Radioallergosorbent Test Inhibition Research Articles

Penicillin and cephalosporin cross-reactivity: role of side chain and synthetic cefadroxil epitopes

BackgroundAnalysis of cross-reactivity is necessary for prescribing safe cephalosporins for penicillin allergic patients. Amoxicillin (AX) is the betalactam most often involved in immediate hypersensitivity reactions (IHRs), and cefadroxil (CX) the most likely cephalosporin to cross-react with AX, since they share the same R1 side chain, unlike cefuroxime (CO), with a structurally different R1. We aimed to analyse cross-reactivity with CX and CO in patients with confirmed IHRs to AX, including sIgE recognition to AX, CX, CO, and novel synthetic determinants of CX.MethodsFifty-four patients with confirmed IHRs to AX based on skin test (ST) and/or drug provocation test (DPT) were included. Serum sIgE to AX and benzylpenicillin was determined by Radioallergosorbent test (RAST). Two potential determinants of CX, involving intact or modified R1 structure, with open betalactam ring, were synthesised and sIgE evaluated by RAST inhibition assay.ResultsTolerance to CX (Group A) was observed in 64.8% cases and cross-reactivity in 35.2% cases (Group B). Cross-reactivity with CO was only found in 1.8% cases from Group B. ST to CX showed a negative predictive value of 94.6%. RAST inhibition assays showed higher recognition to CX as well as to both synthetic determinants (66% of positive cases) in Group B.ConclusionsCross-reactivity with CX in AX allergic patients is 35%, being ST not enough for prediction. R1, although critical for recognition, is not the unique factor. The synthetic determinants of CX, 1-(HOPhG-Ser-Bu) and 2-(pyrazinone) are promising tools for determining in vitro cross-reactivity to CX in AX allergic patients.

Molecular Allergy Diagnostics Refine Characterization of Children Sensitized to Dog Dander

To analyze patterns of immunoglobulin E (IgE) reactivity to dog allergen components in sensitized children and investigate the association between those patterns and clinical symptoms by nasal provocation tests (NPT).Children 10 to 18 years (n = 60) with known sensitization to dog identified by a skin prick test wheal >3 mm or serum-specific IgE >10 kU/L were included.NPTs were performed with a commercial dog extract by using a standardized protocol. The extract was analyzed by radioallergosorbent test inhibition and contained all 6 dog allergens (Can f 1, 2, 3, 4, 5, and 6) used in subsequent analyses. The allergens were present in a wide range of concentrations. Serum-specific IgE levels to dog extract and each of the allergenic components were measured.All but 2 children had allergic rhinitis or conjunctivitis, and 41 out of 60 (68%) reported symptoms with dog exposure. The mean serum-specific IgE level was 11.5 kU/L (interquartile range: 3.15–43.0 kU/L). Sensitization to Can f 1 was most common, followed in frequency by Can f 5, 4, 6, 2, and 3. There was a wide range of IgE levels to the components, with the highest levels to Can f 1, 2, and 5. History of dog-related rhinitis was most common among those sensitized to Can f 3 (94% vs 59% of those not sensitized, P = .012) and Can f 6 (86% vs 63%, P = .007). Similar associations were found for histories of dog-induced asthma. Most patients were sensitized to several components, with great overlap in their sensitization to the proteins belonging to different groups (lipocalins: Can f 1, 2, 4, and 6; serum albumin: Can f 3; and prostatic kallikrein: Can f 5). No single protein group was associated with history of dog-induced airway symptoms, but sensitization to more proteins increased the odds ratio (OR) of dog-induced asthma or rhinitis. Those sensitized to all proteins from all 3 groups were at higher risk of dog-induced rhinitis (OR: 8.36; 95% confidence interval: 1.0–69.5). Forty-two percent (25 out of 60) of patients had positive NPT results, 14 out of 60 (23%) had inconclusive NPTs, and 21 out of 60 (35%) had negative challenges. Those with positive NPT results were significantly more likely to report dog-induced rhinitis and asthma as compared with those with negative NPT results. After adjusting for the effects of different protein groups, only sensitization to the lipocalins (Can f 1, 2, 4, and 6) was associated with positive NPT results. Those sensitized to all 3 groups had the highest OR for a positive NPT result, but as with reported symptoms above, sensitization to increasing numbers of proteins was associated with increasing risk for a positive NPT result.Sensitization to lipocalins and to an increasing number of allergenic dog proteins is associated with dog-induced airway symptoms (dog allergy).“I am not allergic to my dog, but other dogs bother me.” My dog is “hypoallergenic.” How many times have we heard comments like these from patients? Every study that has tried to identify allergenic differences between breeds of dogs has failed to find that elusive hypoallergenic dog. In fact, there is great homology of Can f 1 (the major dog allergen) among breeds. Furthermore, all breeds have comparable amounts of airborne Can f 1 in the homes of dog owners. So, what is going on here? The answer might lie in component-resolved diagnostics (CRD). Commercially available CRDs have already helped our understanding of some food allergies by enhancing our ability to make accurate diagnoses of allergy in sensitized patients. Options for follow-up studies to this one are obvious. Do different breeds of dog produce varying amounts of individual or total lipocalins? Are some patients more highly sensitized to particular proteins or more types of proteins, placing them at differential risk of allergy to certain breeds? Or, are some less highly sensitized, making them more tolerant of different breeds? When those questions are answered and when CRDs for dog allergens are commercially available, we still might not identify a consistently less-allergenic dog. But, we might be able to identify the breeds most likely to be tolerated by a child sensitized to dog.

