1、Designation: D7427 14D7427 16Standard Test Method forImmunological Measurement of Four Principal AllergenicProteins (Hev b 1, 3, 5 and 6.02) in Hevea Natural Rubberand Its Products Derived from Latex1This standard is issued under the fixed designation D7427; the number immediately following the desi
2、gnation indicates 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 imm
3、unological method known as an immunoenzymetric assay to quantify the amount of 4principal Hevea brasiliensis Hev b allergenic proteins Hev b 1, Hev b 3, Hev b 5 and Hev b 6.02 in Hevea natural rubber andits products2 derived from latex using monoclonal antibodies specific for epitopes on these prote
4、ins. Since these assays quantifythe levels of only 4 of the known 14 officially acknowledged allergens potentially present in Hevea natural rubber latex containingproducts, the sum of the four allergen levels shall be viewed as an indicator of the allergen burden and not as a measure of thetotal all
5、ergen content that can be released from the product.1.2 For the purpose of this test method, the range of allergenic protein will be measured in terms of nanogram to microgramquantities per gram or unit surface area of a Hevea natural rubber containing product.1.3 The test method is not designed to
6、evaluate the potential of Hevea natural rubber containing materials to induce or elicitType I (IgE-mediated) hypersensitivity reactions.1.4 This test method should be used under controlled laboratory conditions to detect and quantify the level of 4 allergenicproteins found in Hevea natural rubber co
7、ntaining products. It should not be used to describe, appraise or assess the hazard or riskof these Hevea natural rubber containing materials or products under actual in use conditions.1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in thi
8、s standard.1.6 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 this standard to establish appropriate safety and health practices and determine the applicability of regulatorylimitations prior to use.2. Ref
9、erenced Documents2.1 ASTM Standards:3D1193 Specification for Reagent WaterD4483 Practice for Evaluating Precision for Test Method Standards in the Rubber and Carbon Black Manufacturing IndustriesD4678 Practice for RubberPreparation, Testing, Acceptance, Documentation, and Use of Reference MaterialsD
10、5712 Test Method for Analysis of Aqueous Extractable Protein in Latex, Natural Rubber, and Elastomeric Products Using theModified Lowry MethodD6499 Test Method for The Immunological Measurement of Antigenic Protein in Natural Rubber and its ProductsE691 Practice for Conducting an Interlaboratory Stu
11、dy to Determine the Precision of a Test Method3. Terminology3.1 Definitions:3.1.1 accepted reference value (ARV)value that serves as an agreed upon reference for comparison and which is derived as(1) a theoretical or established value, based on scientific principles, (2) an assigned or certified val
12、ue, based on experimental work1 This test method is under the jurisdiction of ASTM Committee D11 on Rubber and is the direct responsibility of Subcommittee D11.40 on Consumer Rubber Products.Current edition approved Sept. 1, 2014June 1, 2016. Published September 2014July 2016. Originally approved in
13、 2008. Last previous edition approved in 20082014 asD7427 08D7427 14.2. DOI: 10.1520/D7427-14.10.1520/D7427-16.2 This procedure has not been validated for condoms, particularly lubricated condoms, which could contain surfactants or other ingredients that could interfere with theassay.3 For reference
14、dASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer Service at serviceastm.org. For Annual Book of ASTM 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 us
15、er of an ASTM standard an indication of what changes have been made to the previous version. Becauseit may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current versionof the standard a
16、s published by ASTM is to be considered the official document.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1of some national or international organization, or (3) a consensus or certified value, based on collaborative experimental w
17、orkunder the auspices of a scientific or engineering group.3.1.1.1 DiscussionARV is an average industrial reference material (IRM) property or parameter value established by way of a specified test program.In this standard, the ARV as defined in the IRMs for the reference antigens and capture and de
18、tection antibodies is determined byanalyzing a high and low control in an inter-laboratory study and using the assigned values of these high and low controls to verifythat the assay is in control and that the reagents are performing properly.3.1.2 accuracythe closeness of agreement between a test re
