ELISA Inhibition Studies Research Articles

Comparison of sensitization patterns to dust mite allergens between atopic dermatitis patients and dogs, and non-specific reactivity of canine IgE to the storage mite Tyrophagus putrescentiae.

House dust mite is a common cause of atopic dermatitis (AD) both in humans and dogs. Detection of serum IgE to allergens is commonly used to diagnose allergic diseases. However, false-positive reactions due to cross-reactivity and non-specific reactivity may lead to misdiagnosis. We compared human and canine IgE reactivities to mite component allergens. Canine IgE-reactive components of Dermatophagoides farinae and Tyrophagus putrescentiae were identified by tandem mass spectrometry. Recombinant proteins were produced and IgE reactivities to component allergens were assessed by ELISA and inhibition assays using sera from AD patients and dogs. Canine IgE-reactive proteins (Der f 1, Der f 11, Tyr p 4, Tyr p 8, Tyr p 11, Tyr p 28) were identified by proteome analysis. Most patients were sensitized to Der f 1 (93.3%) and Der f 2 (86.7%). Dogs showed high sensitization to Der f 2 (94.1%) and Der f 18 (84.6%). Both patients and dogs showed low IgE binding frequency to Tyr p 8, 43.3% and 4%, respectively. The ELISA inhibition study indicated that canine IgE reactivity to T. putrescentiae is mostly due to non-specific reaction and cross-reaction with D. farinae. Different IgE sensitization patterns were shown between allergic humans and dogs with AD, especially to Der f 18, for the first time in Korea. Furthermore, non-specific canine IgE reactivity to storage mite indicates the possibility of misdiagnoses. Standardizations focused on the major canine allergen content of extracts should be developed. This will allow precision diagnosis and individuated treatments for each patient and atopic dog.

Preferential recognition of epitopes on peroxynitrite-modified alpha-2-macroglobulin by circulating autoantibodies in rheumatoid arthritis patients

Autoimmune responses against post-translationally modified antigens are a hallmark of several autoimmune diseases. In this work, we have studied the changes in alpha-2-macroglobulin (α2M) upon modification by peroxynitrite. Furthermore, we have evaluated the immunogenicity of modified α2M in experimental rabbits and rheumatoid arthritis (RA) patients. Peroxynitrite-modified α2M showed disturbed microenvironment and altered aromatic residues under UV and fluorescence studies. Aggregation, reduction in β-sheet content, production of nitrotyrosine and shift in amide I and II bands were observed in the modified α2M by polyacrylamide gel electrophoresis besides CD and FTIR spectroscopic analysis. The exposure of hydrophobic clusters and changes in contact positions were observed in ANS and ThT binding assays. Immunological studies using ELISA showed peroxynitrite-modified α2M as highly immunogenic producing high titre of specific antibodies in immunized rabbits. Cross-reactivity studies revealed the polyspecificity of the elicited antibodies. Direct binding ELISA and competitive inhibition studies confirmed the presence of circulating antibodies in the sera of RA patients having high specificity towards the peroxynitrite-modified α2M as compared to the native α2M. Sera from healthy (normal) human subjects showed lower binding with the native and modified protein. This study confirms that peroxynitrite induces structural modifications in α2M and makes it immunogenic. The presence of neo-antigenic determinants on modified α2M with enhanced binding for circulating autoantibodies in RA patients could offer new possibilities for diagnosis and etiopathology of the disease. Communicated by Ramaswamy H. Sarma

Heterogeneity in Allergy to Mollusks: A Clinical-Immunological Study in a Population From the North of Spain.

