Multiple 43 kDa allergenic proteins in natural rubber latex

$332 Abstracts

J ALLERGYCLINIMMUNOL JANUARY 2002

1 N~"I Antigen Content but Not Total Protein Correlates With Allerll,l~--g genicity of "Protein-Poor" Latex Gloves C Audo, J Barbara, H Chabane, M Armange, D Lev3, F Leynadier Hospital Tenon, Paris, France INTRODUCTION: Measurement of latex glove allergenicity by RAST-inhibition (RI) has important limitations, Alternatively, allergenicity is estimated as total extractible proteins (TP) using a modified Lowry method (MLA). However, allergen (A1) content correlates poorly with low TP levels. Previously, we found a correlation between antigenic protein (Ag) in extracts from "protein-poor" gloves measured by competitive immunoassay using rabbit antiserum (CIALP) and AI measured by RI but no correlation with TP determined by MLA (Invest Allergol Clin Immunol 1999;9:372). Here, we extend these findings. MATERIALS AND METHODS: Extracts from 62 gloves were assayed for TP by MLA, AI by RI and Ag by CIALP. RESULTS: TP content of the gloves ranged from 30-1250 ~g/g of glove (gog), Ag from 0.1-100 lag/gog and A1 from 0.7-700 units (IR)/gog. Correlations between the measurements are shown in the table. Those on the present study were excellent. When combined with previous results (All gloves), the correlations were also excellent. Results obtained with gloves having > 100 lag TP/gog were also highly correlated. In contrast, neither A I nor Ag content of gloves having < 100 lag TP/gog correlated with TP but there was reasonably good correlation between AI and Ag content of these relatively "protein-poor" gloves: of the 41 extracts with < 100 lag TP/gog, the TP was below the detection limit in 18 and 17 of them had detectable AI and Ag. DISCUSSION: These results confirm previous reports--and the opinion of many--showing that the MLA does not give a valid estimation of latex glove allergenicity, particularly for less allergenic gloves, such as those now being manufactured. Estimation of glove A1 by RI is limited by its dependence on human serum. Assays for latex Ag (LEAP and CIALP) depending on anti-latex antisera produced in animals have been developed as a substitute. CIALP is a competitive immunoassay, with good control of Ag fixation to assay microtiter plates, and it yields results that correlate well with A1 content determined by RI. CONCLUSION: MLA is a poor measure of glove allergenicity. Results obtained with CIALP correlate well with glove allergenicity and not being dependent on human serum could be used more widely to estimate glove allergenicity.

Present study (n=62) All gloves (n=104) TP>100ug/gog(n=63) TP < 100ug/gog (n--41)

l

TP vs AI

TP vs Ag

Ag vs AI

r=0.81,p<0.001 r=-0.77, p < 0.001 r=-0.48,p<0.001 r= -0.08, NS

r=0.79, p<0.001 r=0.75, p < 0.001 r=0.50, p<0.001 r=- -0.29, NS

r=0.87, p<0.001 r=0.89, p < 0.001 r=0.83, p<0.001 r=0.43, p < 0.001

a?.

Concentrations of Natural Rubber Latex (NRL)Allergens Hey IJWLIMI bl, 3, 5 and 602 in NRL Gloves Capable of Causing Positive Skin Prick Test (SPT) Reactions in NRL-Allergic Subjects

