ASTM D7427-2008e2 1875 Standard Test Method for Immunological Measurement of Four Principal Allergenic Proteins (Hev b 1 3 5 and 6 02) in Natural Rubber and Its Products Derived fr.pdf

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1、Designation: D7427 082Standard 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 D7427; the number immediately following the designation indic

2、ates 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.1NOTEFootnote 6 editorially corrected in January 2010.2

3、NOTEAdded Research Report footnote in March 2013.1. Scope1.1 This test method covers an immunological methodknown 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 products2d

4、erived from latex using monoclonal anti-bodies specific for epitopes on these proteins. Since theseassays 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

5、as an indicator of the allergen burden and notas a measure of the total allergen content that can be releasedfrom 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 natura

6、lrubber containing product.1.3 The test method is not designed to evaluate the potentialof natural rubber 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 leve

7、l of 4allergenic proteins found in natural rubber containing products.It should not be used to describe, 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 oth

8、er units of measurement are included in thisstandard.1.6 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 determine the applica-bility o

9、f regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:3D1193 Specification for Reagent WaterD4483 Practice for Evaluating Precision for Test MethodStandards in the Rubber and Carbon Black ManufacturingIndustriesD4678 Practice for RubberPreparation, Testing,Acceptance, Docum

10、entation, and Use of Reference Mate-rialsD5712 Test Method for Analysis of Aqueous ExtractableProtein in Natural Rubber and Its Products Using theModified Lowry MethodD6499 Test Method for The Immunological Measurement ofAntigenic Protein in Natural Rubber and its ProductsE691 Practice for Conductin

11、g an Interlaboratory Study toDetermine the Precision of a Test Method3. Terminology3.1 Definitions: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 assi

12、gned or certified value, based on experi-mental work of some national or international organization, or(3) a consensus or certified value, based on collaborativeexperimental work under the auspices of a scientific orengineering group.1This test method is under the jurisdiction of ASTM Committee D11

13、on Rubberand is the direct responsibility of Subcommittee D11.40 on Consumer RubberProducts.Current edition approved Aug. 1, 2008. Published September 2008. DOI:10.1520/D7427-08E02.2This procedure has not been validated for condoms, particularly lubricatedcondoms, which could contain surfactants or

14、other ingredients that could interferewith the assay.3For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.Copyrigh

15、t ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States13.1.1.1 DiscussionARV is an average industrial referencematerial (IRM) property or parameter value established by wayof a specified test program. In this standard, theARVas definedin the IRMs fo

16、r 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 performing properly.3.1.2 accuracythe

17、 closeness of agreement between a testresult and an accepted reference value.3.1.3 allergensprotein antigens which induce allergic im-mune reactions typically mediated through IgE antibodies.3.1.4 analyteany element, ion, compound, substance,factor, infectious agent, cell, organelle, activity (enzym

18、atic,hormonal, or immunological), or property the presence orabsence, concentration, activity, intensity, or other characteris-tics of which 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 comb

19、ining with antigen.3.1.5.1 DiscussionAny of numerous Y-shaped proteinmolecules produced by B lymphocytes as a primary 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 othe

20、rimmune responses. (See monoclonal antibody.)3.1.6 antigenany substance that can stimulate the produc-tion of antibodies within an organism and combine specificallywith them.3.1.7 background absorbancethe absorbance reading inthe solution resulting from non-specific interactions caused bythe presenc

21、e of chemicals, ions, etc., other than the analytebeing measured.3.1.8 binding capacitywithin the context of this document,refers to the number of Hev b allergen molecules that a primarycapture antibody can bind reproducibly under standardizedassay conditions (pH, ionic strength, protein matrix, tim

22、e,temperature).3.1.9 blocking solutiona non-reactive protein solutionused to prevent nonspecific antibody adsorption and to reducebackground absorbance.3.1.10 calibrationthe standardization of an instrumentsetting or an assay configuration.3.1.11 calibration material/calibratora material (forexample

23、, solution) of known quantitative/qualitative character-istics (for example, concentration, activity, intensity, reactivity)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 recom