Wild Buckwheat Is Unlikely to Pose a Risk to Buckwheat‐Allergic Individuals

Buckwheat (Fagopyrum esculentum) is a commonly allergenic food especially in Asia where buckwheat is more commonly consumed. Wild buckwheat (Polygonum convolvulus, recently changed to Fallopia convolvulus) is an annual weed prevalent in grain-growing areas of the United States. Wild buckwheat is not closely related to edible buckwheat although the seeds do have some physical resemblance. A large shipment of wheat into Japan was halted by the discovery of the adventitious presence of wild buckwheat seeds over possible concerns for buckwheat-allergic consumers. However, IgE-binding was not observed to an extract of wild buckwheat using sera from 3 buckwheat-allergic individuals either by radio-allergosorbent test inhibition or by immunoblotting after protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthermore, the extract of wild buckwheat was not detected in a buckwheat enzyme-linked immunosorbent assay developed with antisera against common buckwheat. Thus, wild buckwheat is highly unlikely to pose any risk to buckwheat-allergic individuals. The common names of plants should not be a factor in the risk assessment for possible cross-allergenicity.

Immunoglobulin E‐mediated hypersensitivity to amoxicillin: in vivo and in vitro comparative studies between an injectable therapeutic compound and a new commercial compound

Skin testing with amoxicillin (AX) is necessary to diagnose immediate hypersensitivity reactions to this β-lactam. A commercial AX (DIA-AX) has recently become available for skin testing. The aim of this study was to compare DIA-AX with the injectable form (INJ-AX) in patients who have well-demonstrated IgE-mediated hypersensitivity to AX. Chemical characterization using high-performance liquid chromatography of both DIA-AX and INJ-AX reagents was performed. Patients diagnosed with an immediate allergic reaction to AX and a positive skin test to INJ-AX (N=55) were re-evaluated within 6 months by performing skin testing with INJ-AX and DIA-AX. Basophil activation test (BAT) and Radioallergosorbent test (RAST) inhibition assay using both reagents were performed in a selected group of patients. The chemical analysis indicated that both DIA-AX and INJ-AX contained an AX compound with a purity above 95%. In the re-evaluation, 53 (96.4%) cases maintained skin test positivity to INJ-AX and were also positive to DIA-AX. Comparison of the papule area between the two reagents showed no significant differences between both reagents. BAT was performed in 30 samples and was positive to both compounds in 15 cases; no patient had a positive result to just one reagent. RAST inhibition studies using three individual cases and a pool of positive sera showed that the percentage inhibition detected with DIA-AX and INJ-AX was parallel and almost exactly the same. This study shows that DIA-AX is equivalent to INJ-AX in terms of skin test response, as well as with in vitro immunochemical and biological tests. DIA-AX can therefore be used in the diagnosis of immediate hypersensitivity reactions.

