1、Designation: D 7427 08Standard Test Method forImmunological Measurement of Four Principal AllergenicProteins (Hev b 1, 3, 5 and 6.02) in Natural Rubber and ItsProducts Derived from Latex1This standard is issued under the fixed designation D 7427; the number immediately following the designation indi
2、cates the year oforiginal adoption or, 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 m
3、ethodknown as an immunoenzymetric assay to quantify the amountof 4 principal Hevea brasiliensis Hev b allergenic proteinsHev b 1, Hev b 3, Hev b 5 and Hev b 6.02 in natural rubberand its products2derived from latex using monoclonal anti-bodies specific for epitopes on these proteins. Since theseassa
4、ys quantify the levels of only 4 of the known 13 officiallyacknowledged allergens potentially present in natural rubberlatex containing products, the sum of the four allergen levelsshall be viewed as an indicator of the allergen burden and notas a measure of the total allergen content that can be re
5、leasedfrom the product.1.2 For the purpose of this test method, the range ofallergenic protein will be measured in terms of nanogram tomicrogram quantities per gram or unit surface area of a naturalrubber containing product.1.3 The test method is not designed to evaluate the potentialof natural rubb
6、er containing materials to induce or elicit TypeI (IgE-mediated) hypersensitivity reactions.1.4 This test method should be used under controlledlaboratory conditions to detect and quantify the level of 4allergenic proteins found in natural rubber containing products.It should not be used to describe
7、, appraise or assess the hazardor risk of these natural rubber containing materials or productsunder actual in use conditions.1.5 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.6 This standard does not purport to address all
8、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 determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:3D 1193 Specification f
9、or Reagent WaterD 4483 Practice for Evaluating Precision for Test MethodStandards in the Rubber and Carbon Black ManufacturingIndustriesD 4678 Practice for RubberPreparation, Testing, Accep-tance, Documentation, and Use of Reference MaterialsD 5712 Test Method for Analysis of Aqueous ExtractableProt
10、ein in Natural Rubber and Its Products Using theModified Lowry MethodD 6499 Test Method for The Immunological Measurementof Antigenic Protein in Natural Rubber and its ProductsE 691 Practice for Conducting an Interlaboratory Study toDetermine the Precision of a Test Method3. Terminology3.1 Definitio
11、ns:3.1.1 accepted reference value (ARV)value that serves asan agreed upon reference for comparison and which is derivedas (1) a theoretical or established value, based on scientificprinciples, (2) an assigned or certified value, based on experi-mental work of some national or international organizat
12、ion, or(3) a consensus or certified value, based on collaborativeexperimental work under the auspices of a scientific orengineering group.3.1.1.1 DiscussionARV is an average industrial referencematerial (IRM) property or parameter value established by wayof a specified test program. In this standard
13、, theARV as definedin the IRMs for the reference antigens and capture anddetection antibodies is determined by analyzing a high and lowcontrol in an inter-laboratory study and using the assignedvalues of these high and low controls to verify that the assay isin control and that the reagents are perf
14、orming properly.3.1.2 accuracythe closeness of agreement between a testresult and an accepted reference value.1This test method is under the jurisdiction of ASTM Committee D11 on Rubberand is the direct responsibility of Subcommittee D11.40 on Consumer RubberProducts.Current edition approved Aug. 1,
15、 2008. Published September 2008.2This procedure has not been validated for condoms, particularly lubricatedcondoms, which could contain surfactants or other ingredients that could interferewith the assay.3For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Se
16、rvice at serviceastm.org. For Annual Book of ASTMStandards volume information, 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.3.1.3 allergensprotein antigens which indu
17、ce allergic im-mune reactions typically mediated through IgE antibodies.3.1.4 analyteany element, ion, compound, substance, fac-tor, infectious agent, cell, organelle, activity (enzymatic, hor-monal, or immunological), or property the presence or absence,concentration, activity, intensity, or other
18、characteristics ofwhich are to be determined.3.1.5 antibodyan immunoglobulin, a protein that is pro-duced as a part of the humoral immune response which iscapable of specifically combining with antigen.3.1.5.1 DiscussionAny of numerous Y-shaped proteinmolecules produced by B lymphocytes as a primary
