- Email: [email protected]
Assessment of IgE allergen specificity among latex-allergic health care workers: review of IgE-binding components of various latex extracts
Assessment of IgE allergen specificity among latex-allergic health care workers: review of IgEbinding components of various latex extracts P S Nielsen, MD*; D Nissen, MSc*; P S Skov, MD, PhD*; G Cieslewicz, MD, PhD†; A Markov, MD, PhD†; A Babakhin, MD, PhD†; I Zakirova, MD†; T Gots, MD, PhD; L M DuBuske, MD‡; A Sheffer, MD‡; and H Nolte, MD, PhD*
Background: Allergic reactions to natural rubber latex have increased during the past 10 years, especially in many health care workers who have high exposure to latex allergens both by direct skin contact and by inhalation of latex particles from powdered gloves. Development of satisfactory diagnostic methods to verify the presence of latex allergy in health care workers requires characterization of the immunoreactive proteins in latex products and identification of specific IgE antibodies in sensitized patients. A number of different latex preparations are now available for in vitro evaluations. Objectives: Utilizing different in vitro methods, this study examines IgE sensitization to components of latex in a selected population of hospital employees, employing a raw natural latex glove extract and various commercial latex extracts. Methods: Two hundred hospital employees exposed to latex were evaluated using an allergy history questionnaire. To further identify sensitized patients, two different specific IgE tests and leukocyte histamine release tests were performed using a panel of latex extracts obtained from different manufacturers. Sodium dodecylsulfate polyacrylamide electrophoresis (SDS-PAGE) profiles were obtained. Sera from 34 subjects suspected to be latex-sensitized were IgE immunoblotted to assess the presence of IgE antibodies directed toward specific latex proteins. Results: Thirty-four participants (17%) were considered sensitized to latex by a positive clinical history in conjunction with positive specific IgE tests (18 individuals) and/or positive histamine release tests (26 individuals). Significant extract differences in both the histamine release response profile and the frequency of positive test results were noted in the histamine release test. Significant individual differences in patients’ latex epitope-specificity were found by IgE immunoblotting, substantiated by sodium dodecylsulfate polyacrylamide profiles revealing differences in protein band patterns among the various extracts. The IgE immunoblots indicated that the majority of patients reacted to proteins with molecular weights of 14, 21, 30 to 35, and 42 kD; the 30 to 35 kD protein being predominant. Seven subjects (22%) of the 34 considered to be latex-sensitized did not reveal binding of specific IgE in immunoblots. One latex extract (Stallergene) with the widest IgE-reacting protein repertoire identified the majority of subjects (63%) as latex sensitive by leukocyte histamine release and also provided the best quantitative histamine release test results. Conclusion: Only by testing with a combination of latex extracts were all sensitized individuals identified. This study demonstrates that currently several in vitro methods may be necessary to detect IgE sensitization to latex. Latex extracts to be employed in future skin tests must contain a wide epitope repertoire of IgE-binding proteins to identify all latex-sensitized individuals. Ann Allergy Asthma Immunol 2000;85:489– 494.
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INTRODUCTION Several proteins contained in non-ammoniated latex sap, ammoniated latex sap, raw latex extract and latex extracts prepared from latex gloves have been proposed as major allergens of latex. The most important latex allergens include Rubber Elongation Factor (58 kD), the homotetramer of a 14.6-kD peptide (Hev b 1) identified by Czuppon.1 Two hevamines of 26 to 30-kD2 have been identified in latex as well as hevein of 14 kD3 and prohevein with molecular weight of 20 kD.4,5 Beezhold also identified a 27-kD natural rubber latex protein in children with spina bifida.6 – 8 Additionally, several cross-reacting allergens have been observed in various fruits and plants such as avocado, banana, and kiwi.9 –11 It is an interesting feature of latex sensitization that different patient populations show unique protein sensitization patterns; eg, spina bifida children develop specific IgE antibodies against proteins of 14 kD,7 while the majority of hospital employees are sensitized to proteins in the range of 30 to 35 kD.6 It has been demonstrated that the manufacturing procedures and handling of raw latex preparation changes the protein profile of latex by hydrolyzing and
* The Reference Laboratory, National University Hospital, Copenhagen, Denmark. † Immunology Research Institute of New England, Fitchburg, Massachusetts. ‡ The Allergy Department, Brigham and Women’s Hospital, Boston, Massachusetts. This study was partly funded by an unrestricted grant from Allergifonden of 1981. Received for publication July 2, 1999. Accepted for publication in revised from May 10, 2000.
