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Impact of biologic variation on latex allergenicity
Correspondence
J ALLERGY CLiN IMMUNOL
145
VOLUME 101, NUMBER 1, PART 1
REFERENCES
1. Spach DH, Liles WC, Campbell G, Quick RE, Anderson DE, Fritsche TR. Tick-borne diseases in the United States. N Engl J Med
quantitative and would introduce yet another variable (i.e., the allergen composition of the article being tested). Hoong Yeet Yeang, PhD Biotechnology and Strategic Research Division Rubber Research Institute of Malaysia 260, lalan Ampang 50450 Kuala Lumpur Malaysia 1/8/85502
1993;329:936-47.
2. Gaud M, Loh R, Stone BF, Thong YH. Allergic reactions to the Australian paralysis tick, Ixodes holocyclus: diagnosis, evaluation by skin tests and radioimmunoassay. Clin Exp Allergy 1989;19:279-83. 3. Gauci M, Stone BF, Thong YH. Detection in allergic individuals of IgE specific for Australian paralysis tick, Ixodes holocyclus. Int Arch Allergy Appl Immunol 1988;85:190-3. 4. Solley GO. Allergy to stinging and biting insects in Queensland. Med J Aust 1990;153:650-4. 5. Grassi J, Didier-Laurent A, Stadler BM. Quantitative determination of total and specific IgE with the use of monoclonal antibodies. J Allergy Clin Immunol 1986;77:808-22.
1/8/85497
Impact of biologic variation on latex allergenicity To the Editor: I read with interest the position statement on the use of standardized allergen extracts (J Allergy Clin Immunol1997; 99:583-6) and recall the article by Hamilton et al. (J Allergy Clin Immunol 1996;98:872-83) on the development of a characterized latex skin-testing reagent. The position statement emphasized the importance of a consistent relationship between the allergen being measured and other allergens present in the extract. Establishing a reference reagent prepared from whole Hevea latex, rather than purified proteins, is no simple task. Latex;. allergy stems not from a single allergen bll't from no less than seven latex proteins (Hev b 1 t.o Hev b 7). These proteins could differ in their relative allergenicity; they certainly vary widely in their relative abundance in natural rubber latex. Hevein (Hev b 6), for example, is the most abundant soluble protein in latex, constituting some 22% of the total soluble latex proteins. This figure is even higher if we include its precursor, prohevein, which is another major latex protein carrying the hevein allergenic epito~es. Thus one allergen in a latex preparation can be of a concentration tens or hundreds of times greater than that of another. A patient who is only marginally allergic to hevein might very well have a strongly positive test response to a whole-latex reference preparation simply because there is so much hevein present. On the other hand, a patient who is allergic to another allergen that is underrepresented in latex might have a negative test response. My associates and I have found statistically significant seasonal differences (up to 25-fold) in the allergenicity of latex from three of the most widely planted Hevea clones. The total level of soluble allergens, as determined by competitive inhibition assays, does not reveal whether the individual latex allergens all follow the same trend in seasonal variation; further complications ensue if they do not. Clearly, it would take more than simple dilution or concentration to adjust a reference reagent prepared from whole latex to match a predetermined level of allergenicity. The reproducibility of Hamilton's latex allergen reagent would likely be adequate if the allergens used were in molar excess (nonlimiting) to the 19E antibody in the skin or blood. Such a reagent would generate a positive or negative test result but would not be quantitative. On the other hand, the use of a whole-latex reference reagent to assay for residual allergens present in a latex product (e.g., by an inhibition assay) needs to be considered critically. Such an assay would need to be
