ASTM D7427-08

IgE-mediated allergic reactions to protein allergens in 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 natural rubber latex.

Since no safe level of Hevea latex allergen exposure is known, avoidance is the primary mode of treating latex allergy.

As a result of investigations conducted by many scientists across the world, thirteen 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 D 1193). 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 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 natural rubber latex products will require separate documentation and validation.

From the historical context, a number of assays have been developed to quantify the level of protein, antigen and allergen in natural rubber latex containing products (see Practices D 4483 and D 4678).

The modified Lowry assay for total protein, Test Method D 5712, 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.

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 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 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.

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 D 6499. It measures latex proteins that elicit immune responses, but it cannot distinguish between latex allergens (IgE inducing) from non-allergenic antigens (non-IgE inducing).

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.

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 natural rubber and its products derived from latex using monoclonal antibodies specific for epitopes on these proteins. Since these assays quantify the levels of only 4 of the known 13 officially acknowledged allergens potentially present in 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 natural rubber containing product.

1.3 The test method is not designed to evaluate the potential of 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 natural rubber containing products. It should not be used to describe, appraise or assess the hazard or risk of these 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 and health practices and determine the applicability of regulatory limitations prior to use.