Cephalosporin and penicillin cross-reactivity in patients allergic to penicillins

Bata-lactam antibiotics are the most commonly used antibiotics which usually cause serious IgE-mediated allergic reactions. Of all bata-lactam antibiotics, penicillins have so far been the best-studied, but the studies of cephalosporins and their cross-reactivity with penicillins are rare. We sought to evaluate the IgE response in vitro and estimate cross-reactivity between penicillins and cephalosporins in patients allergic to penicillins. We studied 87 control subjects and 420 subjects allergic to penicillins. Radioallergosorbent test (RAST) was performed to detect eight types of specific-penicillin IgE and eleven types of specific-cephalosporin IgE. The cross-reactivity and different molecules recognition by IgE were studied with a radioallergosorbent inhibition test. Of 420 patients allergic to penicillins, 95 patients (22.62%) showed specific-cephalosporin IgE positive, 73 patients (17.38%) showed IgEs positive to both penicillins and cephalosporins. In specific-penicillin IgE positive group, the positive rate of specific-cephalosporin IgE was significantly higher than in specific-penicillin IgE negative group (27.14% vs. 14.57%, p < 0.01). In urticaria group, the positive rate of specific-cephalosporin IgE was significantly higher than in other symptoms group (30.65% vs. 8.11%, p < 0.05). The analysis of drugs which have the same or similar side-chains showed that benzylpenicillanyl-IgE (BPA-IgE), ampicillanyl-IgE (APA-IgE), amoxicillanyl-IgE (AXA-IgE) were respectively related to cephalothanyl-IgE (CLA-IgE), cephalexanyl-IgE (CEXA-IgE), cephalexanyl-IgE (CEXA-IgE)in sera of penicillin-allergic patients we studied, and compared with patients who had negative amoxicillin-IgE, the positive rates of specific-ampicillin IgE and specific-cephalexin IgE were significantly higher in patients who had positive amoxicillin-IgE (14.43% vs. 3.72%, 14.00% vs. 2.96%, p < 0.01). Radioallergosorbent test and radioallergosorbent inhibition test confirmed that both nuclear structure and R1 side-chain contribute to IgE recognition. There exists cross-reactivity between cephalosporins and penicillins; patients allergic to several penicillins are more likely to develop allergic reaction to cephalosporins; due to sensitization to the similar structural characteristics (nuclear and R1 side-chain), penicillin-allergic patients may develop cross-allergic reactions with not only first-generation but also third-generation cephalosporins.

Amoxicillin And Tlr Agonists Increase The Maturation Of Dendritic Cells From Patients With Non-immediate Allergy To Betalactams

M O N D A Y 755 Different Patterns Of IgE Recognition To Amoxicillin In Patients With Immediate Hypersensitivity Reactions A. Ariza, C. Mayorga, I. Do~na, F. Gomez, E. Perez-Inestrosa, M. I. Monta~nez, J. L. Rodriguez-Bada, M. A. Guerrero, M. Blanca, M. J. Torres; Research Laboratory F-IMABIS, M alaga, SPAIN, Allergy Service, Carlos Haya Hospital, M alaga, SPAIN, Organic Chemistry Department, Chemistry University, M alaga, SPAIN. RATIONALE: The optimal recognition of penicillin determinants, including amoxicillin, by specific IgE antibodies requires a covalent binding to a carrier molecule. The nature of the carrier and its contribution to the antigenic determinant is not well known. The aim of this study was to evaluate the recognition of different amoxicillin structures by specific IgE antibodies in patients with immediate hypersensitivity reactions. METHODS: Patients (N521) with immediate hypersensitivity reactions to amoxicillin and positive radioallergosorbent test (RAST) were included. RASTwas done using benzylpenicillin and amoxicillin bound to poly-Llysine (BPO-PLL and AXO-PLL). RAST inhibition assays used AXOPLL in the solid phase and amoxicillin, amoxicilloic acid, amoxicillin bound to butilamine (AXO-BA), human serum albumin (AXO-HSA) and dendrimers (AXO-DN) in the fluid phase. RESULTS:We observed two clear defined patterns of IgE recognition: A) Cases (N58) with a higher recognition to free amoxicillin being lower to amoxicilloic acid, AXO-BA, AXO-HSA and AXO-DN; B) Cases (N513) which recognize both free and conjugated amoxicillin in similar degree with low recognition to amoxicilloic acid. Group A corresponds to patients with RAST only positive to AXO-PLL and Group B to those positive to BPO-PLL and AXO-PLL. CONCLUSIONS: These results seems to indicate that IgE recognition is influenced by the binding and nature of the carrier molecule (Group A), with others mainly recognizing the amoxicillin structure independently of the nature of the carrier (Group B). The fact that amoxicilloic acid is not optimally recognized in Group B reinforced the need of conjugation to a carrier in this group of patients.