Timo Palosuo*, Kristiina Turjanmaa§, Vladimir Ovod~, Tytti Kiirkkiiinen~, Katja FriskY, Harri Tapio Alenius~, Timo Reunala§, Markku Kulomaa% Nisse Kalkkinen@, Hely Reinikka-Railo+ *National Public Health Institute, Helsinki, Finland §Tampere University Hospital, Tampere, Finland ~Pb'ITBioTechnology Oyj Plc, Tampere, Finland ~Finnish Institute of Occupational Health, Helsinki, Finland <
ous nationwide medical glove survey carried out in 1994-95. Specific allergen levels were measured in stored extracts of gloves ( 1:5 w/vol) by a novel capture ELISA using monoclonal and polyclonal antibodies and respective purified recombinant or native allergens. The sensitivity of the assay was 2 ng/ml to Hev b5 and Hev b 6.02 and 10 ng/ml to Hev bl and Hev b3. Levels of these 4 allergens were compared both to SPT in the 20 patients and the total allergen levels (allergen units: AU/ml) of the gloves, measured by a human lgE-ELISA inhibition assay. Hevein (Hev b 6.02) was measurable in 17/22 gloves (2 to 22258 ng/ml), Hey b 5 in 17 (2 to 2734 ng/ml), Hev b3 in 10 (10 to 6010 ng/ml) and Hey bl in 3 glove extracts (10 to 327 ng/ml). Correlation between Hev b 6.02 and Hev b5 concentration and SPT reactivity pattern, respectively, in the 20 patients was highly significant. Three of the 22 gloves did not contain measurable amounts of any of these 4 allergens. Hev b 6.02 was found as the only allergen in 3 and Hev b5 alone in 2 gloves, whereas Hev b3 and Hey b 1 were always accompanied by 1-3 other allergens. Similar pattern of distribution has been observed in our further studies in 1999-2001 although the total number of high-allergen gloves has considerably decreased and the presence of Hev bl has turned out to be very rare. Seven of 12 "low-allergen" gloves (<10 AU/ml) contained low amounts of I-2 of the 4 allergens and some patients (median 5) reacted against them in SPT. Yet. due to the common presence of several allergens at the same time in gloves safety levels for each individual allergen can only be assessed using purified allergens as reagents. On the other hand, it could be seen that a majority of the NRL-allergic patients showed positive SPT reactions to gloves when the concentration of Hev b I or Hev b3 reached or exceeded about 50-60 ng/ml, Hev b5 about 40-50, and Hev b 6.02 about 200 ng-/ml. In conclusion, the novel capture ELISA provides an easy tool for accurately quantifying NRL allergens in various materials and by means of SPT in a representative patient material it will be possible to set up recommendations for acceptable threshold levels.

1 029

Multiple 43 kDa Allergenic Proteins in Natural Rubber Latex

Siti Arija M Arif*, Robert G Hamilton§, Faridah Yusof*, Subodh Nimkar~,

Jaap J Beintema~, Hoong-Yeet Yeang* *Rubber Research Institute of Malaysia. Kuala Lumpur, Malaysia §Johns Hopkins University School of Medicine. Baltimore, MA YPE Biosystems, Singapore, Singapore ~University of Groningen, Groningen, Netherlands In early latex allergy research, IgE-binding proteins of about 42 to 46 kDa had frequently been observed on Western-immunoblots following polyacrylamide gel electrophoresis of natural rubber latex extracts. From this, it was inferred that a major allergenic protein of that size was present in the latex. In time, a latex allergen of about 46 kDa, designated Hev b 7, was identified that had partial protein homology to patatin, the major storage protein of potatoes. When the Hev b 7 cDNA was successfully cloned, the recombinant protein was found to be reactive with IgE from latex allergic patients. However, the proportion of latex-allergic patient sera that recognized recombinant Hey b 7 was much lower than that which reacted with the 42-46 kDa protein(s) in natural rubber latex. Native Hev b 7 (Hev b 7.01), a protein of 43.70 kDa that was isolated from latex, gave results similar to that of recombinant Hev b 7. Hence, Hev b 7 alone could not account for the frequently encountered sensitivity to the 42 to 46 kDa latex protein(s). Recently, two other allergenic latex proteins of about 43 kDa were isolated. Unlike Hev b 7.01, which is a cytosolic protein found in the latex C-serum, these two proteins are organelle peptides isolated from the latex B-serum. One of these, Hev b 7.02W. is a homologue of Hev b 7, sharing at least some of its amino acid sequences but having epitope sites dissimilar from those of Hev b 7. IgE binding to Hev b 7.02W is inhibited by patatin, whereas Hev b 7.01 is not similarly inhibited. The second B-serum protein, provisionally referred to as Hev b 7b, has a molecular weight of 42.97 kDa by mass spectrometry. The protein is N-terminal blocked, but partial amino acid sequences were obtained from its peptide fragments after trypsin digestion. A database search did not reveal a sequence match to any known Hevea protein, According to these amino acid sequences, Hev b 7b is unrelated to Hev b 7.01. Hev b 7.02W or patatin. Nevertheless, inhibition experiments with patatin suggested that Hev b 7b shared the patatin IgE epitope.

