S.A.M. Arif

Allergenic proteins of natural rubber latex

As the living cytoplasm of laticiferous cells, Hevea brasiliensis latex is a rich blend of organic substances that include a mélange of proteins. A small number of these proteins have given rise to the problem of latex allergy. The salient characteristics of H. brasiliensis latex allergens that are recognized by the International Union of Immunological Societies (IUIS) are reviewed. These are the proteins associated with the rubber particles, the cytosolic C-serum proteins and the B-serum proteins that originate mainly from the lutoids. Procedures for the isolation and purification of latex allergens are discussed, from latex collection in the field to various preparative approaches adopted in the laboratory. As interest in recombinant latex allergens increases, there is a need to validate recombinant proteins to ascertain equivalence with their native counterparts when used in immunological studies, diagnostics, and immunotherapy.

Specific IgE response to purified and recombinant allergens in latex allergy

BackgroundIn recent years, allergy to natural rubber latex has emerged as a major allergy among certain occupational groups and patients with underlying diseases. The sensitization and development of latex allergy has been attributed to exposure to products containing residual latex proteins. Although improved manufacturing procedures resulted in a considerable reduction of new cases, the potential risk for some patient groups is still great. In addition the prevalent cross-reactivity of latex proteins with other food allergens poses a major concern. A number of purified allergens and a few commercial kits are currently available, but no concerted effort was undertaken to evaluate them.MethodsWe studied 11 purified latex allergens, Hev b 1 to Hev b 10, and Hev b 13 along with several crude allergen extracts and two commercial ImmunoCAP assays to evaluate specific IgE antibody in the sera from latex allergic patients and controls. Health care workers and spina bifida patients with clinical symptoms of latex allergy, spina bifida patients without latex allergy, and non-atopic health care workers have been studied.ResultsThe results suggest that Hev b 2, 5, 6, and 13 together identified over 80 percent health care workers with latex allergy, while Hev b 6 along with Hev b 1 or 3 detected specific IgE antibody in all sera studied from patients with spina bifida and latex allergy. The ImmunoCAP results using both Hev b 5 amplified and non-amplified closely agreed with the clinical diagnosis of latex allergy in health care workers and in spina bifida.ConclusionAlthough the purified allergens and crude extracts reacted diversely with IgE from different patient groups, the results indicated that use of certain combinations of purified recombinant antigens will be useful in commercial kits or in in-house assays for detecting specific IgE antibody in the sera. The results suggest that a combination of Hev b 2, 3, 5, 6, and 13 together detected specific IgE in 80% of the sera from latex allergic patients. Both ImmunoCAPs correctly identified over 95% of latex allergic patients, however, showed reactivity with a few normal control subjects

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.

Purified and recombinant latex proteins stimulate peripheral blood lymphocytes of latex allergic patients.

Background: Natural rubber latex proteins have been implicated in severe allergy in individuals exposed to latex products, particularly health care workers. Until recently, only crude antigens were available to study the immune response in these patients. In recent years a number of relevant allergens have been purified, but few have been used in lymphocyte studies. Hence, to better understand the immunological mechanisms involved in latex allergy, we investigated the response of peripheral blood mononuclear cells (PBMCs) to various purified natural rubber latex allergens. Methods: Using conventional protein purification methods and gene cloning, we have obtained 6 natural rubber latex proteins. We studied allergen–specific IgE levels and PBMC responses to these allergens along with 3 crude latex antigen preparations. Results: Of the 28 latex–allergic health care workers studied, 16 reacted to one or more of the allergens studied, but PBMCs from controls failed to respond to these antigens. Serum IgE to the antigens was detected in 11–90% of the patients. Conclusion: Fifty–seven percent of the latex–allergic patients demonstrated PBMC responses to at least one of the latex allergens tested, but there was no direct correlation between serum IgE levels and PBMC responses. However, since none of the control subjects showed any PBMC stimulation, this may prove useful in determining sensitization to latex. Among the allergens studied, the predominant mononuclear cell responses were directed against Hev b 2, while serum IgE against rHev b 6 was demonstrable in the greatest number of patients. The crude latex allergens were toxic to PBMCs and hence, the purified allergens may be of greater value in demonstrating sensitization of patients to latex allergens.