Lupin allergy and lupin sensitization among patients with suspected food allergy

Lupin, a legume with good nutritional value, is used in food production today, most often in bakery products. Lupin sensitization is often seen among patients with reactions to legumes, but the number of reports describing lupin anaphylaxis is also increasing. To investigate the occurrence of lupin sensitization, cross-reactivity, and lupin allergy among patients with suspected food allergy in Finland, where lupin is a labeled ingredient in few products. The occurrence of positive skin prick test (SPT) reactions to lupin seed flour was studied among 1522 patients with suspected food allergy from November 1, 2005, through December 31, 2007. Clinical histories and diagnostic SPT results were analyzed among patients with positive SPT results to lupin. For 1 patient, ImmunoSpot and lupin radioallergosorbent test inhibition methods were used. Lupin sensitization was shown in 25 of 1522 patients (1.6%), and probable lupin allergy was diagnosed in 7 of 25 patients, in whom the clinical symptoms varied from anaphylaxis and respiratory symptoms to contact urticaria and itchy mouth. Cross-reactions or concurrent reactions to other legumes were seen in 18 of 25 patients. Clinically relevant lupin allergy often occurs in patients without atopic background or other food allergies, although lupin sensitization most commonly seems to represent cross-reactivity to other legumes. The occurrence of lupin allergy in a country where lupin has not been traditionally used is surprisingly common, suggesting that short-term use of modest amounts of lupin can cause serious allergic reactions.

Hypersensitivity reactions to penicillins: studies in a group of patients with negative benzylpenicillin G skin test

Although skin tests are usually employed to evaluate current penicillin allergy status, a negative result does not exclude hypersensitivity. There is a need for accurate in vitro tests to exclude hypersensitivity. A radioallergosorbent test (RAST) is a potentially good supplementary approach, but there is little information on the suitability of this method to diagnose penicillin hypersensitivity in subjects with a negative skin test to benzylpenicillin. A total of 133 patients with a negative skin test to benzylpenicillin G (PG) and all of whom developed allergic reactions to PG were studied. RAST was used to detect eight kinds of specific IgE antibodies to penicillins in serum, which included four kinds of major and minor antigenic determinants to four penicillin drugs. The combination sites for the specific IgE antibodies were studied by RAST inhibition test. The rate of positive reactions for the specific IgE antibodies was 59.40% (79/133). Of the eight kinds of antigenic determinants, the positive rates for specific IgE against the major and minor determinants were 39.10% (52) and 42.86% (57) respectively. Of the four drugs, positive cases only to PG were 10 (7.5%), were significantly fewer than the cross-reacting positive cases (36) to PG (P < 0.01). In the RAST inhibition studies all drugs exhibited good inhibitory potencies, and in some instances the side-chain of the penicillins could induce specific responses with a variable degree of cross-reactivity among the different penicillins. Radioallergosorbent test is a good complementary test in persons who are skin-test negative with PG, and the sensitivity of RAST increases with increasing specificity of IgE antibodies to be detected. 6-APA and the groups, making part of the different side-chains on penicillins, all contributed to the cross-reactivity.