19、 immuneresponse, each molecule and its clones having a unique bindingsite that can combine with the complementary site of anantigen, as on a virus or bacterium, thereby signaling otherimmune responses. (See monoclonal antibody.)3.1.6 antigenany substance that can stimulate the produc-tion of antibod
20、ies within an organism and combine specificallywith them.3.1.7 background absorbancethe absorbance reading inthe solution resulting from non-specific interactions caused bythe presence of chemicals, ions, etc., other than the analytebeing measured.3.1.8 binding capacitywithin the context of this doc
21、ument,refers to the number of Hev b allergen molecules that a primarycapture antibody can bind reproducibly under standardizedassay conditions (pH, ionic strength, protein matrix, time,temperature).3.1.9 blocking solutiona non-reactive protein solutionused to prevent nonspecific antibody adsorption
22、and to reducebackground absorbance.3.1.10 calibrationthe standardization of an instrumentsetting or an assay configuration.3.1.11 calibration material/calibratora material (for ex-ample, solution) of known quantitative/qualitative characteris-tics (for example, concentration, activity, intensity, re
23、activity)used to calibrate, graduate, or adjust a measurement procedureor to compare the response obtained with the response of a testspecimen/sample.3.1.12 concentration rangethe recommended analyteconcentration range in nanograms per mL to micrograms permL that produces an absorbance reading from
24、0.1 to 2.03.0units (depending on the instrument).3.1.13 data reduction algorithma mathematical processthat converts assay-response data (for example, absorbanceunits) into interpolated dose results.3.1.13.1 DiscussionThe doseresponse relationship in theassay is defined by the standard, reference, or
25、 calibration curve.3.1.14 detection limit/limit of detectionthe smallest quan-tity of an analyte that can be reproducibly and a statisticallysignificant manner distinguished from the variance of thebackground, or a zero calibrator in a given assay system.3.1.14.1 DiscussionIt is usually defined at t
26、he 95 %confidence interval and has also been called the lower detectionlimit or positive threshold of the assay; this term is notsynonymous with analytical sensitivity.3.1.15 enzyme linked immunosorbent assay (ELISA)animmunological test method to quantify antigen or antibodylevels using an enzyme as
27、 the detection mechanism.3.1.16 epitope/determinant(1) the minimum molecularstructure of the antigenic site that will react with an antibody;(2) any site on an antigen molecule at which an antibody canbind; the chemical structure of the site determining the specificcombining antibody.3.1.17 IgEhuman
28、 IgE is an immunoglobulin of the ap-proximate molecular weight of 190 000, which exists normallyin monomeric form and constitutes approximately 0.0005 % ofthe total serum immunoglobulins.3.1.17.1 DiscussionIt binds with high affinity to FcR1receptors on mast cells and basophils and FcRII receptors o
29、na number of cells. IgE mediates the production and release ofvasoactive mediators following the binding of allergen.3.1.18 immunoenzymetric assay (IEMA)a two-site non-isotopic immunological test method that employs two antibod-ies, a primary antibody to capture and a secondary enzymeconjugated anti
30、body to detect the analyte of interest.3.1.19 immunoglobulina glycoprotein composed of twoheavy and two light chains that functions as an antibody.Human immunoglobulins have been subdivided into differentisotypes (IgM, IgG, IgA, IgD, IgE), each of which possess aunique set of antigenic markers, phys
31、iochemical properties,and each of which produce a different pattern of effectorfunctions (receptor binding, complement activation, opsoniza-tion).3.1.19.1 DiscussionAll antibodies are immunoglobulins,but it is not certain that all immunoglobulins possess antibodyfunction.3.1.20 industry reference ma
32、terials (IRM)materials thathave been prepared according to a specified production processto generate a uniform lot; the parameters that define the qualityof the lot are evaluated by a specified measurement program.3.1.20.1 DiscussionIRMs are divided into two types ac-cording to the production proces
33、s for generating the material.3.1.21 linearitythe ability (within a given range) of anassay to provide results that are directly proportional to theconcentration amount of the analyte in the test sample.3.1.22 monoclonal antibodyantibody produced by cellscreated through the fusion of an antibody pro
34、ducing cell(B-lymphocyte) with immortal cancer cells.3.1.22.1 DiscussionThis process produces a hybrid (hy-bridoma) that expresses properties of both cells. The cells areall identical since they derive from a single cell and are called“monoclonal.”3.1.23 parallelismextent to which the doseresponse r