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denaturing the proteins.12,13 Prolonged extraction can yield higher amounts of protein, although no major differences in reactivity were found among the two types of extracts. Utilizing the inhibition immunoblot method, Akasawa demonstrated that the reactive IgE proteins differ among different latex extracts. Ammoniated latex and latex glove extract contain more complete immunoreactive repertoires for detecting IgE antibodies.14 Latex extracts for diagnostic use may show variable diagnostic quality and further delineation of allergen content is necessary to prepare a clinically useful latex extract for skin testing. Because of the lack of a commercially available latex skin test extract, this study was undertaken in order to estimate the frequency of IgE-dependent latex sensitization in 200 Brigham and Women’s Hospital employees who were evaluated by clinical history, specific IgE tests and leukocyte histamine release test. In addition, the protein profiles of five different latex extracts were by characterized by sodium dodecylsulfate polyacrylamide gel electrophoresis. Specific-IgE immunoblotting was used to evaluate the latex epitope reactivity in these latex sensitized health care workers. MATERIALS AND METHODS Subjects Two hundred hospital employees filled out a questionnaire reporting symptoms possibly associated with latex allergy. Symptoms included contact urticaria, angioedema, contact dermatitis, asthma, rhinoconjunctivitis, and anaphylaxis. To verify a diagnosis of latex allergy, histamine release tests and specific IgE tests were performed. Latex sensitization was determined by the presence of symptoms combined with either a positive specific IgE test or positive leukocyte histamine release test. Sera from participants with latex sensitization according to the above criteria as well as control sera from eight non-atopic controls with no regular latex exposure were collected for IgE immunoblotting. Human myeloma
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IgE (non-sensitized IgE) was used as a reference in the immunoblot control experiments. Allergens A panel of five different latex extracts was tested. Extracts were supplied from the following manufacturers: Stallergene Latex Lyophilized (166 g protein/mL); Bencard Latex Test Solution (1% in 50% glycerol); Greer Latex non-ammoniated Latex (1 mg protein/mL); ALK skin prick test latex extract (2 mg protein/mL); and Latex glove extract (The Reference Laboratory, Denmark). The latter latex glove extract was prepared from 7.8 g of finely cut latex glove, incubated in 40 mL. Pipes buffer and extracted for 24 hours at 37°C. All extracts were stabilized in a 50% glycerol solution and stored at 4°C. Leukocyte Histamine Release Test Leukocyte histamine release test was performed as previously described with glass microfiber-coated microtiter plates, which selectively bind histamine.15 In this test, 25 L of whole blood was incubated with 25 L of each latex extract at six different concentrations. The stock solution of Stallergene latex extract, ALK latex extract, Bencard latex extract, Greer latex extract and glove extract were diluted 1:10 in Pipes-AMC buffer [10 mL piperazine-N,N⬘-bis(2-ethanesulfonic acid)]; 140 mM sodium acetate; 5 mM potassium acetate; 30 mM Trizma-T-1503; 0.5 mM calcium chloride; and 1 mM magnesium chloride (pH 7.4), followed by 3.5-fold dilutions. Aliquots were run in duplicate and tested at six dilutions predetermined to insure a representative histamine release curve. In addition, all blood samples were incubated with several dilutions of anti-IgE (Behringwerke, Germany). After 1-hour incubation at 37°, the glass microfiber-coated microtiter wells were rinsed and cell debris and proteins were enzymatically removed. Released histamine was coupled to -phthaldedehyde and measured spectrophotofluorometrically with a Gilson fluorometer.16,17 Released histamine was expressed as nanograms of histamine per milliliter and
calculated on the basis of a histamine standard curve.16,17 Pharmacia CAP Tests were performed according to manufacturer’s (Pharmacia-UpJohn, NJ) directions. Test results are reported in classes ranging from 0 to 6. The upper and lower threshold limits for detection of specific IgE are 100 kU/L and 0.35 kU/L. Hytec RAST Tests were performed according to manufacturer’s (Hycor Biomedical, Inc, CA) directions. Test results are reported in classes ranging from 0 to 6. The upper and lower threshold limits are 100 kU/L and 0.35 kU/L. Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis Sodium dodecylsulfate polyacrylamide gel electrophoresis was performed using a 8% to 16% Tris-glycine Novex precast gel running in a Novex Xcell II Mini-cell (El 9001, Novex, San Diego, CA, USA). This system is a modified Laemmli system,18 wherein extracts were boiled 4 minutes with a reducing sample buffer (2.5:1.0) containing 2-mercaptoethanol. For protein characterization, 25 L latex extract was loaded onto a 10-well gel; and for immunoblotting, 400 L latex extract was loaded onto 2D (2-well) gels. Protein sizes were estimated from a prestained marker (Multimark, TM, Multicolored Standard, range 4 to 250 kD, Novex). IgE Immunoblotting Transfer of latex proteins to nitrocellulose membranes (pore size 0.45 M) was performed with Novex X cell II Blot Module and a pre-mixed buffer (Tris-Glycine Transfer Buffer) according to manufacturer’s instructions. Proteins were transferred at 35 V and 100 mA for 90 minutes. For IgE immunoblotting, the nitrocellulose paper was cut into 3 to 5 mm strips and incubated 30 minutes in TBS buffer (0.05 M Tris, 0.15 N NaCl, 0.005 M NaH3, pH 7.4) containing 1% Tween. The membranes were then incubated overnight at room temperature with
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500 L patient sera diluted with 1:10 in TBS-buffer containing 0.1% Tween 20. Normal sera and human myeloma IgE (4000 U/mL) served as negative control samples. After three washings, each membrane was incubated two hours with 500 L alkaline phosphatase conjugated goat anti-human IgE, 1:1000. After washing, bands were visualized by adding 500 L mixture of nitro blue tetrazolium and 5-bromo-4chloro-3-indolyl phosphate (50 mg/mL Promega). Membranes were incubated for a maximum of 30 minutes, and when visible bands appeared, further reaction was inhibited by removing the substrate and adding distilled water. Data Evaluation The histamine release data were evaluated by calculating the area under the curve (AUC) by numerical integration using the formula 1⁄2 c1 ⫹ c2 ⫹ c3 ⫹ c4 ⫹ c5 ⫹ 1⁄2 c6, where “c” is the histamine released (in ng/mL whole blood) at a given concentration (symbolized by the numbers 1 through 6). An AUC of ⱖ 40 ng histamine/mL blood constitutes a positive histamine release response.19 The proteins detected by IgE immunoblotting were divided into three categories; weak/visible, moderate, and strong by visual evaluation. The immunoblot was considered positive when at least one protein band was visible. RESULTS Subjects Thirty-four individuals (17%) of 200 subjects were considered latex sensitized by clinical history and either a positive specific IgE test result (Hytec RAST; n ⫽ 18) and/or a positive leukocyte histamine release test result (n ⫽ 26) (Table 1). Sixty-five (33%) subjects reported latex-associated symptoms without testing positive by the in vitro tests. The rest of the subjects (n ⫽ 101) were not reporting latex-associated symptoms and were negative by in vitro tests. In the latex-sensitized patient group the mean age was 37 years with a female:male ratio of 31:1. Eleven latex sensitized subjects (32%) reported a