Date Palm Pollen Allergoid: Characterization of Its Chemical-Physical and Immunological Properties

Background: Date palm (DP) pollen can cause allergic symptoms in people living in different countries. Specific immunotherapy with allergenic extracts by subcutaneous route is effective to cure allergic people. However, the risk of side effects has led to explore safer therapeutic modalities. The aim of our work was to evaluate IgE cross-reactivity between DP and autochthonous palm (European fan palm, EFP) pollen extracts, to chemically modify DP extract with potassium cyanate in order to obtain an allergoid, and to characterize it. Methods: By radioallergosorbent test inhibition, immunoblotting (IB) and skin prick test, in vitro and in vivo allergenic activities of native and modified DP extracts were compared. By SDS-PAGE and IB, we compared the protein profile and IgE-binding capacity of both native and modified DP, as well as of EFP extracts. By IB inhibition, IgE cross-reactivity of native DP and EFP extracts was evaluated. By ELISA, the capacity of modified DP-induced IgG to react with native DP extract was determined. Results: Radioallergosorbent test inhibition, IB and skin prick test results demonstrated that modified DP was significantly less allergenic than native DP extract. The SDS-PAGE profile showed that potassium cyanate treatment of DP extract did not alter the molecular weight of its components. In addition, no difference was observed between native DP and EFP extracts. Subsequent IB inhibition data evidenced the existence of a strong IgE cross-reactivity between native DP and EFP extracts. ELISA results indicated that the administration of modified DP in mice was able to induce specific IgG also recognizing native DP extract. Conclusions: Modified DP extract (allergoid) seems to be a good candidate for immunotherapy of patients affected by specific allergy.

Clustered sensitivity to fungi: anaphylactic reactions caused by ingestive allergy to yeasts

Respiratory allergy to environmental molds is relatively common, and fungal allergen-specific reactivity seems to cluster in certain persons. However, generalized reactions caused by ingested fungi have seldom been described. To describe a mold-sensitized patient who developed multiple anaphylactic reactions after ingesting a yeast preparation widely used by the food industry as flavoring in, for example, powdered and ready-made sauces. Skin prick tests and serum IgE tests were performed with inhalant and food allergens, including molds and yeasts, 2 pasta sauces consumed by the patient, individual sauce ingredients, and a food-quality yeast extract. Radioallergosorbent test inhibition was used for specificity studies. Skin prick and serum IgE test results were positive to several molds (Cladosporium herbarum, Alternaria alternata, Aspergillus fumigatus, and Penicillium notatum), baker's yeast (Saccharomyces cerevisiae), Malassezia furfur, and champignon and to the 2 pasta sauces, the yeast ingredient, and a food-quality yeast extract. Radioallergosorbent test inhibition studies confirmed that the sauces contain cross-reacting yeast and mold allergens. This patient has a clustered sensitization to fungi characterized by allergy to environmental fungal allergens and to yeast extracts used in the food industry. Yeasts should be considered as possible ingestive allergens in mold-allergic patients.

Skin testing for immediate hypersensitivity to betalactams: comparison between two commercial kits

Skin testing with major and minor determinants of benzylpenicillin is the recommended standard practice to evaluate subjects with immediate hypersensitivity to betalactams. The withdrawal of these products from the market has set us back to the early days, before the introduction of reagents for in vivo testing. To compare a recently released kit of benzylpenicillin conjugated to poly-l-lysine (PPL) and minor determinants mixture (MDM) with the previously existing kit in a positive control group of subjects sensitized to major and/or minor determinants of benzylpenicillin. Skin tests with both kits were made in a group of positive subjects previously diagnosed with immediate hypersensitivity to penicillins and with positive results to PPL and/or MDM and in a negative control group. Radioallergosorbent test (RAST) inhibition assays with a pool of sera and individual samples were carried out to compare the inhibition capacity of PPL and MDM of both kits. Of 22 cases selected from our historical group, 14 were positive: eight to PPL, three to MDM and three to both. These results were equivalent for both kits. RAST inhibition studies showed similar potencies in the inhibition of PPL and MDM. Both tests show similar results in terms of RAST inhibition assays and skin tests sensitivity and specificity in the groups selected. The new assay can be used for the same purpose and indications as the previous test.

