0.1 to 2.0 units.3.1.8 enzyme linked immunosorbent assay (ELISA)an im-munological test method to quantify antigen or antibody levelsusing an enzyme as the detection mechanism.3.1.9 primary antibodythe antibody used first in a se-quence that is specific for the antigen.3.1.10 refe
11、rence solutionthe solution to which the testsample is being compared against.3.1.11 repeatabilitythe variability or test error betweenindependent test results obtained within a single laboratory.3.1.12 reproducibilitythe variability or error between testresults obtained in different laboratories.3.1
12、.13 secondary antibodythe enzyme conjugated anti-body used second in the sequence that is specific for the heavychain of the primary antibody.3.1.14 standard solutionthe preparation of standard ana-lyte used as a reference to which the unknown sample beingmeasured is compared.3.1.15 substratethe mat
13、erial or substance upon which anenzyme reacts.3.1.16 titerthe strength of the antibody solution (forexample, concentration and affinity of antibody).1This specification is under the jurisdiction of ASTM Committee D11 on Rubber, and is the direct responsibility of Subcommittee D11.40 on Consumer Rubb
14、erProducts.Current edition approved June 10, 2003. Published June 2003. Originallyapproved in 2000. Last previous edition approved in 2000 as D 6499 00.2Annual Book of ASTM Standards, Vol 09.01.3Annual Book of ASTM Standards, Vol 09.02.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C70
15、0, West Conshohocken, PA 19428-2959, United States.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).4The ELISA assay is based onpol
16、yclonal 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 theinhibition
17、 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 addedto the i
18、mmobilized 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 which atta
19、ches to the immobilized antigen-boundspecific antibody. Next, the enzyme substrate is added and thereaction of the enzyme on the substrate results in a colorchange. A reduction in the amount of color in comparison to anuninhibited control is an indicator of the amount of antigenpresent in the sample
20、. 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 urticaria afte
21、r 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 of prot
22、ein 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 colori
23、metric 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 not all
24、 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 couldi
25、nterfere in an ELISA assay. However, due to the sensitivity ofthe ELISA assay, 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 consisten
26、t 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 temper
27、ature at37C.7.7 Microtiter Plate Reader, and optional computer for dataanalysis.7.8 ELISA Plate Sealing Tape.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 thr
28、oughoutthe assay.8.1.1 Carbonate Buffer pH 9.6:Na2CO30.795gNaHCO31.465gNaN30.1 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 4C.Alternatively, carbonate buffer capsules can be pu
29、rchased from a com-mercial source.8.1.2 Phosphate-Buffered Saline (PBS), pH 7.4; 10X stock:NaH2PO4.H2O 5.125 gNa2HPO4.7H2O45Dissolve 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
30、of 10X stock1:10 v/v with distilled water to obtain 1X PBS.NOTE 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:8.2.1 Blocking Solutio
31、nPrepare 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 Reference ReagentsThe lyophilized standard refer
32、-ence antigen (StAg) and the reference anti-NRL serum evalu-ated during development of this protocol will be supplied to the4The boldface numbers given in parentheses refer to a list of references at theend of the text.D6499032test users5. Details of the preparation procedure for thestandard antigen
33、 and the protocol for rabbit immunization aredescribed in an ASTM Research Reports for the IndustryReference Material (IRM)6.NOTE 3Do not use frost free freezers which have temperatures thatfluctuate and can result in degradation of proteins, enzyme activity, orantibody reactivity. To reduce possibl
34、e 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 NRL protein is reconstituted withdistilled H2O to
35、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 carbonate buffer for coating the assay plate,as described
36、 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 was produced in rabbits using the same NRL prote
37、in 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 peroxidase(HRP) conjugated anti-rabbit IgG (recommend Sig
38、ma #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 Development SolutionA yellow colored re-action p
39、roduct 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 Practices. Care should be taken when working wit
40、hall 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.10.2 An aqueous buffer of pH 7.4 and a minimum of
41、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 recorded. Whenpossible, the surface area of the device
42、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 be cut intopieces of appropriate size to accommod
43、ate 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 beginning, middleand end of the extraction period
44、(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 must beextracted in polypropylene vessels to redu
45、ce 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 min at not less than500 3 g to remove particulat
46、e 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 days at or below 15C.11. Calibration and Standa
47、rdization11.1 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
48、 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/mL incarbonate buffer, pH 9.612.2.2 To coat the Nunc assay plate, place 100 L of StAgsolution (3 g/mL) in carbonate
49、buffer, pH 9.6 into all wells ofthe plate. Cover the plate with ELISA plate sealing tape andincubate for2hat37C, wash one time with T-PBS, andremove the contents of the plate. If the procedure is to becontinued the next day, store the empty plate at 20Covernight. Alternatively, the plate may be incubated with thecoating antigen overnight at 4C and washed one time beforeproceeding to 12.3. The plate must be covered with the sealingtape during this step and all subsequent incubation steps.5The sole source of supply of the refer
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