Allergy to mollusks has been the focus of fewer studies than allergy to crustaceans. Furthermore, allergy to mollusks is less well characterized. Objectives: To describe the clinical characteristics of mollusk-allergic patients, to identify the responsible allergens, and to assess cross-reactivity. We performed a prospective multicenter study including 45 patients with mollusk allergy, which was diagnosed based on a suggestive clinical history and a positive skin test result with the agent involved. Fractions were identified using SDS-PAGE and immunoblotting. The proteins responsible were subsequently identified using mass spectrometry. ELISA inhibition studies were performed with mollusks, dust mites, and crustaceans. We found that 25 patients (55%) were allergic to cephalopods, 14 (31%) to bivalves, and 11 (24%) to gastropods. Limpet was the third most frequent cause of allergy (15% of cases). In 31 patients (69%), the manifestation was systemic; 10 (22%) exhibited oral allergy syndrome, and 7 (15%) experienced contact urticaria. Most major allergens were found between 27 kDa and 47 kDa. ELISA inhibition assays revealed a high degree of inhibition of cephalopods and bivalves from all the groups of mollusks, mites, and crustaceans. Mass spectrometry identified tropomyosin, actin, and myosin as the major allergens. Cephalopods, especially squid, are the mollusks that most frequently trigger allergic symptoms. The very frequent occurrence of allergy to limpets is striking, given their low consumption in our area. It is worth highlighting the heterogeneity observed, exemplified by the gastropods. Tropomyosin appears to be responsible for the high cross-reactivity found between mollusks, mites, and crustaceans. Three new mollusk allergens were also identified, namely, actin, enolase, and a putative C1q domain-containing protein.

Identification of the major allergenic epitopes of Eriocheir sinensis roe hemocyanin: A novel tool for food allergy diagnoses.

Crab meat and roe are highly nutritious delicacies in China. While extensive research has been conducted for allergens derived from crab-meat, data relevant to the allergenic potential of crab roe derived proteins, of which hemocyanin is a principal contender, are almost entirely absent. Using bioinformatics prediction and IgE-binding assays, the three principal immunodominant epitopes of hemocyanin were identified and then combined as a single recombinant fusion protein (rHc). This together with the full-length recombinant protein (Hc) were expressed in Escherichia coli and subsequently identified by SDS-PAGE and immunoblotting. Ninety-five percent of our patients were found to carry rHc-specific IgE antibodies by ELISA. Dot-blot inhibition, together with ELISA inhibition studies, showed that pre-incubation of patient sera with the recombinant epitope protein could inhibit26% to 63% (mean: 50%) of IgE binding to immobilized, full-length Hc and the dose-response curve represents as a sigmoid shape. The recombinant protein (rHc) represents a versatile biologic tool with which to diagnose and investigate therapies for E. sinensis allergy.

Allergen of the Month—Annual Wormwood

Allergen of the Month—Annual Wormwood

In vitro evaluation of allergen potencies of commercial house dust mite sublingual immunotherapy reagents.

PurposeThe clinical efficacy of allergen-immunotherapy is known to be dose dependent. However, optimal maintenance dosage has not yet been determined for sublingual immunotherapy (SLIT). Furthermore, since companies adopt their own units for expression of allergenicity, the allergen concentrations of individual reagents cannot be compared easily. We sought to measure and compare the allergenicities of 3 commercially available house dust mite (HDM) SLIT regents and a subcutaneous immunotherapy reagent.MethodsWe measured the HDM allergenic potency of the maintenance dosages of three SLIT reagents: Staloral® (300 index of reactivity [IR] /mL, recommended maintenance dosage [MD]: 120 IR), SLITone® (1,000 standard therapeutic unit [STU]/mL, recommended MD: 200 STU), Wolwopharma® (100 µg/mL, recommended MD: 20 µg), and subcutaneous immunotherapy regents of Hollister-Stier (10,000 allergy unit [AU] /mL). The allergenic potency was assessed by measuring the total protein concentrations, mite group 1 and 2 allergens using 2-site ELISA, and an inhibition test against IgE specific to Dermatophagoides farinae and Dermatophagoides pteronyssinus.ResultsThe protein content of the Wolwopharma® reagent was 1.5-261.4 times higher than that of the other 2 SLIT reagents. The concentration of group 1 major allergens in Staloral® (132.03 µg/mL) was 33- to 44.5-fold higher than in SLITone® (4.00 µg/mL) and Wolwopharma® (2.97 µg/mL). The concentration of group 2 major allergen was also 8.9- to 10.5-fold higher in Staloral® (15.7 µg/mL) than in SLITone® (1.8 µg/mL) or Wolwopharma® (1.5 µg/mL). An ELISA inhibition study against HDM-specific IgE showed that the allergen potency of Staloral® reagent is 8.5-fold and 21-fold higher than that of SLITone® or Wolwopharma®, respectively. The differences between the maintenance dosages are further exaggerated by the differences in the recommended volumes of SLIT reagents.ConclusionsThe allergen potencies of commercially available HDM SLIT reagents are markedly different. Consensus regarding the optimal allergen concentration for SLIT reagents used to treat HDM respiratory allergies is needed.