EuropeanPolistesvenom allergy

The American Polistes species venom mixture--that of P. annularis, P. fuscatus, P. metricus and P. exclamans--was the only commercially available mixture for diagnosis and therapy until 1996. However, these species of Polistes are not present in Europe, where P. dominulus and P. gallicus and to a lesser extent P. nimphus are widespread. The aim of this study was to assess the allergenic differences among the commercial American mix, P. dominulus and P. gallicus venom in European patients and therefore to verify if this mixture is suitable for diagnosis in these patients. We carried out skin tests, radioallergosorbent tests (RAST) and RAST inhibition in Italian patients with adverse reactions to Polistes stings. RAST inhibition results demonstrated that cross-reactivity between the American and European species is only partial and that P. dominulus and P. gallicus venoms have exclusive allergens. Skin tests and direct RAST confirmed these results and also showed that European Polistes venom is more suitable than the American mix in Italian patients. Moreover, we found a high rate of cross-reactivity between P. dominulus and P. gallicus. To conclude, P. dominulus and/or P. gallicus venoms are necessary for diagnosis and therefore in the therapy of European patients.

An antibody produced in tobacco expressing a hybrid β-1,4-galactosyltransferase is essentially devoid of plant carbohydrate epitopes

N-glycosylation of a mAb may have a major impact on its therapeutic merits. Here, we demonstrate that expression of a hybrid enzyme (called xylGalT), consisting of the N-terminal domain of Arabidopsis thaliana xylosyltransferase and the catalytic domain of human beta-1,4-galactosyltransferase I (GalT), in tobacco causes a sharp reduction of N-glycans with potentially immunogenic core-bound xylose (Xyl) and fucose (Fuc) residues as shown by Western blot and MALDI-TOF MS analysis. A radioallergosorbent test inhibition assay with proteins purified from leaves of WT and these transgenic tobacco plants using sera from allergic patients suggests a significant reduction of potential immunogenicity of xylGalT proteins. A mAb purified from leaves of plants expressing xylGalT displayed an N-glycan profile that featured high levels of galactose, undetectable xylose, and a trace of fucose. Hence, a transgenic plant expressing the hybrid GalT might yield more effective and safer monoclonals for therapeutic purposes than WT plants and even transgenic plants expressing the unchanged GalT.

Allergy to jackfruit: a novel example of Bet v 1-related food allergy.

Jackfruit allergy has been reported just once. It is unknown whether this food allergy is caused by direct sensitization or cross-sensitization to pollen allergens. Establish whether jackfruit allergy is linked to birchpollen allergy. Two jackfruit allergic patients and five patients with birchpollen-related apple allergy were recruited. Sensitization to pollen and plant foods was assessed by skin prick test (SPT), radio-allergosorbent test (RAST) and immunoblot. RAST analysis was performed for Bet v 1 and Mal d 1. Cross-reactivity was evaluated by RAST and immunoblot-inhibition. Biological activity of immunoglobulin E (IgE) was measured by basophil histamine release. Allergy to jackfruit was evaluated by double-blind placebo-controlled food challenge (DBPCFC) or open challenge (OC). In both patients DBPCFC confirmed the reported jackfruit allergy. SPT was 41 and 27 mm2 and specific IgE to jackfruit was 5.9 and 0.8 IU/ml, respectively. Immunoblot analysis revealed IgE reactivity at Mr of approximately 17 kDa. The Bet v 1-related nature of this allergen in jackfruit was demonstrated by RAST and immunoblot inhibition. To assess whether jackfruit allergy might be common in patients with combined birchpollen-fruit allergy, five such patients underwent an OC with jackfruit. All five had OA-like symptoms. Jackfruit allergy can be added to the list of birchpollen-related food allergies. Increased consumption of this fruit will result in a rise in allergic reactions.

Immunoblot and radioallergosorbent test inhibition studies of allergenic cross-reactivity of the predatory mite Amblyseius cucumeris with the house dust mite Dermatophagoides pteronyssinus