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history of previous atopy. The most common symptoms attributed to latex exposure was rhinitis (39%), followed by asthma (33%), dermatitis (28%), systemic urticaria (20%), contact urticaria (15%), conjunctivitis (13%), angioedema (4%), and anaphylaxis (4%). Fifty-four percent had two symptoms and 15% had three or more latex-related symptoms. Rhinitis (68%), asthma (53%), urticaria/angioedema (53%), dermatitis (42%), and conjunctivitis (21%) were noted in those having multiple latex-associated symptoms. Leukocyte Histamine Release Test Leukocyte histamine release test results (Fig 1) showed that the Stallergene latex extract produced the highest amount of histamine release (mean 62 ng/mL histamine) when evaluated by the AUC, followed by histamine release from latex glove extract (mean 50 ng/mL). The non-ammoniated latex produced a mean histamine release of 44 ng/mL. The lowest histamine release values were obtained from the Bencard extract (mean 24 ng/mL) and the ALK skin test extract (mean 22 ng/mL). The mean AUC of anti-IgEinduced histamine release was 46 ng/ mL. These results correlated well with the frequency distribution of leukocyte histamine release positive test results among the various latex extracts (Table 1). Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis Separation of latex proteins by sodium dodecylsulfate polyacrylamide gel electrophoresis (Fig 2) revealed a broad molecular weight range for latex proteins (14 to 120 kD). The predominant pro-
teins in both the ALK extract and the latex glove extracts were of molecular weights 14 kD, 30 to 35 kD, and 51 kD. Sodium dodecylsulfate polyacrylamide gel electrophoresis of the Stallergene extract revealed highly stained bands at 30 to 35 kD and 42 kD. Similarly, a wide range of proteins were shown in the non-ammoniated latex extract (6 to 120 kD), the predominant proteins being of 30 kD, 50 kD, and 56 kD (Fig 2). No protein bands were detected in the Bencard extract. Frequency Distribution of Test Results Twenty-six of the latex-sensitized hospital employees (76%) demonstrated leukocyte histamine release to one or more latex extracts (Table 1). Thirteen latex-sensitized individuals (38%) elicited positive specific IgE test results (Pharmacia CAP); 18 (53%) were positive by Hytec RAST test; 25 (78%) in IgE immunoblotting. IgE binding was not observed in eight non-atopic controls who had been immunoblotted; similarly, IgE binding was not observed when using myeloma-IgE (400 IU/mL) as control. Although no stained bands were observed using the Bencard latex extract by sodium dodecylsulfate polyacrylamide gel electrophoresis, the same extract induced a histamine release response in eight patients (31%) with a mean AUC of 25 ng/mL histamine (Fig 2 and Table 1). Immunoblotting with Bencard latex extract was not performed due to the absence of proteins in sodium dodecylsulfate polyacrylamide gel electrophoresis.
Table 1. Frequency Distribution of Latex-Positive Test Results in 200 Health Care Workers (N ⫽ 34/200; 0.17)
Total number of test positives Latex allergen Stallergene Greer Latex glove Bencard ALK
Histamine Release N (%)
Pharmacia CAP N (%)
Hycor RAST N (%)
26 (76%)
13 (38%)
18 (53%)
20 (77%) 13 (50%) 11 (42%) 8 (31%) 5 (19%)
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IgE-Immunoblotting Analysis The most frequent IgE-binding components of the various latex extracts are summarized in Table 2. It is observed that the Stallergene extract, the glove extract and the ALK skin prick test extract revealed the highest frequency of IgE binding (16, 31, and 9 reactions, respectively). IgE immunoblotting with sera from 34 latex-sensitized individuals demonstrated the highest incidence of reactions with IgE-binding proteins of 30 to 35 kD (34 IgE-binding reactions, when tested with all latex extracts). Additionally, 14 and 42 kD proteins showed frequent IgE binding (five IgE-binding reactions, when tested with three extracts). The results of seven patients considered latex-sensitized by the above defined criteria did not reveal binding to latex proteins in IgE-immunoblotting. Five of these seven patients demonstrated leukocyte histamine release, and three had measurable levels of specific levels of IgE both by Pharmacia CAP and Hycor RAST. One patient tested positive for all tests, while four patients tested positive only in the histamine release test. Latex Glove Extract IgE-immunoblotting with the glove extract showed that the 30 –35 kD protein bound IgE in 25 subjects (74%). In addition, two subjects showed binding to the 14 kD, 42 kD, and 51 kD proteins, thus revealing an epitope repertoire in the lower molecular range (14 to 51 kD). Stallergene Latex Extract Immunoblotting with Stallergene latex extract revealed a broad spectrum of IgE-binding proteins (14 kD, 21 kD, 30 to 35 kD, 42 kD, 48 kD, and 56 to 60 kD); only one to four subjects reacting to each protein. The epitope repertoire for this extract was demonstrated to be in the lower molecular range (14 to 60 kD). Non-Ammoniated Latex Extract IgE-binding latex proteins in the Greer latex extract were found in the higher molecular range (39 to 72 kD). IgE
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Figure 1. Histamine release in ng/mL evaluated by the AUC for Bencard latex extract, Stallergene latex extract, latex glove extract, Geer latex extract and ALK skin prick test extract (ALK).
Figure 2. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) results for Stallergene latex extract (SG), latex glove extract (LG), non-ammoniated latex extract, Greer (NAL), latex sap (SAP), ALK latex skin prick test solution and Bencard latex extract.
binding was exhibited in eleven participants, eight of whom reacted to the 56 to 60-kD protein. Latex Skin Prick Test Extract In contrast to the above described extracts, the ALK latex extract demonstrated an epitope repertoire in the higher molecular range (30 to 64 kD). Proteins with IgE-binding properties were 30 to 35 kD, 51 kD, 56 to 60 kD, and 64 kD. As determined by immunoblotting, four subjects were sensitized to the 30 to 35 kD protein, three to the 51-kD protein, and one each to the two proteins of the highest molecular weights (56 to 60 kD and 64 kD).