19、sult and an accepted reference value.3.1.3 allergensprotein antigens which induce allergic immune reactions typically mediated through IgE antibodies.3.1.4 analyteany element, ion, compound, substance, factor, infectious agent, cell, organelle, activity (enzymatic, hormonal,or immunological), or pro
20、perty the presence or absence, concentration, activity, intensity, or other characteristics of which are tobe determined.3.1.5 antibodyan immunoglobulin, a protein that is produced as a part of the humoral immune response which is capable ofspecifically combining with antigen.3.1.5.1 DiscussionAny o
21、f numerous Y-shaped protein molecules produced by B lymphocytes as a primary immune response, each molecule and itsclones having a unique binding site that can combine with the complementary site of an antigen, as on a virus or bacterium, therebysignaling other immune responses. (See monoclonal anti
22、body.)3.1.6 antigenany substance that can stimulate the production of antibodies within an organism and combine specifically withthem.3.1.7 background absorbancethe absorbance reading in the solution resulting from non-specific interactions caused by thepresence of chemicals, ions, etc., other than
23、the analyte being 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 standardized assay conditions (pH, ionic strength, protein matrix, time, temperature).3.1.9 blocking solut
24、iona non-reactive protein solution used to prevent nonspecific antibody adsorption and to reducebackground absorbance.3.1.10 calibrationthe standardization of an instrument setting or an assay configuration.3.1.11 calibration material/calibratora material (for example, solution) of known quantitativ
25、e/qualitative characteristics (forexample, concentration, activity, intensity, reactivity) used to calibrate, graduate, or adjust a measurement procedure or to comparethe response obtained with the response of a test specimen/sample.3.1.12 concentration rangethe recommended analyte concentration ran
26、ge in nanograms per mL to micrograms per mL thatproduces an absorbance reading from 0.1 to 2.03.0 units (depending on the instrument).3.1.13 data reduction algorithma mathematical process that converts assay-response data (for example, absorbance units) intointerpolated dose results.3.1.13.1 Discuss
27、ionThe doseresponse relationship in the assay is defined by the standard, reference, or calibration curve.3.1.14 detection limit/limit of detectionthe smallest quantity of an analyte that can be reproducibly and a statisticallysignificant manner distinguished from the variance of the background, or
28、a zero calibrator in a given assay system.3.1.14.1 DiscussionIt is usually defined at the 95 % confidence interval and has also been called the lower detection limit or positive threshold of theassay; this term is not synonymous with analytical sensitivity.3.1.15 enzyme linked immunosorbent assay (E
29、LISA)an immunological test method to quantify antigen or antibody levelsusing an enzyme as the detection mechanism.D7427 1623.1.16 epitope/determinant(1) the minimum molecular structure of the antigenic site that will react with an antibody; (2) anysite on an antigen molecule at which an antibody ca
30、n bind; the chemical structure of the site determining the specific combiningantibody.3.1.17 IgEhuman IgE is an immunoglobulin of the approximate molecular weight of 190 000, which exists normally inmonomeric form and constitutes approximately 0.0005 % of the total serum immunoglobulins.3.1.17.1 Dis
31、cussionIt (IgE) binds with high affinity to FcR1 receptors on mast cells and basophils and FcRII receptors on a number of cells. IgEmediates the release of vasoactive mediators following the binding of allergen.3.1.18 immunoenzymetric assay (IEMA)a two-site non-isotopic immunological test method tha
32、t employs two antibodies, aprimary antibody to capture and a secondary enzyme conjugated antibody to detect the analyte of interest.3.1.19 immunoglobulina glycoprotein composed of two heavy and two light chains that functions as an antibody. Humanimmunoglobulins have been subdivided into different i
33、sotypes (IgM, IgG, IgA, IgD, IgE), each of which possess a unique set ofantigenic markers, physiochemical properties, and each of which produce a different pattern of effector functions (receptor binding,complement activation, opsonization).3.1.19.1 DiscussionAll antibodies are immunoglobulins, but
34、it is not certain that all immunoglobulins possess antibody function.3.1.20 industry reference materials (IRM)materials that have been prepared according to a specified production process togenerate a uniform lot; the parameters that define the quality of the lot are evaluated by a specified measure