In 1999, an extensive study among bell pepper growers showed that a predatory mite, Amblyseius cucumeris, is a potentially relevant source of occupational allergens because 23% of the population had positive skin prick test reactions. To investigate whether cross-reactivity between A. cucumeris and Dermatophagoides pteronyssinus is responsible for the cosensitization to both mite species found in 58.7% of A. cucumeris-sensitized greenhouse workers. Fifteen serum samples from greenhouse workers with work-related inhalant allergy and a positive radioallergosorbent test (RAST) reaction to A. cucumeris or D. pteronyssinus were selected for immunoblot analysis using extracts of both mites. A subselection (n = 5) was used for RAST and immunoblot inhibition to investigate potential cross-reactivity. On immunoblot, 2 distinct patterns were observed: one pattern showed common protein bands in A. cucumeris and D. pteronyssinus blots suggestive of cross-reactivity between A. cucumeris and D. pteronyssinus and the other pattern showed no shared protein bands. Dermatophagoides pteronyssinus RAST inhibition with A. cucumeris extract was low in 4 serum samples (<25% inhibition) and nearly absent in 1 serum sample; A. cucumeris RAST inhibition with D. pteronyssinus extract was high in 1 serum sample (75% inhibition), low in 2 serum samples (35% and <15% inhibition), and absent in 2 serum samples. These results were confirmed by immunoblot inhibition experiments. Amblyseius cucumeris, a new occupational allergen, has species-specific antigens and common antigens that are cross-reactive with the house dust mite D. pteronyssinus.

Cloning and expression of a major allergen from Cupressus arizonica pollen, Cup a 3, a PR-5 protein expressed under polluted environment.

This paper describes the cloning and expression of the Cupressus arizonica pollen protein Cup a 3. In addition, we present its modulation under polluted environmental conditions. Species of the Cupressaceae family are important because of their high sensitization prevalence. Cup a 3 cloning is based on the sequence of the homologous protein Jun a 3. Cup a 3 was expressed with good yield in the methylotropic yeast Pichia pastoris. Recombinant Cup a 3 (rCup a 3) contains 199 amino acids, 10 potential phosphorylation sites and no glycosylation sites. By immunoblot 63% of cypress allergic patients had specific immunoglobulin E antibodies against rCup a 3 (n = 104). This major allergen is homologous to members of the pathogenesis-related proteins (PR-5 group) and contributes to the overall allergenicity of C. arizonica pollen. Our results show that the increased expression of Cup a 3 is dependent on the pollution in the area where the pollen has been collected, being higher under polluted conditions. Cup a 3 is a PR-5 protein derived from C. arizonica pollen. The expression of the protein under polluted conditions has a direct incidence on the pollen allergenicity, as has been demonstrated by skin tests and Radioallergosorbent test inhibition.

Effects of SDS, PBS, Coca's and NH4HCO3 solutions on the fluorescence values of radioallergosorbent test inhibition

Rationale Radioallergosorbent Test (RAST) inhibition usually is the key technique to assess total allergenic activity of an extract. The purpose of this paper is to explore effects of some re-dissolvent solutions on fluorescence values of RAST inhibition. Methods Dermatophagoides pterronyssinus allergen immunoCAP and UniCAP 100 System were the product of Pharmacia Diagnostics, Sweden. The Mixed sera pool consisted of 12 Dermatophagoides pterronyssinus allergic patients sera, RAST 4-6 classes. Results Percentages of fluorescence values decreased: 62.9%, 54.1%, 43.5%, 6.7%, 3.7%, 2.6%, 2.2%, 1.4% respectively, when SDS Concentrations were 2%, 1%, 0.5%, 0.25%, 0.1%, 0.05%, and 0.01%. PBS in 0.1M, 0.01M concentrations decreased the fluorescence values 2.9%, 0.9% respectively. Coca's and 1.5M NH4HCO3 solutions reduced down the fluorescence values 3.4%, 4.5% respectively. Conclusions SDS is one of the most common used surfactants when people extract allergen and re-dissolvent prepared allergen precipitation for RAST inhibition. It showed that the fluorescence values decreased more than 5% when SDS concentration equal to 0.25% or higher. However, the fluorescence values decreased less than 3.7% when SDS concentration equal to 0.1% or lower. The reason may be that 0.23% is SDS Critical Micelle Concentration (CMC) in water, and SDS can combine with protein to form micelle. So SDS separated the allergen from solid-phase when its concentration higher than 0.23%. Therefore, people must be careful when they use surfactants like SDS as re-dissolvent agent to improve protein resolution in RAST inhibition. To these solutions (PBS, Coca's and 1.5M NH4HCO3)in common used concentrations, it illustrated that the fluorescence values decreased less than 5%.