High prevalence of lipid transfer protein sensitization in apple allergic patients with systemic symptoms.

BackgroundApple allergy manifests as two main groups of clinical entities reflecting different patterns of allergen sensitization: oral allergy syndrome (OAS) and generalized symptoms (GS).ObjectiveWe analysed the sensitization profile to a wide panel of different components of food allergens (rMal d 1, Mal d 2, rMal d 3, rMal d 4, rPru p 3, rBet v 1 and Pho d 2) for a population of Mediterranean patients with OAS and GS to apple.MethodsPatients (N = 81) with a history of apple allergy that could be confirmed by positive prick-prick test and/or double-blind-placebo-controlled food challenge (DBPCFC), were included. Skin prick test (SPT) and ELISA were performed using a panel of inhalant, fruit and nut allergens. ELISA and ELISA inhibition studies were performed in order to analyse the sensitization patterns.ResultsThirty-five cases (43.2%) had OAS and 46 (56.8%) GS. SPT showed a significantly higher number of positive results with peach, cherry and hazelnut in those with GS. ELISA showed a significantly high percentage of positive cases to rMal d 3, rMal d 4, rPru p 3 and Pho d 2 in patients with OAS and GS compared to controls, and to rBet v 1 in patients with OAS vs controls and between OAS and GS patients. Three different patterns of recognition were detected: positive to LTP (rMal d 3 or rPru p 3), positive to profilin (rMal d 4 and Pho d 2), or positive to both. There were also patients with rMal d 1 recognition who showed cross-reactivity to rBet v 1.ConclusionIn an apple allergy population with a high incidence of pollinosis different patterns of sensitization may occur. LTP is most often involved in those with GS. Profilin, though more prevalent in patients with OAS, has been shown to sensitise patients with both types of symptoms.

Identification of thaumatin-like protein and aspartyl protease as new major allergens in lettuce (Lactuca sativa).

Today, about 2-8% of the population of Western countries exhibits some type of food allergy whose impact ranges from localized symptoms confined to the oral mucosa to severe anaphylactic reactions. Consumed worldwide, lettuce is a Compositae family vegetable that can elicit allergic reactions. To date, however, only one lipid transfer protein has been described in allergic reaction to lettuce. The aim of this study was to identify potential new allergens involved in lettuce allergy. Sera from 42 Spanish lettuce-allergic patients were obtained from patients recruited at the outpatient clinic. IgE-binding proteins were detected by SDS-PAGE and immunoblotting. Molecular characterization of IgE-binding bands was performed by MS. Thaumatin was purified using the Agilent 3100 OFFGEL system. The IgE-binding bands recognized in the sera of more than 50% of patients were identified as lipid transfer protein (9 kDa), a thaumatin-like protein (26 kDa), and an aspartyl protease (35 and 45 kDa). ELISA inhibition studies were performed to confirm the IgE reactivity of the purified allergen. Two new major lettuce allergens-a thaumatin-like protein and an aspartyl protease-have been identified and characterized. These allergens may be used to improve both diagnosis and treatment of lettuce-allergic patients.