Reply To the Editor: Dr. Yeang's letter highlights the important issues of allergenic heterogeneity and seasonal variability of natural rubber latex (latex) source materials. We and others obtain latex in a variety of formulations (frozen or in glycerin) from a spectrum of tree clones (mbst commonly clone 600) and from a number of countries in Southeast Asia (most frequently Malaysia). We then process them in different ways to manufacture latex skin-testing and in vitro allergosorbent reagents. Therefore in addition to the inherent biologic variation related to differences in the content of the individual allergenic proteins caused by seasonal differences, these other factors also influence inter-lot variability of individual allergenic components. Unfortunately, we have no control over the relative amounts of the seven known allergenic latex proteins listed in Table I in the initial source materials or over the amounts that survive processing into the final reagents. We can only ensure that they are prepared with the best source materials available and the most reproducible methods (ultracentrifugation, filtration, and covalent coupling) and that their composition is effectively characterized. Our testing includes Food and Drug Administration (FDA)-required safety and sterility tests, a total protein inhibition ELISA with an FDA-derived reference serum for relative allergen content, sodium dodecylsulfate-polyacrylamide gel electrophoresis with silver stain for molecular weight assessment, and Western blot analysis with an FDA reference extract and IgE anti-latex containing serum for allergen assessment. Attention is given to making sure that Hev b 1 (rubber elongation factor) is present. Unfortunately, not one of these qualifying tests is perfect, but as a group, they provide a benchmark for the presence of the major allergens listed in Table I. Our final latex preparations are defined as "characterized" rather than "standardized" reagents.! To address the issue of variation, we have attempted to develop skin testing materials that (1) qualitatively contain all the known latex allergens and (2) are used so that each allergenic constituent is (theoretically) in molar excess to specific IgE bound to mast cells. For serologic reagents, the latex-containing allergosorbents are designed to be in molar excess to latex-specific IgE antibodie5 in the blood. Phase 1 and 2 clinical study data2 indicate that the diagnostic sensitivity and specificity of a candidate nonammoniated latex puncture skintesting reagent are 96% and 100%, respectively. Our ongoing multicenter study uses a glove provocation test 3 to clarify mismatches between the clinical history and skin test or serologic test results. The goals of a diagnostic latex skin test and serologic assay are the accurate detection of the presence of latex-specific IgE in the skin and blood, respectively. Researchers may also attempt to quantify the relative amount of IgE antibody in the skin or blood by titration analyses, but these data have limited utility. To provide further supporting data that we are including all known allergens in our diagnostic latex preparations, it would be helpful if we could quantify the
J ALLERGY CLiN IMMUNOL
145
VOLUME 101, NUMBER 1, PART 1
REFERENCES
1. Spach DH, Liles WC, Campbell G, Quick RE, Anderson DE, Fritsche TR. Tick-borne diseases in the United States. N Engl J Med
quantitative and would introduce yet another variable (i.e., the allergen composition of the article being tested). Hoong Yeet Yeang, PhD Biotechnology and Strategic Research Division Rubber Research Institute of Malaysia 260, lalan Ampang 50450 Kuala Lumpur Malaysia 1/8/85502
1993;329:936-47.
2. Gaud M, Loh R, Stone BF, Thong YH. Allergic reactions to the Australian paralysis tick, Ixodes holocyclus: diagnosis, evaluation by skin tests and radioimmunoassay. Clin Exp Allergy 1989;19:279-83. 3. Gauci M, Stone BF, Thong YH. Detection in allergic individuals of IgE specific for Australian paralysis tick, Ixodes holocyclus. Int Arch Allergy Appl Immunol 1988;85:190-3. 4. Solley GO. Allergy to stinging and biting insects in Queensland. Med J Aust 1990;153:650-4. 5. Grassi J, Didier-Laurent A, Stadler BM. Quantitative determination of total and specific IgE with the use of monoclonal antibodies. J Allergy Clin Immunol 1986;77:808-22.