Low prevalence of latex sensitivity in South African spina bifida children in Cape Town.

Spina bifida children have a high prevalence of latex allergy in studies reported from Europe and the USA. This study investigated the prevalence of latex allergy in a cohort of 24 spina bifida children at the Red Cross Childrens Hospital from Cape Town, South Africa. The children were investigated using a detailed questionnaire, skin prick tests (ALK‐Abello), ImmunoCap RASTs, Western blotting and ELISA, using the purified latex proteins Hev b1 and Hev b3 and whole latex preparation. A low overall prevalence of latex sensitization of 16.7% was found in the children. Children who were sensitive reacted to water insoluble to Hev b1 and Hev b3 proteins. The low prevalence of latex sensitization in the South African children may not be entirely explained by stringent latex avoidance. The children were from a low socioeconomic social status and ‘hygiene’ and other factors should be considered.

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.

Generation of allergen-enriched protein fractions of hevea brasiliensis latex for in vitro and in vivo diagnosis

Background: The latex of Hevea brasiliensis trees contains a complex proteome that includes a range of allergenic proteins. Current latex extracts that are used for the diagnosis of latex allergy still lack important allergens. We aimed to devise a production process for an improved reagent that would ideally contain the complete latex allergome. Methods: Latex C-serum was fractionated by ammonium sulfate precipitation, and B- and C-serum proteins were then separated by anion exchange chromatography. Proteins eluting within defined salt concentration ranges were pooled into six final fractions. Fractions were evaluated by two-dimensional electrophoresis and subsequent IgE immunoblot for their spectrum of allergens. The presence of the most important latex allergens in the fractions was checked by Western blot analyses. Each fraction was further evaluated by skin prick test (SPT). Results: Reproducibility of the preparation method was demonstrated with two batches of latex. Comparison of latex B- and C-serum to the six fractions showed a remarkable increase in the number of detectable allergens in the fractions. The presence of the latex allergens Hev b 1–8 and Hev b 13 in the fractions was demonstrated. In SPTs, the fractions produced wheal-and-flare reactions comparable to commercial latex extracts. Conclusions: This method provides reproducible latex protein fractions of high allergen content for the diagnosis of latex allergy.

A molecular and in silico characterization of Hev b 4, a glycosylated latex allergen.

Hev b 4 is a heavily glycosylated latex allergen with seven attached N-glycans, comprising of both oligomannose and complex type structures. Treatment with a mixture of N-glycosidase A and N-glycosidase F resulted in lowering Hev b 4 protein on SDS-gel from 53 to 55kDa to circa 40kDa, this being comparable to the 38.53kDa mass predicted by its cDNA. In Western-immunoblots, the enzymatically deglycosylated Hev b 4 showed negligible binding to IgE from latex allergic patients; the results indicated that IgE essentially binds to Hev b 4 via its N-glycan moiety. Structural modelling of the Hev b 4 was carried out based on the template protein and carbohydrate crystal coordinates of rhamnogalacturonan acetylesterase (PDB ID 1DEO). We managed to link four N-glycan structures on to the Hev b 4 model; the glycans were scattered over the surface of the model. The structural and functional features of Hev b 4 could prove useful to elucidate its exposed epitopes which are important for IgE binding.

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.

Erratum: Allergen concentration in natural rubber latex (Clinical and Experimental Allergy (2006) 36 (1078-86))

Allergen concentration in natural rubber latex H.-Y. Yeang, R. G. Hamilton, D. I. Bernstein, S. A. M. Arif, K. -S. Chow, Y. -H. Loke, M. Raulf-Heimsoth, S. Wagner, H. Breiteneder and R. E. Biagini Biotechnology and Strategic Research Unit, Rubber Research Institute of Malaysia, Malaysian Rubber Board, Malaysia, Johns Hopkins Asthma and Allergy Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA, Division of Allergy/Immunology, University of Cincinnati College of Medicine, Cincinnati, OH, USA, Allergy/Immunology Division, Institute of Occupational Medicine, Bochum, Germany, Department of Pathophysiology, Medical University of Vienna, Austria and Biomonitoring and Health Assessment Branch, National Institute for Occupational Safety and Health, Cincinnati, OH, USA