IgE Specific to Oxycodone Identified by ELISA Inhibition in a Young Woman with Symptoms of Anaphylaxis Following Oral Ingestion: First Case Report of Oxycodone-Mediated Anaphylaxis

RationaleReactions to opioid analgesics are rarely IgE mediated but usually the result of histamine release. Morphine, codeine, pethidine, fentanyl, and tramadol have been implicated in case reports of Type I IgE-mediated reactions. Here we present a young woman with anaphylaxis and specific IgE to oxycodone.MethodsOxycodone ELISA inhibition study against patient serum and controls by Wilford Hall Allergy Clinic Laboratory.ResultsThis patient with a history of chronic lower back pain and asthma ingested one dose of Percocet (acetaminophen 325mg, oxycodone 5mg). Within 60 minutes, she developed generalized urticaria, dysphonia, and dyspnea. She denied wheezing and her characteristic symptoms of asthma exacerbation. In the ED she had increasing tongue swelling, decreased respiratory effort, and muffled voice; symptoms worsened despite epinephrine 0.3mg intramuscular then 0.1mg intravenous, dexamethasone 10mg IV, diphenhydramine 25mg IV, and ranitidine 50mg IV. She was intubated by anesthesiology for airway protection and transferred to the ICU. Patient extubated 12 hours after intubation, discharged within 48 hours with an Epipen, diagnosed with Percocet allergy, and instructed to avoid all opiates. Serum tryptase (8 hrs after exposure) 3ng/mL, C1 esterase inhibitor 95%, C3 99.6mg/dL, C4 16.17mg/dL, and IgG 831mg/dL. ELISA for oxycodone was run with 100μg oxycodone per well and found an average 47% inhibition of IgE binding with 100μg/100μL of a 1:3 dilution of serum.ConclusionsWe believe this is the first reported case of Type I IgE-mediated anaphylaxis to oxycodone. The use of ELISA to detect oxycodone IgE may be instructional for other diagnostic studies of suspected opioid anaphylaxis. RationaleReactions to opioid analgesics are rarely IgE mediated but usually the result of histamine release. Morphine, codeine, pethidine, fentanyl, and tramadol have been implicated in case reports of Type I IgE-mediated reactions. Here we present a young woman with anaphylaxis and specific IgE to oxycodone. Reactions to opioid analgesics are rarely IgE mediated but usually the result of histamine release. Morphine, codeine, pethidine, fentanyl, and tramadol have been implicated in case reports of Type I IgE-mediated reactions. Here we present a young woman with anaphylaxis and specific IgE to oxycodone. MethodsOxycodone ELISA inhibition study against patient serum and controls by Wilford Hall Allergy Clinic Laboratory. Oxycodone ELISA inhibition study against patient serum and controls by Wilford Hall Allergy Clinic Laboratory. ResultsThis patient with a history of chronic lower back pain and asthma ingested one dose of Percocet (acetaminophen 325mg, oxycodone 5mg). Within 60 minutes, she developed generalized urticaria, dysphonia, and dyspnea. She denied wheezing and her characteristic symptoms of asthma exacerbation. In the ED she had increasing tongue swelling, decreased respiratory effort, and muffled voice; symptoms worsened despite epinephrine 0.3mg intramuscular then 0.1mg intravenous, dexamethasone 10mg IV, diphenhydramine 25mg IV, and ranitidine 50mg IV. She was intubated by anesthesiology for airway protection and transferred to the ICU. Patient extubated 12 hours after intubation, discharged within 48 hours with an Epipen, diagnosed with Percocet allergy, and instructed to avoid all opiates. Serum tryptase (8 hrs after exposure) 3ng/mL, C1 esterase inhibitor 95%, C3 99.6mg/dL, C4 16.17mg/dL, and IgG 831mg/dL. ELISA for oxycodone was run with 100μg oxycodone per well and found an average 47% inhibition of IgE binding with 100μg/100μL of a 1:3 dilution of serum. This patient with a history of chronic lower back pain and asthma ingested one dose of Percocet (acetaminophen 325mg, oxycodone 5mg). Within 60 minutes, she developed generalized urticaria, dysphonia, and dyspnea. She denied wheezing and her characteristic symptoms of asthma exacerbation. In the ED she had increasing tongue swelling, decreased respiratory effort, and muffled voice; symptoms worsened despite epinephrine 0.3mg intramuscular then 0.1mg intravenous, dexamethasone 10mg IV, diphenhydramine 25mg IV, and ranitidine 50mg IV. She was intubated by anesthesiology for airway protection and transferred to the ICU. Patient extubated 12 hours after intubation, discharged within 48 hours with an Epipen, diagnosed with Percocet allergy, and instructed to avoid all opiates. Serum tryptase (8 hrs after exposure) 3ng/mL, C1 esterase inhibitor 95%, C3 99.6mg/dL, C4 16.17mg/dL, and IgG 831mg/dL. ELISA for oxycodone was run with 100μg oxycodone per well and found an average 47% inhibition of IgE binding with 100μg/100μL of a 1:3 dilution of serum. ConclusionsWe believe this is the first reported case of Type I IgE-mediated anaphylaxis to oxycodone. The use of ELISA to detect oxycodone IgE may be instructional for other diagnostic studies of suspected opioid anaphylaxis. We believe this is the first reported case of Type I IgE-mediated anaphylaxis to oxycodone. The use of ELISA to detect oxycodone IgE may be instructional for other diagnostic studies of suspected opioid anaphylaxis.