Bencard Latex Extract IgE immunoblotting with the Bencard latex extract was not performed as sodium dodecylsulfate polyacrylamide gel electrophoresis showed the lack of sufficient quantities of proteins. DISCUSSION Development of satisfactory methods for diagnosing latex allergy requires both characterization of the immunoreactive proteins in the latex products and the identification of specific IgE antibodies in sensitized individuals. This study demonstrates significant differences in protein profiles among
Table 2. Frequency of Patient Sera Reacting with IgE-Binding Components in the Various Latex Extract Preparations kD
14
21
30–35
42
48
Stallergene Greer Glove ALK
3
4
2 3 25 4
3
1
2
2
51
56–60
64
3 8 2
3
1
1
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various latex extracts as well as significant individual differences in sensitization patterns. It is well documented that latex content varies considerably among latex products as a result of dissimilar manufacturing procedures.12–14 The high individual variance in patient reactivity may perhaps be explained by exposure to different latex products such as different brands of manufactured latex gloves. Latex extracts were characterized by a large number of proteins; however, when evaluated by the frequency of reaction, the majority of these proteins were minor allergens and of less importance. Several latex proteins have been identified and suggested as major allergens; eg, rubber elongation factor, which exists in a tetrameric form in latex.1– 8 In this study, however, only the 30 to 35 kD (Hev b 2?) protein affected more than 50% of the population and then only when tested by the Stallergene extract. The high number of immunoblot reactions in the 30 to 35 molecular weight range (74%) may have been nonspecific reactions, but none of the eight non-atopic controls, as well as the remaining 26% of the study population, demonstrated IgE binding to this protein. Amino acid composition and sequence of the IgE binding proteins identified in this study were not performed; therefore, the IUIS nomenclature cannot be applied with certainty. It is possible, however, that the 14 kD, 30 to 35, 42, 48, and 56 to 60 kD protein are Hev b 1, Hev b 6.03 (14.6 and 14.0 kD), Hev b 2 (35.1 kD), Hev b 7 (42.9 kD), Hev b 4 (48, 50 to 57 kD) latex allergens, respectively. When analyzing the sensitization to latex by histamine release from leukocytes, it was observed that eight individuals (24%) showed positive histamine release without detection of specific IgE antibodies either by Pharmacia CAP or by Hycor RAST. In six of these participants, immunoblotting revealed IgE binding, indicating lack of analytical sensitivity by these specific IgE tests. One explanation is that the latex extract used by Pharmacia
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CAP and Hycor RAST may be entirely different from the test extracts obtained for this study. The discrepancy, probably, reflects variability due to extract composition rather than analytical method. In three subjects with a negative histamine release response, the presence of IgE antibodies was verified by both specific IgE tests, Pharmacia CAP, and Hycor RAST. It is hypothesized that these antibodies were incapable of binding the protein sufficiently in order to activate basophils. Seven patients who were diagnosed as latexsensitized by clinical history and specific-IgE testing did not exhibit binding of specific IgE in immunoblotting. A possible explanation may be that biologically important immunoreactive proteins are denatured or degraded by the sodium dodecylsulfate polyacrylamide gel electrophoresis, resulting in loss of IgE-binding capacity. An alternative hypothesis is that some allergens may consist of immunoreactive glycoproteins, causing IgE binding which cannot be detected in either immunoblotting or by specific IgE tests. The Bencard latex extract produced eight positive histamine release responses, but demonstrated no protein bands in sodium dodecylsulfate polyacrylamide gel electrophoresis. Possibly, the amounts of protein were too low or the extract consisted of mainly glycoproteins in which the protein components were not detectable in immunoblotting. Alternatively, the protein fraction could have been degraded by proteolytic enzymes, although two different batch numbers were tested with identical results. In conclusion, health care workers exposed to latex exhibited a high frequency of latex sensitization, possibly due to a high degree of exposure. The results of this investigation indicate that the development of latex sensitization may involve different allergenspecific epitopes, illustrated by the different sensitization patterns among participants. Leukocyte histamine release test allows testing with a variety of different latex sources, an advantage
when elucidating possible differences in extracts. None of the latex extracts tested has currently been able to identify all sensitized subjects due perhaps to a large variety in epitopes as well as different analytical sensitivity by various methods used to demonstrate IgE-sensitization. Before undertaking diagnostic studies of various allergy tests it would be helpful to delineate the optimal allergen profile of latex extracts for clinical testing. Until then a combination of allergy tests may therefore be necessary to determine an individual’s susceptibility to latex. ACKNOWLEDGEMENTS Dr R Esch from Greer Laboratories is thanked for providing the non-ammoniated latex preparation and Dr L Jacobsen and T Wikborg from ALKAbello in Denmark for providing the latex material from ALK. It must be emphasized that latex extracts from Greer and ALK-Abello have been reformulated after this study was finished. Thus, the extract characteristics and performance data of this study may not reflect the currently available extracts from the above manufacturers. REFERENCES 1. Czuppon AB, Chen Z, Rennert S, et al. The rubber elongation factor of rubber trees (Hevea Brasiliensis) is the major allergen in latex. J Allergy Clin Immunol 1993;92:690 – 697. 2. Beezhold D, Sussman G. Characterization and identification of latex aller gens [abstract]. J Allergy Clin Immunol 1994;93:182. 3. Alenius H, Palosuo T, Kelly K, et al. IgE reactivity to 14 kD and 27 kD natural rubber proteins in latex-allergic children with spina bifida and other congenital anomalies. Int Arch Allergy Immunol 1993;102:61– 66. 4. Alenius H, Kalkkinen N, Lukka M, et al. Prohevein from the rubber tree (Hevea Brasiliensis) is a major latex allergen. Clin Exp Allergy 1995;24: 659 – 665. 5. Alenius H, Kalkkinen N, Yip E, et al. Significance of rubber elongation factor as a latex allergen. Int Arch Allergy Immunol 1996;109:362–368. 6. Beezhold DH, Sussman GL, Kostyal
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7.
8.
9.
10.
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D, et al. Identification of a 46 kD latex protein in health care workers. Exp Immunol 1994;98:408 – 413. Alenius H, Kalkkinen N, Lukka M, et al. Purification and partial amino acid sequencing of a 27 kD natural rubber allergen recognized by latex allergic children with spina bifida. Int Arch Allergy Immunol 1995;106:258 –262. Ahlroth M, Alenius H, Turjanmaa K, et al. Cross reacting allergens in natural rubber latex and avocado. J Allergy Clin Immunol 1995;96:167–173. Ceuppens JL, van Durme P, DoomsGoosens A. Latex allergy in patients with allergy to fruit [Letter]. Lancet 1992;339 – 493. Ma¨kinen-Kiljunen S. Banana allergy in patients with immediate type hyper-
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12. 13.