1/8/85497
Impact of biologic variation on latex allergenicity To the Editor: I read with interest the position statement on the use of standardized allergen extracts (J Allergy Clin Immunol1997; 99:583-6) and recall the article by Hamilton et al. (J Allergy Clin Immunol 1996;98:872-83) on the development of a characterized latex skin-testing reagent. The position statement emphasized the importance of a consistent relationship between the allergen being measured and other allergens present in the extract. Establishing a reference reagent prepared from whole Hevea latex, rather than purified proteins, is no simple task. Latex;. allergy stems not from a single allergen bll't from no less than seven latex proteins (Hev b 1 t.o Hev b 7). These proteins could differ in their relative allergenicity; they certainly vary widely in their relative abundance in natural rubber latex. Hevein (Hev b 6), for example, is the most abundant soluble protein in latex, constituting some 22% of the total soluble latex proteins. This figure is even higher if we include its precursor, prohevein, which is another major latex protein carrying the hevein allergenic epito~es. Thus one allergen in a latex preparation can be of a concentration tens or hundreds of times greater than that of another. A patient who is only marginally allergic to hevein might very well have a strongly positive test response to a whole-latex reference preparation simply because there is so much hevein present. On the other hand, a patient who is allergic to another allergen that is underrepresented in latex might have a negative test response. My associates and I have found statistically significant seasonal differences (up to 25-fold) in the allergenicity of latex from three of the most widely planted Hevea clones. The total level of soluble allergens, as determined by competitive inhibition assays, does not reveal whether the individual latex allergens all follow the same trend in seasonal variation; further complications ensue if they do not. Clearly, it would take more than simple dilution or concentration to adjust a reference reagent prepared from whole latex to match a predetermined level of allergenicity. The reproducibility of Hamilton's latex allergen reagent would likely be adequate if the allergens used were in molar excess (nonlimiting) to the 19E antibody in the skin or blood. Such a reagent would generate a positive or negative test result but would not be quantitative. On the other hand, the use of a whole-latex reference reagent to assay for residual allergens present in a latex product (e.g., by an inhibition assay) needs to be considered critically. Such an assay would need to be
Reply To the Editor: Dr. Yeang's letter highlights the important issues of allergenic heterogeneity and seasonal variability of natural rubber latex (latex) source materials. We and others obtain latex in a variety of formulations (frozen or in glycerin) from a spectrum of tree clones (mbst commonly clone 600) and from a number of countries in Southeast Asia (most frequently Malaysia). We then process them in different ways to manufacture latex skin-testing and in vitro allergosorbent reagents. Therefore in addition to the inherent biologic variation related to differences in the content of the individual allergenic proteins caused by seasonal differences, these other factors also influence inter-lot variability of individual allergenic components. Unfortunately, we have no control over the relative amounts of the seven known allergenic latex proteins listed in Table I in the initial source materials or over the amounts that survive processing into the final reagents. We can only ensure that they are prepared with the best source materials available and the most reproducible methods (ultracentrifugation, filtration, and covalent coupling) and that their composition is effectively characterized. Our testing includes Food and Drug Administration (FDA)-required safety and sterility tests, a total protein inhibition ELISA with an FDA-derived reference serum for relative allergen content, sodium dodecylsulfate-polyacrylamide gel electrophoresis with silver stain for molecular weight assessment, and Western blot analysis with an FDA reference extract and IgE anti-latex containing serum for allergen assessment. Attention is given to making sure that Hev b 1 (rubber elongation factor) is present. Unfortunately, not one of these qualifying tests is perfect, but as a group, they provide a benchmark for the presence of the major allergens listed in Table I. Our final latex preparations are defined as "characterized" rather than "standardized" reagents.! To address the issue of variation, we have attempted to develop skin testing materials that (1) qualitatively contain all the known latex allergens and (2) are used so that each allergenic constituent is (theoretically) in molar excess to specific IgE bound to mast cells. For serologic reagents, the latex-containing allergosorbents are designed to be in molar excess to latex-specific IgE antibodie5 in the blood. Phase 1 and 2 clinical study data2 indicate that the diagnostic sensitivity and specificity of a candidate nonammoniated latex puncture skintesting reagent are 96% and 100%, respectively. Our ongoing multicenter study uses a glove provocation test 3 to clarify mismatches between the clinical history and skin test or serologic test results. The goals of a diagnostic latex skin test and serologic assay are the accurate detection of the presence of latex-specific IgE in the skin and blood, respectively. Researchers may also attempt to quantify the relative amount of IgE antibody in the skin or blood by titration analyses, but these data have limited utility. To provide further supporting data that we are including all known allergens in our diagnostic latex preparations, it would be helpful if we could quantify the