J ALLERGY CLIN IMMUNOL VOLUME 109, NUMBER 1

In serologic experiments, both Hev b 7.02W and Hev b 7b were recognized by IgE in serum from over two-thirds of latex allergic patients. In addition, skin prick tests carried out with Hey b 7b revealed that a similar proportion of latex-allergic patients were sensitized to this protein. Unless appropriate precautions are taken during protein isolation and purification from fresh latex, mixtures of these 43 kDa proteins are likely to be obtained. The presence of multiple allergens of closely similar size may create confusion in diagnostic tests to determine which latex allergens patients are sensitized to.

f~f,H'~ Identification of IgE-Binding Amino Acids on the Conforma11,/11,/11,/tional Epitopesof Hevein (Hev b 602) Piia Karisola*, Jari Mikkola§, Timo Reunala¥, Kristiina Tutjanmaa~, Nisse Kalkkinen~, Timo Palosuo¢~, Markku Kulomaa*, Harri Tapio Alenius§ *University of Jyvaskyl~i, Jyv~iskyl~i, Finland §Finnish Institute of Occupational Health, Helsinki, Finland ¥Tampere University Hospital, Tampere, Finland ~University of Helsinki, Helsinki, Finland <~National Public Health Institute, Helsinki, Finland Hevein (Hev b 6.02), a major small molecular weight (4.7 kDa) allergen in natural rubber latex (NRL) is recognized by IgE of approximately 70 % of latex allergic patients. Hevein is an advantageous molecule for studies of conformational B cell epitopes, since its 3D structure has been solved, and it can also be considered as a good candidate for studies aimed at allergen specific immunotherapy. We have previously localized conformational IgE-binding areas of hevein to its N- and C- termini by transferring terminal or core regions of hevein to a nonallergenic, structurally homologous adaptor protein (AMP). In order to confirm the results from our AMP-study and attest the importance of structural epitopes, we produced chimeras containing AMP N- and C-termini within hevein core. Thereafter, we designed and constructed site-specific mutants by QuickChange or megaprimer method to detect and locate lgE-binding epitopes of herein at amino acid level to those potential antigenic sites. The recombinant proteins were produced in insect cells (Sf9) and weights were determined by mass spectroscopy. A chicken biotinbinding protein, avidin, was fused to the N-terminus of the hevein protein to facilitate effective isolation of the recombinant protein. An ELISA-inhibition method and skin prick test (SPT) provided the linkage from structure modification to immunological responses. Introduction of the N- and C-terminal parts of AMP to hevein resulted in a remarkable decrease (> 50 %) in the IgE-binding capacity. The terminal regions were scanned with single point mutations that were chosen site-specifically from the naturally occurring hevein-like domains (by "evolutionary approach"). Most of the substitutions caused a decrease in IgE-binding activities at least with some patients. A multifold mutated molecule that shows minimal or no IgE binding but retains the T cell epitopes was constructed by combining the most effective mutations. In conclusion, we have been able to transfer structurally similar, nonallergenic parts of AMP to the IgE-epitope regions of the originally highly allergenic hevein protein. The B cell epitopes have been confirmed to localize in the terminal parts of the hevein. They seem to be assisted by amino acid residues that are distant in protein primary structure but proximal in the 3D-fold of tile allergen. Homologues to the naturally occurring allergen can be used as a hyposensitizing agent to reduce or diminish the strength of allergic reaction. Selected mutants will be used for tests of immunotherapy of NRL allergy.