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sensitivity to natural rubber latex: characterization of cross-reacting antibodies and allergens. J Allergy Clin Immunol 1994;93:990 –996. Akasawa A, Hsieh L, Lin Y. Serum reactivities to latex proteins (Hevea brasiliensis). J Allergy Clin Immunol 1995;95:1196 –1205. Slater JE, Chhabra SK. Latex allergens. J Allergy Clin Immunol 1992;89: 673– 678. Fink JN, Kelly KJ, Elms N, et al. Comparative studies of latex extracts used in skin testing. Ann Allergy 1996;76: 149 –152. Akasawa A, Hsieh LS, Lin Y. Comparison of latex specific IgE binding among non ammoniated latex, ammoniated latex and latex glove allergenic
extracts by ELISA and immunoblot inhibition. J Allergy Clin Immunol 1996; 97(5):1116 –1120. 15. Nolte H. The clinical utility of basophil histamine release. Allergy Proceedings 1993;14:251–254. 16. Stahl Skov P, Mosbech H, Norn S, et al. Sensitive glass microfibre-based histamine analysis for allergy testing in washed blood cells. Allergy 1985; 40:213–217. Request for reprints should be addressed to: Hendrik Nolte, MD, PhD Allergy Consult, Llc 152 Simsbury Road Avon, CT 06001 E-mail: [email protected]
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Background: Allergic reactions to natural rubber latex have increased during the past 10 years, especially in many health care workers who have high exposure to latex allergens both by direct skin contact and by inhalation of latex particles from powdered gloves. Development of satisfactory diagnostic methods to verify the presence of latex allergy in health care workers requires characterization of the immunoreactive proteins in latex products and identification of specific IgE antibodies in sensitized patients. A number of different latex preparations are now available for in vitro evaluations. Objectives: Utilizing different in vitro methods, this study examines IgE sensitization to components of latex in a selected population of hospital employees, employing a raw natural latex glove extract and various commercial latex extracts. Methods: Two hundred hospital employees exposed to latex were evaluated using an allergy history questionnaire. To further identify sensitized patients, two different specific IgE tests and leukocyte histamine release tests were performed using a panel of latex extracts obtained from different manufacturers. Sodium dodecylsulfate polyacrylamide electrophoresis (SDS-PAGE) profiles were obtained. Sera from 34 subjects suspected to be latex-sensitized were IgE immunoblotted to assess the presence of IgE antibodies directed toward specific latex proteins. Results: Thirty-four participants (17%) were considered sensitized to latex by a positive clinical history in conjunction with positive specific IgE tests (18 individuals) and/or positive histamine release tests (26 individuals). Significant extract differences in both the histamine release response profile and the frequency of positive test results were noted in the histamine release test. Significant individual differences in patients’ latex epitope-specificity were found by IgE immunoblotting, substantiated by sodium dodecylsulfate polyacrylamide profiles revealing differences in protein band patterns among the various extracts. The IgE immunoblots indicated that the majority of patients reacted to proteins with molecular weights of 14, 21, 30 to 35, and 42 kD; the 30 to 35 kD protein being predominant. Seven subjects (22%) of the 34 considered to be latex-sensitized did not reveal binding of specific IgE in immunoblots. One latex extract (Stallergene) with the widest IgE-reacting protein repertoire identified the majority of subjects (63%) as latex sensitive by leukocyte histamine release and also provided the best quantitative histamine release test results. Conclusion: Only by testing with a combination of latex extracts were all sensitized individuals identified. This study demonstrates that currently several in vitro methods may be necessary to detect IgE sensitization to latex. Latex extracts to be employed in future skin tests must contain a wide epitope repertoire of IgE-binding proteins to identify all latex-sensitized individuals. Ann Allergy Asthma Immunol 2000;85:489– 494.
VOLUME 85, DECEMBER, 2000
INTRODUCTION Several proteins contained in non-ammoniated latex sap, ammoniated latex sap, raw latex extract and latex extracts prepared from latex gloves have been proposed as major allergens of latex. The most important latex allergens include Rubber Elongation Factor (58 kD), the homotetramer of a 14.6-kD peptide (Hev b 1) identified by Czuppon.1 Two hevamines of 26 to 30-kD2 have been identified in latex as well as hevein of 14 kD3 and prohevein with molecular weight of 20 kD.4,5 Beezhold also identified a 27-kD natural rubber latex protein in children with spina bifida.6 – 8 Additionally, several cross-reacting allergens have been observed in various fruits and plants such as avocado, banana, and kiwi.9 –11 It is an interesting feature of latex sensitization that different patient populations show unique protein sensitization patterns; eg, spina bifida children develop specific IgE antibodies against proteins of 14 kD,7 while the majority of hospital employees are sensitized to proteins in the range of 30 to 35 kD.6 It has been demonstrated that the manufacturing procedures and handling of raw latex preparation changes the protein profile of latex by hydrolyzing and
* The Reference Laboratory, National University Hospital, Copenhagen, Denmark. † Immunology Research Institute of New England, Fitchburg, Massachusetts. ‡ The Allergy Department, Brigham and Women’s Hospital, Boston, Massachusetts. This study was partly funded by an unrestricted grant from Allergifonden of 1981. Received for publication July 2, 1999. Accepted for publication in revised from May 10, 2000.
