Quantitative analysis of immunoglobulin E reactivity profiles in patients allergic or sensitized to natural rubber latex (Hevea brasiliensis).

BACKGROUND: Characterized native and recombinant Hevea brasiliensis (rHev b) natural rubber latex (NRL) allergens are available to assess patient allergen sensitization profiles. OBJECTIVE: Quantification of individual IgE responses to the spectrum of documented NRL allergens and evaluation of cross-reactive carbohydrate determinants (CCDs) for more definitive diagnosis. METHODS: Sera of 104 healthcare workers (HCW; 51 German, 21 Portuguese, 32 American), 31 spina bifida patients (SB; 11 German, 20 Portuguese) and 10 Portuguese with multiple surgeries (MS) were analysed for allergen-specific IgE antibody (sIgE) to NRL, single Hev b allergens and CCDs with ImmunoCAP technology. RESULTS: In all patient groups rHev b 5-sIgE concentrations were the most pronounced. Hev b 2, 5, 6.01 and 13 were identified as the major allergens in HCW and combined with Hev b 1 and Hev b 3 in SB. In MS Hev b 1 displayed an intermediate relevance. Different sIgE antibody levels to native Hevea brasiliensis (nHev b) 2 and rHev b 6.01 allowed discrimination of SB with clinical relevant latex allergy vs. those with latex sensitization. Sensitization profiles of German, Portuguese and American patients were equivalent. rHev b 5, 6.01 and nHev b 13 combined detected 100% of the latex-allergic HCW and 80.1% of the SB. Only 8.3% of the sera showed sIgE response to CCDs. CONCLUSIONS: Hev b 1, 2, 5, 6.01 and 13 were identified as the major Hev b allergens and they should be present in standardized latex extracts and in vitro allergosorbents. CCDs are only of minor relevance in patients with clinical relevant latex allergy. Component-resolved diagnostic analyses for latex allergy set the stage for an allergen-directed immunotherapy strategy.

Percutaneous reactivity to natural rubber latex proteins persists in health-care workers following avoidance of natural rubber latex.

BACKGROUND: Long-term avoidance of natural rubber latex [Hevea brasiliensis (Hev b)] is currently recommended for health-care workers (HCWs) with established natural rubber latex (NRL) allergy. Percutaneous sensitivity to eight Hev b NRL allergens was evaluated in HCWs in 2000. To date, no studies have evaluated the longitudinal effects of NRL avoidance on percutaneous sensitivity to NRL allergens. OBJECTIVE: The aims of this study were to evaluate changes in percutaneous reactivity to non-ammoniated latex (NAL) and NRL allergens in HCWs 5 years after a recommendation to avoid NRL and to evaluate factors that predict the persistence of in vivo sensitivity to NAL and NRL allergens. METHODS: Skin prick testing was performed with NAL, seven NRL allergens (Hev b 1, 2, 3, 4, 6.01, 7.01, and 13), and recombinant Hev b 5 (rHev b 5) in 34 HCWs who were initially evaluated in 2000 for occupationally related NRL allergy. Serial 10-fold dilutions of NAL and NRL allergens were employed in skin testing. Sera from the HCWs were assayed for latex and enhanced latex (rHev b 5-enriched allergosorbent)-specific IgE antibodies using the ImmunoCAP assay. RESULTS: The prevalence of work-related symptoms significantly decreased between 2000 and 2005 with avoidance of NRL (P<0.05). A >/=100-fold reduction in percutaneous sensitivity to Hev b 2 and Hev b 7 was less likely in those with prior history of systemic reactions to NRL (P=0.0053), reported history of reaction to cross-reactive foods (P=0.014), continued local reactions to NRL gloves (P<0.0001), or high NRL glove exposure since the initial study (P=0.0075). The diagnostic sensitivity and specificity of the latex-specific IgE serology was 54% and 87.5%, respectively, in comparison with NAL skin tests. The addition of rHev b 5 to the ImmunoCAP (enhanced latex) allergosorbent altered the diagnostic sensitivity and specificity of the ImmunoCAP to 77% and 75%, respectively. CONCLUSION: While symptoms may resolve quickly with NRL avoidance therapy, detectable IgE indicating continued sensitization remains beyond 5 years, and thus continued avoidance of NRL should be recommended.