35、e-lationship between two materials (that is, calibrator versusunknown specimens) is constant for the examined range ofconcentrations.3.1.23.1 DiscussionParallelism is a property (and a re-quirement) of quantitative immunoassays in which the calibra-tor and test sera produce parallel doseresponse cur
36、ves.3.1.24 precisionthe closeness of agreement between in-dependent test results obtained under prescribed conditions;agreement between replicate measurements.D74270823.1.24.1 DiscussionPrecision has no numerical value butis expressed in terms of imprecisionthe standard deviation(SD) or the coeffici
37、ent of variation (CV: SD/mean) of theresults in a set of replicate measurements.3.1.25 precision profilethe precision of an assay acrossthe analyte concentration range of interest.3.1.25.1 DiscussionA precision profile is constructed bydetermining the standard deviation (or coefficient of variation)
38、of replicate measurements (within assays, between assays, orbetween specimen dilutions within an assay) spanning theentire analyte concentration range, albeit without the exactknowledge of the true analyte concentration that is contained inthe serum specimens. When the CVdose(Y-axis) is graphedagain
39、st the dose (X-axis), a precision profile plot is generated.The precision profile is also referred to as the “imprecisionprofile” by some investigators.3.1.26 primary antibodythe antibody used first in anassay sequence that is specific for the antigen and is sometimesreferred to as the capture antib
40、ody that binds the analyte ofinterest from a biological specimen.3.1.27 proficiency testing (PT)an independent (non-manufacturer sponsored) program in which challenge speci-mens are sent to participating laboratories to be evaluated inassays that measure a spectrum of analytes.3.1.28 qualitative ass
41、ayan assay system that produces anindication of the presence or absence of an analyte but does notprovide a precise estimate of the concentration of that analyte.3.1.28.1 DiscussionApositive test result implies only thatthe assay signal exceeds the analytical threshold or positivecutoff point that h
42、as been set to obtain an arbitrary combinationof diagnostic sensitivity and specificity.3.1.29 quantitative assayan assay system that producesan accurate and reproducible estimate of the concentration ofan analyte in the test specimen.3.1.29.1 DiscussionIts analysis involves interpolationfrom a cali
43、bration curve, which is referenced to a readilyavailable standard reference preparation.3.1.30 quality control responselevel of analyte producedby an assay for a quality control specimen that has a previouslydefined analyte concentration range as defined by the manu-facturer.3.1.30.1 DiscussionAssay
44、 performance was evaluated bydetermining the agreement in Hev b 1, 3, 5 or 6.02 levelsobtained for two quality control extracts containing a high orlow level of each Hev b allergen, following analysis in multiplelaboratories participating in the multi-center study.3.1.31 reference solutionthe soluti
45、on against which thetest sample is being compared.3.1.32 relative standard deviation (RSD)the coefficient ofvariation which is the standard deviation divided by the mean.3.1.33 repeatabilityprecision under conditions where in-dependent test results are obtained with the same method onidentical test
46、items in the same laboratory by the same operatorusing the same equipment within short intervals of time.3.1.34 repeatability limit (r)the value below which theabsolute difference between two individual test results obtainedunder repeatability conditions may be expected to occur with aprobability of
47、 approximately 0.95 (95 %).3.1.34.1 DiscussionThe repeatability limit is 2.8(1.96 square root of 2) times the repeatability standarddeviation. This multiplier is independent of the size of theinter-laboratory study.3.1.35 reproducibilityprecision obtained under conditionswhere test results are obtai
48、ned with the same method onidentical test items in different laboratories with differentoperators using different equipment.3.1.36 reproducibility limit (R)the value below which theabsolute difference between two test results obtained underreproducibility conditions may be expected to occur with apr
49、obability of approximately 0.95 (95 %).3.1.36.1 DiscussionThe reproducibility limit is 2.8(1.96 square root of 2) times the reproducibility standarddeviation. This multiplier is independent of the number oflaboratories participating.3.1.37 secondary antibodyin an IEMA, it is the enzymeconjugated antibody used second in a sequence that is specificfor the analyte or interest and that completes the sandwich ofthe analyte.3.1.38 standard solutionthe preparation containing a stan-dard analyte that is used as a refere