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denaturing the proteins.12,13 Prolonged extraction can yield higher amounts of protein, although no major differences in reactivity were found among the two types of extracts. Utilizing the inhibition immunoblot method, Akasawa demonstrated that the reactive IgE proteins differ among different latex extracts. Ammoniated latex and latex glove extract contain more complete immunoreactive repertoires for detecting IgE antibodies.14 Latex extracts for diagnostic use may show variable diagnostic quality and further delineation of allergen content is necessary to prepare a clinically useful latex extract for skin testing. Because of the lack of a commercially available latex skin test extract, this study was undertaken in order to estimate the frequency of IgE-dependent latex sensitization in 200 Brigham and Women’s Hospital employees who were evaluated by clinical history, specific IgE tests and leukocyte histamine release test. In addition, the protein profiles of five different latex extracts were by characterized by sodium dodecylsulfate polyacrylamide gel electrophoresis. Specific-IgE immunoblotting was used to evaluate the latex epitope reactivity in these latex sensitized health care workers. MATERIALS AND METHODS Subjects Two hundred hospital employees filled out a questionnaire reporting symptoms possibly associated with latex allergy. Symptoms included contact urticaria, angioedema, contact dermatitis, asthma, rhinoconjunctivitis, and anaphylaxis. To verify a diagnosis of latex allergy, histamine release tests and specific IgE tests were performed. Latex sensitization was determined by the presence of symptoms combined with either a positive specific IgE test or positive leukocyte histamine release test. Sera from participants with latex sensitization according to the above criteria as well as control sera from eight non-atopic controls with no regular latex exposure were collected for IgE immunoblotting. Human myeloma
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IgE (non-sensitized IgE) was used as a reference in the immunoblot control experiments. Allergens A panel of five different latex extracts was tested. Extracts were supplied from the following manufacturers: Stallergene Latex Lyophilized (166 g protein/mL); Bencard Latex Test Solution (1% in 50% glycerol); Greer Latex non-ammoniated Latex (1 mg protein/mL); ALK skin prick test latex extract (2 mg protein/mL); and Latex glove extract (The Reference Laboratory, Denmark). The latter latex glove extract was prepared from 7.8 g of finely cut latex glove, incubated in 40 mL. Pipes buffer and extracted for 24 hours at 37°C. All extracts were stabilized in a 50% glycerol solution and stored at 4°C. Leukocyte Histamine Release Test Leukocyte histamine release test was performed as previously described with glass microfiber-coated microtiter plates, which selectively bind histamine.15 In this test, 25 L of whole blood was incubated with 25 L of each latex extract at six different concentrations. The stock solution of Stallergene latex extract, ALK latex extract, Bencard latex extract, Greer latex extract and glove extract were diluted 1:10 in Pipes-AMC buffer [10 mL piperazine-N,N⬘-bis(2-ethanesulfonic acid)]; 140 mM sodium acetate; 5 mM potassium acetate; 30 mM Trizma-T-1503; 0.5 mM calcium chloride; and 1 mM magnesium chloride (pH 7.4), followed by 3.5-fold dilutions. Aliquots were run in duplicate and tested at six dilutions predetermined to insure a representative histamine release curve. In addition, all blood samples were incubated with several dilutions of anti-IgE (Behringwerke, Germany). After 1-hour incubation at 37°, the glass microfiber-coated microtiter wells were rinsed and cell debris and proteins were enzymatically removed. Released histamine was coupled to -phthaldedehyde and measured spectrophotofluorometrically with a Gilson fluorometer.16,17 Released histamine was expressed as nanograms of histamine per milliliter and
calculated on the basis of a histamine standard curve.16,17 Pharmacia CAP Tests were performed according to manufacturer’s (Pharmacia-UpJohn, NJ) directions. Test results are reported in classes ranging from 0 to 6. The upper and lower threshold limits for detection of specific IgE are 100 kU/L and 0.35 kU/L. Hytec RAST Tests were performed according to manufacturer’s (Hycor Biomedical, Inc, CA) directions. Test results are reported in classes ranging from 0 to 6. The upper and lower threshold limits are 100 kU/L and 0.35 kU/L. Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis Sodium dodecylsulfate polyacrylamide gel electrophoresis was performed using a 8% to 16% Tris-glycine Novex precast gel running in a Novex Xcell II Mini-cell (El 9001, Novex, San Diego, CA, USA). This system is a modified Laemmli system,18 wherein extracts were boiled 4 minutes with a reducing sample buffer (2.5:1.0) containing 2-mercaptoethanol. For protein characterization, 25 L latex extract was loaded onto a 10-well gel; and for immunoblotting, 400 L latex extract was loaded onto 2D (2-well) gels. Protein sizes were estimated from a prestained marker (Multimark, TM, Multicolored Standard, range 4 to 250 kD, Novex). IgE Immunoblotting Transfer of latex proteins to nitrocellulose membranes (pore size 0.45 M) was performed with Novex X cell II Blot Module and a pre-mixed buffer (Tris-Glycine Transfer Buffer) according to manufacturer’s instructions. Proteins were transferred at 35 V and 100 mA for 90 minutes. For IgE immunoblotting, the nitrocellulose paper was cut into 3 to 5 mm strips and incubated 30 minutes in TBS buffer (0.05 M Tris, 0.15 N NaCl, 0.005 M NaH3, pH 7.4) containing 1% Tween. The membranes were then incubated overnight at room temperature with
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500 L patient sera diluted with 1:10 in TBS-buffer containing 0.1% Tween 20. Normal sera and human myeloma IgE (4000 U/mL) served as negative control samples. After three washings, each membrane was incubated two hours with 500 L alkaline phosphatase conjugated goat anti-human IgE, 1:1000. After washing, bands were visualized by adding 500 L mixture of nitro blue tetrazolium and 5-bromo-4chloro-3-indolyl phosphate (50 mg/mL Promega). Membranes were incubated for a maximum of 30 minutes, and when visible bands appeared, further reaction was inhibited by removing the substrate and adding distilled water. Data Evaluation The histamine release data were evaluated by calculating the area under the curve (AUC) by numerical integration using the formula 1⁄2 c1 ⫹ c2 ⫹ c3 ⫹ c4 ⫹ c5 ⫹ 1⁄2 c6, where “c” is the histamine released (in ng/mL whole blood) at a given concentration (symbolized by the numbers 1 through 6). An AUC of ⱖ 40 ng histamine/mL blood constitutes a positive histamine release response.19 The proteins detected by IgE immunoblotting were divided into three categories; weak/visible, moderate, and strong by visual evaluation. The immunoblot was considered positive when at least one protein band was visible. RESULTS Subjects Thirty-four individuals (17%) of 200 subjects were considered latex sensitized by clinical history and either a positive specific IgE test result (Hytec RAST; n ⫽ 18) and/or a positive leukocyte histamine release test result (n ⫽ 26) (Table 1). Sixty-five (33%) subjects reported latex-associated symptoms without testing positive by the in vitro tests. The rest of the subjects (n ⫽ 101) were not reporting latex-associated symptoms and were negative by in vitro tests. In the latex-sensitized patient group the mean age was 37 years with a female:male ratio of 31:1. Eleven latex sensitized subjects (32%) reported a
