1.1 本试验方法包括一种称为免疫酶测定的免疫学方法，用于量化4种主要成分的含量 巴西橡胶树 橡胶树天然橡胶及其制品中的过敏蛋白[Hev b 1、Hev b 3、Hev b 5和Hev b 6.02] 2. 使用针对这些蛋白质表位的单克隆抗体从乳胶中提取。由于这些分析仅量化了含有橡胶树天然胶乳的产品中可能存在的已知14种官方公认过敏原中的4种，因此四种过敏原水平的总和应视为过敏原负担的指标，而不是衡量产品中可释放的总过敏原含量。 1.2 在本试验方法中，过敏性蛋白质的范围将以每克或含有橡胶树天然橡胶的产品的单位表面积的纳克到微克数量来测量。 1.3 本试验方法的目的不是评估含有橡胶树天然橡胶的材料诱导或诱发I型（IgE介导）超敏反应的可能性。 1.4 本试验方法应在受控实验室条件下使用，以检测和量化在含橡胶树天然橡胶的产品中发现的4种过敏蛋白的水平。它不应用于描述、评估或评估这些含有橡胶树天然橡胶的材料或产品在实际使用条件下的危害或风险。 1.5 以国际单位制表示的数值应视为标准值。本标准不包括其他计量单位。 1.6 本标准并非旨在解决与其使用相关的所有安全问题（如有）。 本标准的用户有责任在使用前制定适当的安全、健康和环境实践，并确定监管限制的适用性。 1.7 本国际标准是根据世界贸易组织技术性贸易壁垒（TBT）委员会发布的《关于制定国际标准、指南和建议的原则的决定》中确立的国际公认标准化原则制定的。 ====意义和用途====== 5.1 IgE介导的过敏反应的蛋白质过敏原的橡胶树天然胶乳来自 巴西橡胶树 tree于20世纪90年代出现，是一种偶尔出现过敏症状的疾病。 据报道，暴露于橡胶树天然胶乳产品的乳胶过敏个体出现了荨麻疹、尿毒症、鼻炎、哮喘和过敏反应等症状。 5.2 由于没有已知的橡胶树乳胶过敏原暴露的安全水平，避免是治疗乳胶过敏的主要方式。 5.3 由于世界各地许多科学家进行的调查，迄今为止，国际免疫学会联合会（IUIS）过敏原命名小组委员会已识别出14种乳胶过敏原，并将其归类为Hev b 1至Hev b 13( 表1 )（见规范 D1193 ). 由于研究人群、IgE抗体测定方法和分析中用作校准品和质量控制试剂的Hev b过敏原的质量不同，许多报告中报告的这些过敏性Hev b蛋白质的致敏率不同。 然而，大多数研究一致认为，对于因多次手术或长时间使用乳胶导管而通过粘膜接触暴露的个体（例如，脊柱裂儿童），Hev b 1和Hev b 3是重要的过敏原。此外，进行致敏研究的研究人员也同意，Hev b 5和Hev b 6.02是重要的过敏原，可能引起暴露于橡胶树天然胶乳的遗传易感性个体的致敏 ( 2- 4. ) . 在这些临床研究的基础上，开发了这四种过敏蛋白（即Hev b 1、Hev b 3、Hev b 5和Hev b 6.02）的检测方法，因此它们是本标准的主题。采用免疫酶测定试剂和标准蛋白质来量化其他乳胶过敏原（Hev b 1、3、5和6除外）。 02）在橡胶树天然胶乳产品的提取物中，需要单独的文件和验证。 （A） PR=发病机制相关。 5.3.1 从历史背景来看，已经开发了许多分析方法来量化含橡胶树天然胶乳产品中的蛋白质、抗原和过敏原水平（见实践） D4483 和 D4678 ). 5.3.2 这个 改良Lowry 总蛋白的测定，试验方法 D5712 ，是这种类型的第一次检测。它评估总蛋白水平，作为含乳胶产品过敏性的间接指标。本试验不区分过敏性蛋白质和非过敏性蛋白质。 5.3.3 待开发的第二种检测方法涉及在实验室中使用人乳胶特异性IgE抗体 竞争抑制免疫分析 评估橡胶树天然橡胶产品提取物整体过敏效力的格式 ( 5. , 6. ) . 提取物与含有乳胶特异性IgE抗体的人血清孵育，然后将该混合物与固相乳胶过敏吸附剂孵育。乳胶过敏蛋白（如果存在）与溶液中的乳胶特异性IgE抗体结合，从而抑制IgE抗体与乳胶过敏吸附剂结合。然后对过敏吸附结合的IgE进行量化，IgE结合的竞争抑制程度是衡量乳胶过敏原的一个指标。虽然该试验提供了可从产品中提取的橡胶树天然橡胶过敏原的过敏性或水平的估计，但难以获得可重复的大量含乳胶特异性IgE的人血清，阻碍了该试验的广泛使用。 因此，本试验未作为ASTM标准提出。 5.3.4 第三种测定设计类似于基于人IgE的竞争抑制免疫测定，但它使用兔抗血清而不是含有IgE抗乳胶的人血清。这个 竞争抑制酶联免疫吸附试验 （ELISA）已被用作测试方法 D6499 . 它测量引起免疫反应的乳胶蛋白，但无法区分乳胶过敏原（IgE诱导）和非过敏性抗原（非IgE诱导）。 5.3.5 作为本标准主题的最新测定是 双位点免疫酶法（IEMA） 它使用不溶性捕获抗体结合乳胶产品提取物中的一种Hev b过敏蛋白，并使用第二种酶标记的检测抗体检测结合的过敏原。 根据已知过敏原构建的参考曲线插值光密度响应。在与本标准制定相关的国际合作研究中，研究了用于Hev b 1、3、5和6.02免疫酶测定的试剂的性能特征，结果见第节 15 通过 17 .