Abstracts

$333

1031 cosidaseHeV b 4 Heavy Peptide Identified as Latex Cyanogenic GluE Sunderasan*, Keng-See Chow*, Malcolm A Ward§, Hoong-Yeet Yeang* *Rubber Research Institute of Malaysia, Kuala Lumpur, Malaysia §GlaxoSmithKline, Stevenage, UK The latex protein Hev b 4 comprises a triplet of allergenic peptides of molecular weight 50 to 57 kDa. The three peptides share a common characteristic in remaining soluble only in the presence of high ionic concentration (e.g. 0.2M NaCI and higher). Dilution or dialysis induces precipitation of all three peptides that might have related biochemical functions in the latex, and that might be components of a protein complex. Hev b 4 has been shown to be allergenic by IgE-binding assays and in skin prick tests. The heaviest component of the Hev b 4 triplet, a 57 kDa peptide, was isolated from the latex B-serum by dialysis-induced protein precipitation and Sephacryl gel filtration. Further purification was carried out by preparative SDS-polyacrylamide gel electrophoresis. Following trypsin digestion, the 57 kDa peptide was scanned using a nano-electrospray tandem mass spectrometry (ESI-MS) and amino acid sequencing was carried out by MS/MS. Partial amino acid sequences of the protein matched published sequences of several plant glucosidases, including those of cyanogenic glucosidases. Enzymatic assays confirmed that the protein possessed glucosidase activity, the enzyme activity being further demonstrated by isozyme staining of the protein after electrophoretic separation on native polyacrylamide gels. Enzymic activity was also observed when linamarin was used as the specific enzymic substrate, indicating that the protein was a cyanogenic glucosidase (linamarase) as suggested by the partial amino acid sequences. A partial cDNA sequence with homology to plant glucosidases was obtained by reverse transcription-PCR using sense and antisense strand degenerate primers. The 5"/3"RACE is being employed to generate the complete cDNA of H. brasiliensis latex glucosidase.

~'la'~a) Prevalence of Arabic and Karaya Gum Allergy in tatex-Allerl VeJP L- gic Patients Dean T Chiang*, Kenneth T Kim§ *UC Irvine, Orange, CA §Allergy, Asthma, and Respiratory Care Center, Incorporated, Long Beach, CA There have been suggestions that karaya and arabic gums, commonly used in food, pharmaceutical, and textile industries, may have cross reacting epitopes with natural latex rubber. To address this issue, we analyzed serum samples of 116 patients with latex allergy and a control group of 95 in whom latex allergy was excluded to assess the prevalence of specific IgE antibodies to arabic and karaya gums. Latex allergy was defined by positive history of IgE mediated reactions to contact with latex and positive skin prick test to latex extract and/or positive in vitro test (AlaSTAT and/or Pharmacia CAP). Specific IgE to karaya and arabic gums was detected by using the Pharmacia CAP assays for these two gums. We detected IgE antibodies to arabic gum in 12 (10%) patients in the latex allergic group and 6 (6.3%) patients in the non-latex allergic group (p=.417). lgE antibodies to karaya gum was found in 17 (15%) patients in the latex allergic group and 14 (15 %) in the non-latex allergic group (p=.417). IgE antibodies to both gums were found in 11 (9.5%) patients in the latex allergic group and 6 (6.3%) patients in the non-latex allergic group (p=.566), Our study reveals that there is no statistically significant difference in the prevalence of specific lgE antibodies to arabic gum and karaya gum in latex allergic patients and non-latex allergic patients.

Latex Allergic (116) Non-Latex Allergic (95) p-value

Arabic

Karaya

Both

12 (10%) 6 (6.3%) .417

17 (15%) 14 (15%) .417

11 (9.5%) 6 (6.3%) .566