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history of previous atopy. The most common symptoms attributed to latex exposure was rhinitis (39%), followed by asthma (33%), dermatitis (28%), systemic urticaria (20%), contact urticaria (15%), conjunctivitis (13%), angioedema (4%), and anaphylaxis (4%). Fifty-four percent had two symptoms and 15% had three or more latex-related symptoms. Rhinitis (68%), asthma (53%), urticaria/angioedema (53%), dermatitis (42%), and conjunctivitis (21%) were noted in those having multiple latex-associated symptoms. Leukocyte Histamine Release Test Leukocyte histamine release test results (Fig 1) showed that the Stallergene latex extract produced the highest amount of histamine release (mean 62 ng/mL histamine) when evaluated by the AUC, followed by histamine release from latex glove extract (mean 50 ng/mL). The non-ammoniated latex produced a mean histamine release of 44 ng/mL. The lowest histamine release values were obtained from the Bencard extract (mean 24 ng/mL) and the ALK skin test extract (mean 22 ng/mL). The mean AUC of anti-IgEinduced histamine release was 46 ng/ mL. These results correlated well with the frequency distribution of leukocyte histamine release positive test results among the various latex extracts (Table 1). Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis Separation of latex proteins by sodium dodecylsulfate polyacrylamide gel electrophoresis (Fig 2) revealed a broad molecular weight range for latex proteins (14 to 120 kD). The predominant pro-
teins in both the ALK extract and the latex glove extracts were of molecular weights 14 kD, 30 to 35 kD, and 51 kD. Sodium dodecylsulfate polyacrylamide gel electrophoresis of the Stallergene extract revealed highly stained bands at 30 to 35 kD and 42 kD. Similarly, a wide range of proteins were shown in the non-ammoniated latex extract (6 to 120 kD), the predominant proteins being of 30 kD, 50 kD, and 56 kD (Fig 2). No protein bands were detected in the Bencard extract. Frequency Distribution of Test Results Twenty-six of the latex-sensitized hospital employees (76%) demonstrated leukocyte histamine release to one or more latex extracts (Table 1). Thirteen latex-sensitized individuals (38%) elicited positive specific IgE test results (Pharmacia CAP); 18 (53%) were positive by Hytec RAST test; 25 (78%) in IgE immunoblotting. IgE binding was not observed in eight non-atopic controls who had been immunoblotted; similarly, IgE binding was not observed when using myeloma-IgE (400 IU/mL) as control. Although no stained bands were observed using the Bencard latex extract by sodium dodecylsulfate polyacrylamide gel electrophoresis, the same extract induced a histamine release response in eight patients (31%) with a mean AUC of 25 ng/mL histamine (Fig 2 and Table 1). Immunoblotting with Bencard latex extract was not performed due to the absence of proteins in sodium dodecylsulfate polyacrylamide gel electrophoresis.
Table 1. Frequency Distribution of Latex-Positive Test Results in 200 Health Care Workers (N ⫽ 34/200; 0.17)
Total number of test positives Latex allergen Stallergene Greer Latex glove Bencard ALK
Histamine Release N (%)
Pharmacia CAP N (%)
Hycor RAST N (%)
26 (76%)
13 (38%)
18 (53%)
20 (77%) 13 (50%) 11 (42%) 8 (31%) 5 (19%)
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IgE-Immunoblotting Analysis The most frequent IgE-binding components of the various latex extracts are summarized in Table 2. It is observed that the Stallergene extract, the glove extract and the ALK skin prick test extract revealed the highest frequency of IgE binding (16, 31, and 9 reactions, respectively). IgE immunoblotting with sera from 34 latex-sensitized individuals demonstrated the highest incidence of reactions with IgE-binding proteins of 30 to 35 kD (34 IgE-binding reactions, when tested with all latex extracts). Additionally, 14 and 42 kD proteins showed frequent IgE binding (five IgE-binding reactions, when tested with three extracts). The results of seven patients considered latex-sensitized by the above defined criteria did not reveal binding to latex proteins in IgE-immunoblotting. Five of these seven patients demonstrated leukocyte histamine release, and three had measurable levels of specific levels of IgE both by Pharmacia CAP and Hycor RAST. One patient tested positive for all tests, while four patients tested positive only in the histamine release test. Latex Glove Extract IgE-immunoblotting with the glove extract showed that the 30 –35 kD protein bound IgE in 25 subjects (74%). In addition, two subjects showed binding to the 14 kD, 42 kD, and 51 kD proteins, thus revealing an epitope repertoire in the lower molecular range (14 to 51 kD). Stallergene Latex Extract Immunoblotting with Stallergene latex extract revealed a broad spectrum of IgE-binding proteins (14 kD, 21 kD, 30 to 35 kD, 42 kD, 48 kD, and 56 to 60 kD); only one to four subjects reacting to each protein. The epitope repertoire for this extract was demonstrated to be in the lower molecular range (14 to 60 kD). Non-Ammoniated Latex Extract IgE-binding latex proteins in the Greer latex extract were found in the higher molecular range (39 to 72 kD). IgE
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Figure 1. Histamine release in ng/mL evaluated by the AUC for Bencard latex extract, Stallergene latex extract, latex glove extract, Geer latex extract and ALK skin prick test extract (ALK).
Figure 2. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) results for Stallergene latex extract (SG), latex glove extract (LG), non-ammoniated latex extract, Greer (NAL), latex sap (SAP), ALK latex skin prick test solution and Bencard latex extract.
binding was exhibited in eleven participants, eight of whom reacted to the 56 to 60-kD protein. Latex Skin Prick Test Extract In contrast to the above described extracts, the ALK latex extract demonstrated an epitope repertoire in the higher molecular range (30 to 64 kD). Proteins with IgE-binding properties were 30 to 35 kD, 51 kD, 56 to 60 kD, and 64 kD. As determined by immunoblotting, four subjects were sensitized to the 30 to 35 kD protein, three to the 51-kD protein, and one each to the two proteins of the highest molecular weights (56 to 60 kD and 64 kD).