1.1 This test method covers an immunological method known as an immunoenzymetric assay to quantify the amount of 4 principal Hevea brasiliensis [Hev b] allergenic proteins [Hev b 1, Hev b 3, Hev b 5 and Hev b 6.02] in Hevea natural rubber and its products 2 derived from latex using monoclonal antibodies specific for epitopes on these proteins. Since these assays quantify the levels of only 4 of the known 14 officially acknowledged allergens potentially present in Hevea natural rubber latex containing products, the sum of the four allergen levels shall be viewed as an indicator of the allergen burden and not as a measure of the total allergen 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 microgram quantities per gram or unit surface area of a Hevea natural rubber containing product. 1.3 The test method is not designed to evaluate the potential of Hevea natural rubber containing materials to induce or elicit Type 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 allergenic proteins found in Hevea natural rubber containing products. It should not be used to describe, appraise or assess the hazard or risk of 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 this standard. 1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. 1.7 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee. ====== Significance And Use ====== 5.1 IgE-mediated allergic reactions to protein allergens in Hevea natural rubber latex derived from the Hevea brasiliensis tree emerged in the 1990s as a concern with occasional allergic manifestations. Symptoms encompassing hives, uriticaria, rhinitis, asthma and anaphylaxis have all been reported in latex allergic individuals exposed to products derived from Hevea natural rubber latex. 5.2 Since no safe level of Hevea latex allergen exposure is known, avoidance is the primary mode of treating latex allergy. 5.3 As a result of investigations conducted by many scientists across the world, fourteen latex allergens have so far been identified and categorized by the Allergen Nomenclature Sub-Committee of the International Union of Immunological Societies (IUIS) as Hev b 1 to Hev b 13 ( Table 1 ) (see Specification D1193 ). Reported sensitization rates for these allergenic Hev b proteins vary among the many reports as a result of differences in the study populations, IgE antibody assay methods and the quality of the Hev b allergens used as calibrators and quality control reagents in the analysis. Most studies, however, agree that Hev b 1 and Hev b 3 are important allergens for individuals (for example, children with spina bifida) who are exposed through mucosal contact as a result of multiple surgeries or latex catheter use for an extended period of time. Additionally, investigators performing sensitization studies also agree that Hev b 5 and Hev b 6.02 are important allergens that may elicit sensitization in genetically-predisposed individuals who are exposed to Hevea natural rubber latex ( 2- 4 ) . On the basis of these clinical studies, assays for these four allergenic proteins (that is, Hev b 1, Hev b 3, Hev b 5 and Hev b 6.02) have been developed and they are thus the subject of this standard. Adoption of immunoenzymetric assay reagents and standard proteins needed to quantify other latex allergens (other than Hev b 1, 3, 5, and 6.02) in extracts of Hevea natural rubber latex products will require separate documentation and validation. (A) PR = pathogenesis-related. 5.3.1 From the historical context, a number of assays have been developed to quantify the level of protein, antigen and allergen in Hevea natural rubber latex containing products (see Practices D4483 and D4678 ). 5.3.2 The modified Lowry assay for total protein, Test Method D5712 , was the first assay of this type. It assesses the level of total protein as an indirect indicator of allergenicity of latex-containing products. This assay does not discriminate between the allergenic and non-allergenic proteins. 5.3.3 The second assay to be developed involved the use of human latex-specific IgE antibody in a competitive inhibition immunoassay format to estimate the overall allergenic potency of a Hevea natural rubber product extract ( 5 , 6 ) . The extract is incubated with human serum containing latex-specific IgE antibody and then this mixture is incubated with a solid phase latex allergosorbent. Latex allergenic proteins, if they are present, bind to the latex-specific IgE antibody in solution and they thus inhibit IgE antibody binding onto the latex allergosorbent. Allergosorbent bound IgE is then quantified and the extent of competitive inhibition of IgE binding is a measure of latex allergens. While this assay provides an estimate of the allergenicity or level of Hevea natural rubber allergens extractable from a product, difficulty in procuring reproducible lots of latex specific IgE containing human serum has precluded widespread use of this assay. For this reason, this assay has not been put forth as an ASTM standard. 5.3.4 A third assay design is similar to the human IgE based competitive inhibition immunoassay, but it employs rabbit antiserum instead of human serum containing IgE anti-latex. The competitive inhibition enzyme linked immunosorbent assay (ELISA) has been adopted as Test Method D6499 . It measures latex proteins that elicit immune responses, but it cannot distinguish between latex allergens (IgE inducing) from non-allergenic antigens (non-IgE inducing). 5.3.5 The most recent assay, which is the subject of this standard, is the two-site immunoenzymetric assay (IEMA) which uses an insolubilized capture antibody to bind one of Hev b allergenic proteins from a latex product extract, and a second enzyme labeled detection antibody to detect bound allergens. Optical density responses are interpolated from reference curves constructed with known allergens. The performance characteristics of the reagents used in immunoenzymetric assays for Hev b 1, 3, 5 and 6.02 were investigated in the international collaborative study associated with the development of this standard and results are provided in Sections 15 through 17 .