Characterization of Human IgE and Mouse IgG1 Responses to Allergens in Mosquito Species

IgE‐mediated allergic reactions caused by mosquito bites are a common problem all over the world. This study was undertaken to determine IgE levels in subjects, to elucidate human IgE and mouse IgG1 binding patterns and to investigate the cross‐reactivity of salivary gland antigens with three mosquitoes. Mosquito larvae of Aedes togoi, Culex tritaeniorhynchus and Culex pipiens pallens were collected and maintained in the laboratory. Salivary gland extracts (SGE) and whole‐body extracts (WBE) were prepared from female mosquitoes of each species. The 9 sera out of 12 with positive skin reactions to SGE of A. togoi by skin prick test showed significantly higher anti‐mosquito SGE IgE levels than in those without skin reactions. Protein band patterns of the SGE and WBE of the three species were different from one another. There were specific mouse IgG1 reactions to the bands of 30.5, 33, 37 and 57.5 kD in the SGE of A. togoi. The ELISA inhibition studies disclosed almost no crossreactivities between A. togoi, C. tritaeniorhynchus and C. pipiens pallens. Immunoblot analysis disclosed that allergenic proteins in the SGE of mosquitoes and their patterns were remarkably similar between human and mouse sera to the SGE of A. togoi. Species shared allergens may not exist among the three mosquito species prevalent in Korea.

Occupational asthma caused by IgE-mediated sensitization to multiple woods

Wood is a natural material that is able to trigger rhinitis and asthma in exposed subjects in occupational settings. This has been described with both soft and hard woods.1,2 Involvement of both low- and high-molecular-weight allergens has been reported, and the relevance of these is related with the wood type.1 There are cases where protein may be the responsible allergen. Crossreactivity between obeche and ramin woods3 and between obeche and latex4 has been shown. However, to the best of our knowledge, this is the first report of a multiple IgE-mediated sensitization to different woods that caused occupational respiratory symptoms in the same worker.

The accumulation of inflammatory cells in synovial sheath and epitenon during adhesion formation in healing rat flexor tendons.