Bencard Latex Extract IgE immunoblotting with the Bencard latex extract was not performed as sodium dodecylsulfate polyacrylamide gel electrophoresis showed the lack of sufficient quantities of proteins. DISCUSSION Development of satisfactory methods for diagnosing latex allergy requires both characterization of the immunoreactive proteins in the latex products and the identification of specific IgE antibodies in sensitized individuals. This study demonstrates significant differences in protein profiles among
Table 2. Frequency of Patient Sera Reacting with IgE-Binding Components in the Various Latex Extract Preparations kD
14
21
30–35
42
48
Stallergene Greer Glove ALK
3
4
2 3 25 4
3
1
2
2
51
56–60
64
3 8 2
3
1
1
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various latex extracts as well as significant individual differences in sensitization patterns. It is well documented that latex content varies considerably among latex products as a result of dissimilar manufacturing procedures.12–14 The high individual variance in patient reactivity may perhaps be explained by exposure to different latex products such as different brands of manufactured latex gloves. Latex extracts were characterized by a large number of proteins; however, when evaluated by the frequency of reaction, the majority of these proteins were minor allergens and of less importance. Several latex proteins have been identified and suggested as major allergens; eg, rubber elongation factor, which exists in a tetrameric form in latex.1– 8 In this study, however, only the 30 to 35 kD (Hev b 2?) protein affected more than 50% of the population and then only when tested by the Stallergene extract. The high number of immunoblot reactions in the 30 to 35 molecular weight range (74%) may have been nonspecific reactions, but none of the eight non-atopic controls, as well as the remaining 26% of the study population, demonstrated IgE binding to this protein. Amino acid composition and sequence of the IgE binding proteins identified in this study were not performed; therefore, the IUIS nomenclature cannot be applied with certainty. It is possible, however, that the 14 kD, 30 to 35, 42, 48, and 56 to 60 kD protein are Hev b 1, Hev b 6.03 (14.6 and 14.0 kD), Hev b 2 (35.1 kD), Hev b 7 (42.9 kD), Hev b 4 (48, 50 to 57 kD) latex allergens, respectively. When analyzing the sensitization to latex by histamine release from leukocytes, it was observed that eight individuals (24%) showed positive histamine release without detection of specific IgE antibodies either by Pharmacia CAP or by Hycor RAST. In six of these participants, immunoblotting revealed IgE binding, indicating lack of analytical sensitivity by these specific IgE tests. One explanation is that the latex extract used by Pharmacia
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CAP and Hycor RAST may be entirely different from the test extracts obtained for this study. The discrepancy, probably, reflects variability due to extract composition rather than analytical method. In three subjects with a negative histamine release response, the presence of IgE antibodies was verified by both specific IgE tests, Pharmacia CAP, and Hycor RAST. It is hypothesized that these antibodies were incapable of binding the protein sufficiently in order to activate basophils. Seven patients who were diagnosed as latexsensitized by clinical history and specific-IgE testing did not exhibit binding of specific IgE in immunoblotting. A possible explanation may be that biologically important immunoreactive proteins are denatured or degraded by the sodium dodecylsulfate polyacrylamide gel electrophoresis, resulting in loss of IgE-binding capacity. An alternative hypothesis is that some allergens may consist of immunoreactive glycoproteins, causing IgE binding which cannot be detected in either immunoblotting or by specific IgE tests. The Bencard latex extract produced eight positive histamine release responses, but demonstrated no protein bands in sodium dodecylsulfate polyacrylamide gel electrophoresis. Possibly, the amounts of protein were too low or the extract consisted of mainly glycoproteins in which the protein components were not detectable in immunoblotting. Alternatively, the protein fraction could have been degraded by proteolytic enzymes, although two different batch numbers were tested with identical results. In conclusion, health care workers exposed to latex exhibited a high frequency of latex sensitization, possibly due to a high degree of exposure. The results of this investigation indicate that the development of latex sensitization may involve different allergenspecific epitopes, illustrated by the different sensitization patterns among participants. Leukocyte histamine release test allows testing with a variety of different latex sources, an advantage
when elucidating possible differences in extracts. None of the latex extracts tested has currently been able to identify all sensitized subjects due perhaps to a large variety in epitopes as well as different analytical sensitivity by various methods used to demonstrate IgE-sensitization. Before undertaking diagnostic studies of various allergy tests it would be helpful to delineate the optimal allergen profile of latex extracts for clinical testing. Until then a combination of allergy tests may therefore be necessary to determine an individual’s susceptibility to latex. ACKNOWLEDGEMENTS Dr R Esch from Greer Laboratories is thanked for providing the non-ammoniated latex preparation and Dr L Jacobsen and T Wikborg from ALKAbello in Denmark for providing the latex material from ALK. It must be emphasized that latex extracts from Greer and ALK-Abello have been reformulated after this study was finished. Thus, the extract characteristics and performance data of this study may not reflect the currently available extracts from the above manufacturers. REFERENCES 1. Czuppon AB, Chen Z, Rennert S, et al. The rubber elongation factor of rubber trees (Hevea Brasiliensis) is the major allergen in latex. J Allergy Clin Immunol 1993;92:690 – 697. 2. Beezhold D, Sussman G. Characterization and identification of latex aller gens [abstract]. J Allergy Clin Immunol 1994;93:182. 3. Alenius H, Palosuo T, Kelly K, et al. IgE reactivity to 14 kD and 27 kD natural rubber proteins in latex-allergic children with spina bifida and other congenital anomalies. Int Arch Allergy Immunol 1993;102:61– 66. 4. Alenius H, Kalkkinen N, Lukka M, et al. Prohevein from the rubber tree (Hevea Brasiliensis) is a major latex allergen. Clin Exp Allergy 1995;24: 659 – 665. 5. Alenius H, Kalkkinen N, Yip E, et al. Significance of rubber elongation factor as a latex allergen. Int Arch Allergy Immunol 1996;109:362–368. 6. Beezhold DH, Sussman GL, Kostyal
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sensitivity to natural rubber latex: characterization of cross-reacting antibodies and allergens. J Allergy Clin Immunol 1994;93:990 –996. Akasawa A, Hsieh L, Lin Y. Serum reactivities to latex proteins (Hevea brasiliensis). J Allergy Clin Immunol 1995;95:1196 –1205. Slater JE, Chhabra SK. Latex allergens. J Allergy Clin Immunol 1992;89: 673– 678. Fink JN, Kelly KJ, Elms N, et al. Comparative studies of latex extracts used in skin testing. Ann Allergy 1996;76: 149 –152. Akasawa A, Hsieh LS, Lin Y. Comparison of latex specific IgE binding among non ammoniated latex, ammoniated latex and latex glove allergenic
extracts by ELISA and immunoblot inhibition. J Allergy Clin Immunol 1996; 97(5):1116 –1120. 15. Nolte H. The clinical utility of basophil histamine release. Allergy Proceedings 1993;14:251–254. 16. Stahl Skov P, Mosbech H, Norn S, et al. Sensitive glass microfibre-based histamine analysis for allergy testing in washed blood cells. Allergy 1985; 40:213–217. Request for reprints should be addressed to: Hendrik Nolte, MD, PhD Allergy Consult, Llc 152 Simsbury Road Avon, CT 06001 E-mail: [email protected]
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