Allergen concentration in natural rubber latex.

BACKGROUND: Hevea brasiliensis latex serum is commonly used as the in vivo and in vitro reference antigen for latex allergy diagnosis as it contains the full complement of latex allergens. OBJECTIVE: This study quantifies the concentrations of the significant allergens in latex serum and examines its suitability as an antigen source in latex allergy diagnosis and immunotherapy. METHODS: The serum phase was extracted from centrifuged latex that was repeatedly freeze-thawed or glycerinated. Quantitation of latex allergens was performed by two-site immunoenzymetric assays. The abundance of RNA transcripts of the latex allergens was estimated from the number of their clones in an Expressed Sequence Tags library. RESULTS: The latex allergens, Hev b 1, 2, 3, 4, 5, 6, 7 and 13, were detected in freeze-thawed and glycerinated latex serum at levels ranging from 75 (Hev b 6) to 0.06 nmol/mg total proteins (Hev b 4). Hev b 6 content in the latex was up to a thousand times higher than the other seven latex allergens, depending on source and/or preparation procedure. Allergen concentration was reflected in the abundance of mRNA transcripts. When used as the antigen, latex serum may bias the outcome of latex allergy diagnostic tests towards sensitization to Hev b 6. Tests that make use of latex serum may fail to detect latex-specific IgE reactivity in subjects who are sensitized only to allergens that are present at low concentrations. CONCLUSION: Latex allergy diagnostics and immunotherapy that use whole latex serum as the antigen source may not be optimal because of the marked imbalance of its constituent allergens.

Molecular cloning and immunoglobulin E reactivity of a natural rubber latex lecithinase homologue, the major allergenic component of Hev b 4.

BACKGROUND: Hev b 4 is an allergenic natural rubber latex (NRL) protein complex that is reactive in skin prick tests and in vitro immunoassays. On SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Hev b 4 is discerned predominantly at 53-55 kDa together with a 57 kDa minor component previously identified as a cyanogenic glucosidase. Of the 13 NRL allergens recognized by the International Union of Immunological Societies, the 53-55 kDa Hev b 4 major protein is the only candidate that lacks complete cDNA and protein sequence information. OBJECTIVE: We sought to clone the transcript encoding the Hev b 4 major protein, and characterize the native protein and its recombinant form in relation to IgE binding. METHODS: The 5'/3' rapid amplification of cDNA ends method was employed to obtain the complete cDNA of the Hev b 4 major protein. A recombinant form of the protein was over-expressed in Escherichia coli. The native Hev b 4 major protein was deglycosylated by trifluoromethane sulphonic acid. Western immunoblots of the native, deglycosylated and recombinant proteins were performed using both polyclonal antibodies and sera from latex-allergic patients. RESULTS: The cDNA encoding the Hev b 4 major protein was cloned. Its open reading frame matched lecithinases in the conserved domain database and contained 10 predicted glycosylation sites. Detection of glycans on the Hev b 4 lecithinase homologue confirmed it to be a glycoprotein. The deglycosylated lecithinase homologue was discerned at 40 kDa on SDS-PAGE, this being comparable to the 38.53 kDa mass predicted by its cDNA. Deglycosylation of the lecithinase homologue resulted in the loss of IgE recognition, although reactivity to polyclonal rabbit anti-Hev b 4 was retained. IgE from latex-allergic patients also failed to recognize the non-glycosylated E. coli recombinant lecithinase homologue. CONCLUSION: The IgE epitopes of the Hev b 4 lecithinase homologue reside mainly in its carbohydrate moiety, which also account for the discrepancy between the observed molecular weight of the protein and the value calculated from its cDNA.