The accumulation of inflammatory cells in synovial tissue was studied using indirect immunofluorescence assays on cell cultures and frozen tissue sections of healing rat digital flexor tendons. Flexor tendons were collected from rats 3, 7 and 14 days after crush injury. Tendon sheath and epithenon cells were isolated by sequential enzymic digestion and cultured for 2 days. Subpopulations of synovial and inflammatory cells were identified with MoAbs against cell surface glycoproteins present on B lymphocytes (CD45), T lymphocytes (CD2, CD4, CD8), macrophages (CD14) and endothelial cells. A phagocytosis assay was also used to identify macrophages. We report a substantial increase in the number of T lymphocytes (mainly helper/inducer) and phagocytotic cells with monocyte/macrophage surface markers in tendon sheath and epitenon 3 days after crush injury. The infiltration of inflammatory cells into synovial sheath and epitenon preceded an increase in fibronectin production by tendon cells which was seen 7 days after injury. To study the interaction between T lymphocytes and synovial cells in vitro, we established synovial fibroblast-like type B cell cultures and used stimulated and non-stimulated T lymphocytes in cell binding assays. We observed increased adhesiveness between unstimulated synovial cells and synovial cells previously cultured with activated and non-activated T lymphocytes. ELISA inhibition studies have shown an increase in fibronectin production by synovial fibroblasts co-cultured with stimulated CD4+ T lymphocytes. We suggest that the presence of inflammatory cells in synovial sheath and epitenon during tendon healing induces synovial fibroblasts and epitenon cells to increase their production of fibronectin, which provides a scaffold for subsequent adhesion formation.

Allergenicity and cross‐reactivity of Senecio pollen: identification of novel allergens using the immunoproteomics approach

The genus Senecio is the largest genus of the family Asteraceae (Compositae). The allergenicity of Senecio has not been assessed previously. The aim of this study was to investigate the allergens of Senecio jacobea pollen and to determine their immunological characteristics and clinical relevance. Fifty patients with rhinoconjunctivitis and a positive skin prick test (SPT) to Senecio were recruited. The clinical relevance of this pollen was assessed by means of a nasal provocation test (NPT). Allergens were characterized by one-dimensional electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis and immunoblotting. Furthermore, characterization and identification of the allergens were performed by mass spectrometry (MS). In vitro inhibition tests were performed to evaluate cross-reactivity with other pollen. Three predominant allergens, both in the intensity of reaction and the frequency of recognition by human-allergic sera, were 59 (60%), 42 (50%) and 31 kDa (50%). The two-dimensional analysis allowed the identification of several allergens. One spot around 42 kDa was identified as a protein homologous to pectate lyase and three other spots were homologous to malate dehydrogenase by MS. S. jacobea proteins showed cross-reactivity with other proteins of the Asteraceae family and also with Parietaria judaica. This was demonstrated by immunoblotting and ELISA inhibition studies. S. jacobea constitute a newly discovered allergenic source. It shows cross-reactivity with other members of the Asteraceae plant family as well as with P. judaica.

Cross‐reactivity between pollen allergens from common Pooideae grasses and rye

SummaryIn Germany, specific pollen extracts are currently used for immunotherapy of rye (Secale cereale) allergy. Like most common grasses, S. cereale belongs to the Pooideae subfamily. Using sera from grass pollen‐allergic patients, immunoblotting and ELISA inhibition studies demonstrate that a five‐grass allergenic extract from Anthoxanthum odoratum, Dactylis glomerata, Lolium perenne, Poa pratensis and Phleum pratense inhibits over 90% of IgE binding to rye allergens. This result confirms the high degree of homology between allergens from S. cereale and common grass species. We conclude that a five‐grass pollen mixture is appropriate for desensitization of patients who are allergic to rye pollen without the need for additional rye pollen extracts.

Gräserpollenextrakte eignen sich auch zur Hyposensibilisierung einer Roggenpollenallergie