Allergic reactions to jackfruit in birch pollen allergic patients

Rationale Confirm allergy to jackfruit in 2 patients with an according personal history and characterize allergens and cross-reactivity of specific IgE antibodies to (birch) pollen and food allergens. In addition, determine the prevalence of jackfruit allergy in a group (n=5) of birch pollen (BP) and food allergic patients. Methods All patients underwent skin prick testing (SPT). Two patients were tested in a double-blind placebo-controlled food challenge (DBPCFC) and the additional 5 underwent an open challenge (OC) with jackfruit. Specific IgE was measured with the radio-allergosorbent test (RAST). RAST and immunoblot (inhibitions) were carried out. In the 2 jackfruit allergic patients, basophil histamine release was measured with several allergens. Results Both patients had positive SPT responses to jackfruit and BP and reacted with oral allergy symptoms (OAS) in the DBPCFC. The 5 selected patients reacted with OAS in the OC. Specific IgE of patient 1 to jackfruit was completely inhibited by BP extract and natural (n)Bet v 1 in RAST and immunoblot inhibition. In addition, this patient showed histamine release upon stimulation with jackfruit, apple or BP extracts and nBet v 1. Conclusions Sensitization to jackfruit allergy is due to the cross-reactive BP allergen Bet v 1. The prevalence of jackfruit allergy in the selected group of 5 patients was 100%, but jackfruit is not regularly consumed in Northern Europe, explaining why jackfruit allergy is not common.

Immunologic characterization of the recombinant fire ant venom allergen Sol i 3.

Individuals sensitized to fire ant stings show immunoglobulin (Ig)E antibodies against the venom protein Sol i 3. We determined the full-length complementary DNA (cDNA) sequence of this protein and expressed recombinant Sol i 3 in immunogenic form. The complete cDNA of Sol i 3 was obtained by reverse transcription polymerase chain reaction (RT-PCR) and PCR + 1 reactions using gene-specific oligonucleotides, and oligonucleotides designed from the amino acid sequence of this protein. The encoding cDNA is 705 bp in length corresponding to 235 amino acids. The first 22 amino acids are a leader sequence. The protein with an added C-terminal hexahistidine tag was expressed in insect cells using a baculovirus system. The recombinant protein was secreted into the supernatant and affinity purified with a cobalt chelating resin. The recombinant fire ant venom allergen Sol i 3 showed similar IgE binding activity to the native protein in radioallergosorbent test (RAST) and RAST inhibition assays. It was produced in both a glycosylated and an unglycosylated form. A three-dimensional reconstruction of Sol i 3 was compared with the experimentally determined structure of the related allergen Ves v 5. This model is supported by results of circular dichroism spectroscopy.

23. Clinical laboratory assessment of IgE-dependent hypersensitivity

This chapter reviews clinical and laboratory analyses that aid in the diagnosis and management of human allergic (IgE-dependent) diseases. The diagnostic algorithm for immediate-type hypersensitivity begins with a thorough clinical history and physical examination. Once signs and symptoms compatible with an allergic disorder have been identified, a skin test and/or blood test for allergen-specific IgE antibodies may serve as primary confirmation to strengthen the diagnosis. Puncture and intradermal skin testing provide a biologically relevant immediate-type hypersensitivity response in the skin, with resultant wheal and flare reactions within 15 minutes of allergen application. Bleeding, dermatographism, and antihistamines may confound the quality of the skin test. Allergenspecific IgE antibody may also be detected in the blood using a radioallergosorbent test (RAST). Nonisotopic “secondgeneration” RAST-type assays have evolved to provide more quantitative, sensitive, precise IgE antibody results. In vivo provocation tests may serve as secondary confirmatory tests when the clinical history is discordant with a primary IgE antibody test result. The multiallergen screen is a qualitative RAST-type assay that detects specific IgE antibody to approximately 15 allergens that evoke a large majority of aeroallergen or food-related allergic disorders. Other useful serological assays performed in the diagnostic allergy laboratory include total serum IgE, Hymenoptera venom-specific IgG antibody, IgG precipitins for organic dusts, mast cell tryptases, and the venom RAST inhibition test. Above all, in vivo or laboratory confirmatory test results that are inconsistent with the clinical history should be repeated as for any laboratory assessment. (J Allergy Clin Immunol 2003;111:S687-701.)