35、ment program.3.1.20.1 DiscussionIRMs are divided into two types according to the production process for generating the material.3.1.21 linearitythe ability (within a given range) of an assay to provide results that are directly proportional to theconcentration amount of the analyte in the test sampl
36、e.3.1.22 monoclonal antibodyantibody produced by cells created through the fusion of an antibody producing cell(B-lymphocyte) with immortal cancer cells.3.1.22.1 DiscussionThis fusion process produces a hybrid (hybridoma) that expresses properties of both cells. The cells are all identical since the
37、yderive from a single cell and are called “monoclonal.”3.1.23 parallelismextent to which the doseresponse relationship between two materials (that is, calibrator versus unknownspecimens) is constant for the examined range of concentrations.3.1.23.1 DiscussionParallelism is a property (and a requirem
38、ent) of quantitative immunoassays in which the calibrator and test sera produce paralleldoseresponse curves.3.1.24 precisionthe closeness of agreement between independent test results obtained under prescribed conditions; agreementbetween replicate measurements.3.1.24.1 DiscussionPrecision has no nu
39、merical value but is expressed in terms of imprecisionthe standard deviation (SD) or the coefficient ofvariation (CV: SD/mean) of the results in a set of replicate measurements.3.1.25 precision profilethe precision of an assay across the analyte concentration range of interest.3.1.25.1 DiscussionA p
40、recision profile is constructed by determining the standard deviation (or coefficient of variation) of replicate measurementsD7427 163(within assays, between assays, or between specimen dilutions within an assay) spanning the entire analyte concentration range,albeit without the exact knowledge of t
41、he true analyte concentration that is contained in the serum specimens. When theCVdose (Y-axis) is graphed against the dose (X-axis), a precision profile plot is generated. The precision profile is also referred toas the “imprecision profile” by some investigators.3.1.26 primary antibodythe antibody
42、 used first in an assay sequence that is specific for the antigen and is sometimes referredto as the capture antibody that binds the analyte of interest from a biological specimen.3.1.27 proficiency testing (PT)an independent (non-manufacturer sponsored) program in which challenge specimens are sent
43、to participating laboratories to be evaluated in assays that measure a spectrum of analytes.3.1.28 qualitative assayan assay system that produces an indication of the presence or absence of an analyte but does notprovide a precise estimate of the concentration of that analyte.3.1.28.1 DiscussionA po
44、sitive test result implies only that the assay signal exceeds the analytical threshold or positive cutoff point that has been setto obtain an arbitrary combination of diagnostic sensitivity and specificity.3.1.29 quantitative assayan assay system that produces an accurate and reproducible estimate o
45、f the concentration of ananalyte in the test specimen.3.1.29.1 DiscussionIts (quantitative assay) analysis involves interpolation from a calibration curve, which is referenced to a readily available standardreference preparation.3.1.30 quality control responselevel of analyte produced by an assay fo
46、r a quality control specimen that has a previouslydefined analyte concentration range as defined by the manufacturer.3.1.30.1 DiscussionAssay performance was evaluated by determining the agreement in Hev b 1, 3, 5 or 6.02 levels obtained for two quality controlextracts containing a high or low level
47、 of each Hev b allergen, following analysis in multiple laboratories participating in themulti-center study.3.1.31 reference solutionthe solution against which the test sample is being compared.3.1.32 relative standard deviation (RSD)the coefficient of variation which is the standard deviation divid
48、ed by the mean.3.1.33 repeatabilityprecision under conditions where independent test results are obtained with the same method on identicaltest items in the same laboratory by the same operator using the same equipment within short intervals of time.3.1.34 repeatability limit (r)the value below whic
49、h the absolute difference between two individual test results obtained underrepeatability conditions may be expected to occur with a probability of approximately 0.95 (95 %).3.1.34.1 DiscussionThe repeatability limit is 2.8 (1.96 square root of 2) times the repeatability standard deviation. This multiplier is independentof the size of the inter-laboratory study.3.1.35 reproducibilityprecision obtained under conditions where test results are obtained with the same method on identicaltest items in different lab