In Germany, specific immunotherapy of patients with allergy against rye pollens (Secale cereale) is frequently done with different grass pollen extracts. We reassess the practice of applying rye allergens as single extract or in an individual allergen mixture in the context of the availability of a 5-grass tablet vaccine as speciality based on Anthoxanthum odoratum, Dactylis glomerata, Lolium perenne, Poa pratensis, Phleum pratense. Phylogenetically, Secale cereale shows a strong similarity to the most frequent grasses in Northern and Middle Europe. This similarity applies also to the allergen profile. In 3 studies with 256 grass pollen-allergic patients, the sensitization against rye as well as against other in Northern or Middle Europe frequently found grasses could be shown in all patients. Using sera from 15 grass pollen-allergic patients, it could be shown by immunoblot and ELISA inhibition studies that a 5-grass extract inhibits > 90% of IgE binding to rye allergens. The high cross-immunogenicity between Secale cereale and common grasses is reflected in medical practice in many European countries. Patients with rye allergy are treated with grass pollen extracts. According to the high cross-reactivity between grasses and rye-specific immunotherapy of rye allergy seems to be feasible also with a grass pollen extract. Related to existing data this is independent whether the therapeutic extract includes a single grass, a mixture of 5 grasses or other mixtures.

Severe allergic reactions to guinea pig

BackgroundAllergic sensitization and reactions to guinea pig (Cavia porcellus) have been well documented in laboratory animal handlers, primarily manifesting as rhinitis, conjunctivitis, and asthma. Severe allergic reactions, however, are rare.MethodsWe report two patients with severe allergic reactions following non-occupational exposure to guinea pigs. The first patient, an 11-year-old female, developed ocular, nasal, skin and laryngeal edema symptoms immediately after handling a guinea pig. The second patient, a 24-year-old female, developed symptoms of isolated laryngeal edema after cleaning a guinea pig cage. Percutaneous skin testing, RAST, ELISA and ELISA inhibition testing with guinea pig extract were performed.ResultsBoth patients had IgE-mediated allergy to guinea pig confirmed by ELISA and either RAST or skin testing. ELISA inhibition studies confirmed the specificity of the IgE reactivity to guinea pig.ConclusionSevere IgE-mediated reactions can occur following non-occupational guinea pig exposure. Physicians should be aware of this possibility.

Evaluation of Cross-Reactivity between Holoptelea integrifolia and Parietaria judaica

Background:Holoptelea integrifolia and Parietaria judaica belong to the family Urticaceae, but are geographically distantly located. H. integrifolia is an important pollen allergen of India and sensitizes almost 10% of the atopic population in Delhi. P. judaica, on the other hand, is a very dominant pollen allergen of the Mediterranean region, sensitizing almost 80% of the allergic population. Since both these important pollen allergens belong to the family Urticaceae, the objective of the present study was to assess cross-reactivity between these two pollen allergens from different geographical regions. Methods: Cross-reactivity between these two pollen allergens was assessed on the basis of skin prick tests and ELISA, ELISA inhibition and immunoblot inhibition studies. Results: Out of 44 atopic Indian patients skin prick tested with H. integrifolia extract, 34% were found to be sensitized. All the patients sensitized to H. integrifolia also showed varying degrees of skin positivity to P. judaica pollen extract. ELISA and ELISA inhibition studies suggested strong cross-reactivity between H. integrifolia and P. judaica pollen. Immunoblot inhibition studies revealed that 14-, 16-, 28-, 38-, 42- and 46-kDa proteins are the cross-reactive proteins in H. integrifolia and P. judaica. However, Par j 1, the major allergen of P. judaica, is absent in H. integrifolia pollen. Conclusion:H. integrifolia and P. judaica pollens share cross-reactive as well as unique epitopes. The major allergen of P. judaica, Par j 1, seems to be absent in H. integrifolia pollen allergen.

Recognition of allergenic determinants to cefaclor in patients with cefaclor-induced anaphylaxis