24、mended analyte con-centration range in nanograms per mL to micrograms per mLthat produces an absorbance reading from 0.1 to 2.03.0 units(depending on the instrument).3.1.13 data reduction algorithma mathematical processthat converts assay-response data (for example, absorbanceunits) into interpolate

25、d dose results.3.1.13.1 DiscussionThe doseresponse relationship in theassay is defined by the standard, reference, or 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

26、variance of thebackground, or a zero calibrator in a given assay system.3.1.14.1 DiscussionIt is usually defined at the 95 % con-fidence 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

27、 linked immunosorbent assay (ELISA)animmunological test method to quantify antigen or antibodylevels using an enzyme as 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 w

28、hich an antibody canbind; the chemical structure of the site determining the specificcombining antibody.3.1.17 IgEhuman 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 immunoglob

29、ulins.3.1.17.1 DiscussionIt binds with high affinity to FcR1receptors on mast cells and basophils and FcRII receptors ona 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 immu

30、nological test method that employs twoantibodies, a primary antibody to capture and a secondaryenzyme conjugated antibody 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 subd

31、ivided into differentisotypes (IgM, IgG, IgA, IgD, IgE), each of which possess aunique set of antigenic markers, physiochemical properties,and each of which produce a different pattern of effectorfunctions (receptor binding, complement activation, opsoniza-tion).3.1.19.1 DiscussionAll antibodies are

32、 immunoglobulins,but it is not certain that all immunoglobulins possess antibodyfunction.3.1.20 industry reference materials (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

33、 specified measurement program.3.1.20.1 DiscussionIRMs are divided into two types ac-cording to the production process 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

34、 in the test sample.3.1.22 monoclonal antibodyantibody produced by cellscreated through the fusion of an antibody producing 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

35、 since they derive from a single cell and are called“monoclonal.”D7427 08223.1.23 parallelismextent to which the doseresponse re-lationship between two materials (that is, calibrator versusunknown specimens) is constant for the examined range ofconcentrations.3.1.23.1 DiscussionParallelism is a prop

36、erty (and a re-quirement) of quantitative immunoassays in which the calibra-tor and test sera produce parallel doseresponse curves.3.1.24 precisionthe closeness of agreement between inde-pendent test results obtained under prescribed conditions;agreement between replicate measurements.3.1.24.1 Discu

37、ssionPrecision has no numerical value butis expressed in terms of imprecisionthe standard deviation(SD) or the coefficient of variation (CV: SD/mean) of theresults in a set of replicate measurements.3.1.25 precision profilethe precision of an assay across theanalyte concentration range of interest.3

38、.1.25.1 DiscussionA precision profile is constructed bydetermining the standard deviation (or coefficient of variation)of replicate measurements (within assays, between assays, orbetween specimen dilutions within an assay) spanning theentire analyte concentration range, albeit without the exactknowl

39、edge of the true analyte concentration that is contained inthe serum specimens. When the CVdose(Y-axis) is graphedagainst 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 an

40、tibody used first in an assaysequence that is specific for the antigen and is sometimesreferred to as the capture antibody 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

41、 sent to participating laboratories to be evaluated inassays that measure a spectrum of analytes.3.1.28 qualitative assayan 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 Discussion

42、Apositive test result implies only thatthe assay signal exceeds the analytical threshold or positivecutoff point that has been set to obtain an arbitrary combinationof diagnostic sensitivity and specificity.3.1.29 quantitative assayan assay system that produces anaccurate and reproducible estimate o

43、f the concentration of ananalyte in the test specimen.3.1.29.1 DiscussionIts analysis involves interpolationfrom a calibration 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 spe

44、cimen that has a previouslydefined analyte concentration range as defined by the manu-facturer.3.1.30.1 DiscussionAssay 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,

45、 following analysis in multiplelaboratories participating in the multi-center study.3.1.31 reference solutionthe solution 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 repe

46、atabilityprecision under conditions where in-dependent test results are obtained with the same method onidentical test 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 b

47、etween two individual test results obtainedunder repeatability conditions may be expected to occur with aprobability of 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

48、size of theinter-laboratory study.3.1.35 reproducibilityprecision obtained under conditionswhere test results are obtained 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 the

49、absolute difference between two test results obtained underreproducibility conditions may be expected to occur with aprobability 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 complet