Rationale It has been known that beta-lactam antibiotics such as cefaclor may cause IgE-mediated anaphylactic reaction. However antigenic determinants of these drugs were unclear. The purpose of this study was to identify the chemical structure of antigen recognized by specific IgE antibodies to cefaclor-human serum albumin (HSA) conjugate in sera of anaphylaxis patients. Methods A total of four subjects with anaphylaxis to cefaclor were studied. Cefaclor-HSA conjugate was prepared, and serum specific IgE to cefaclor-HSA conjugate was detected by ELISA. In addition, ELISA inhibition study was done with various concentrations of inhibitors including cefaclor-HSA, free cefaclor, cephalexin, cefadroxil, ampicillin, cefotriaxone, and cefotaxime. Results Three (75%) showed high serum specific IgE to cefaclor-HAS, when positive cut-off was determined from mean + 3 SD of absorbance values of the negative control. ELISA inhibition test with three individual sera showed two different patterns; In patient LS, significant dose dependant inhibitions (up to 92%) were noted with additions of cefaclor-HSA and free cefaclor, and lesser inhibitions (up to 74%) were noted with cephalexin which is sharing with aminobenzyl side chain and two ring structure. minimal inhibitions were noted with other four compounds. In patient KJ and YK, marked dose dependent inhibitions were noted with additions of cefaclor-HSA and free cefaclor, while minimal inhibitions with other five compounds. Conclusion These finding suggest that an entire cefaclor molecule with little tolerance for change at R2 (-cl) was preferentially recognized pattern in patients with anaphylaxis to this drug.

Expression of recombinant Bla g 7 in Pichia pastoris and comparison of IgE reactivity with native counterpart

Rationale Tropomyosins derived from invertebrates are a well-known pan allergen. However, the allergenicity of the recombinant tropomyosins is not coherent. The aim of this study was to compare the IgE-binding reactivities of native and recombinant German cockroach tropomyosins. Methods Native tropomyosin was purified by ammonium sulfate fractionation, hydroxyapatite column chromatography, and gel elution. Recombinant tropomyosin was expressed in Pichia pastoris. The allergenicity of native and recombinant tropomyosin was compared by ELISA inhibition study. Results Native German cockroach tropomyosin showed 17.6% IgE-binding reactivity from German cockroach sensitized sera. Recombinant tropomyosin was produced without fusion protein and the N-terminus was blocked as a native counterpart. Nevertheless, the IgE binding reactivity was comparable to that of native tropomyosin over the concentration range of 1 to 1000 ng/ml concentration in the ELISA inhibition test. Conclusions Recombinant German cockroach tropomyosin that was improved in terms of allergenicity relative to the recombinant protein expressed in Escherichia coli. Other factors, other than the structural differences of native and recombinant proteins, could influence on the various IgE reactivities of tropomyosins.

Parietaria Profilin Shows Only Limited Cross-Reactivity with Birch and Grass Profilins

Background: In patients showing multiple reactivity to seasonal airborne allergens, a skin prick test (SPT) with Parietaria pollen scores frequently negative, suggesting that profilin from Parietaria might not share IgE-binding epitopes with profilin from botanically unrelated airborne allergens. This study investigated the immunologic cross-reactivity between profilins from Parietaria, grass and birch pollen. Methods: 36 patients hypersensitive to birch profilin, Bet v 2, underwent SPT with Parietaria, and IgE to both whole Parietaria pollen and Phleum profilin were sought in their sera by ELISA. In ELISA inhibition studies, IgE reactivity to Phleum profilin was measured before and after absorption of sera from Parietaria reactors with both whole Parietaria pollen extract, and Par j 1/Par j 2 fraction. Further, their IgE reactivity to whole Parietaria pollen was measured before and after absorption with the Par j 1/Par j 2 fraction. Results: All sera showed IgE reactivity to Phleum profilin. Only 14/36 (39%) patients had a positive SPT to Parietaria and 17/36 (47%) showed IgE to Parietaria. Absorption of sera from Parietaria reactors with whole Parietaria extract caused a marked reduction in IgE reactivity to grass profilin; in contrast, absorption of sera with the Par j 1/Par j 2 fraction did not exert any inhibitory effect on IgE reactivity to grass profilin. Absorption of sera with the Par j 1/Par j 2 fraction markedly reduced IgE reactivity to whole Parietaria extract in 8/9 cases. Conclusion: Less than 50% of patients sensitized to birch and grass profilin cross-react to Parietaria profilin. In most cases, cross-reactors are hypersensitive to major, specific Parietaria allergens as well. This findings may be of practical relevance when the prescription of specific immunotherapy is considered.