Complex diagnosis of IgE mediated allergy by in vivo and in vitro methods (original) (raw)
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Journal of Allergy and Clinical Immunology, 1998
Current diagnostics for grass pollen allergy are composed of mixtures of pollen of different grass species. Their complex composition hampers accurate standardization. The aim of the study was to investigate whether mixtures of grass pollen extracts can be replaced by a single pollen species and whether a single pollen species can be replaced by a limited number of purified natural or recombinant major allergens. Sera (n = 800) were selected on the basis of a general suspicion for inhalant allergy and tested in a RAST for IgE reactivity with pollen from 17 different grass species. Cross-reactivity of IgE responses was studied by means of RAST inhibition. Sera with positive test results for grass pollen were tested in a RAST for natural Lol p 1 and Lol p 5 and recombinant Phl p 1 and Phl p 5. Specific IgE antibodies against one or more of the 17 pollen species were detected in 209 of 800 sera (26.1%). The highest responses were observed against Poa pratensis followed by Festuca rubra, Phleum pratense, and Dactylis glomerata. IgE responses were clearly lower (approximately by a factor of 5) against only three species (Phragmites communis, Cynodon dactylon, and Zea mays). With the exception of a few low-responder sera, no sera were found to have negative test results to the high responder species and positive results to any of the other species. Sera with positive test results for grass pollen (n = 154) were tested with purified Lol p 1 and Lol p 5. IgE anti-Lol p 1 and Lol p 5 accounted for an average of 81% +/- 7% of total anti-grass pollen IgE. For 14 sera (all with low anti-grass pollen IgE titers), a RAST with purified allergens resulted in a false-negative diagnosis for grass pollen allergy. With recombinant Phl p 1 and Phl p 5, the mean IgE reactivity was 57% +/- 6% of the anti-grass pollen IgE response (n = 141), with 13 false-negative results. One grass species is sufficient for in vitro diagnosis of grass pollen allergy. With purified natural Lol p 1 and Lol p 5, greater than 90% of grass-positive sera is detected. Around 80% of the IgE response to grass pollen is directed to these major allergens. Recombinant allergens, produced in Escherichia coli, did not equal the IgE-binding capacity of their natural counterparts.
European annals of allergy and clinical immunology, 2023
Background. In the diagnostic work up of allergy, determining allergen component-specific immunoglobulin E (IgE) is important for diagnosis, prognosis and choice of treatment. The purpose of this study was to evaluate the performance of the immunoblotting assay (Euroline) in detection of IgE antibodies against timothy grass and birch pollen allergen components compared to fluorescent enzyme assay (ImmunoCAP, Phadia 250). Methods. A total of 128 serum samples from patients allergic to timothy grass and birch pollen were analyzed. The levels of IgE antibodies to timothy grass and birch pollen were measured using Euroline DPA-Dx pollen 1 and ImmunoCAP assay. The two methods were then compared on binary (positive vs negative), semi-quantitative (IgE classes) and quantitative (concentration) levels. The two methods were also compared to results from skin prick testing. Results. The Euroline method showed a positive percentage agreement of 93% and negative percentage agreement of 94% with an overall accuracy of 94% when compared to Im-munoCAP. Kappa analysis showed moderate strength of agreement between the methods in determining IgE classes for 7/11 components tested. All components showed a positive correlation when analysed using Spearman's rank correlation. Conclusions. Overall, we found that there is good correlation between the Euroline and ImmunoCAP methods in measuring IgE sensitization. Impact statement Use of the Euroline method may be recommended as an alternative routine clinical allergy diagnostic work up to determine sensitization profiles to timothy grass and birch pollen.
Appropriateness of reducing the number of pollen allergens to three
World Allergy Organization Journal, 2007
Background: Skin prick testing (SPT) is an essential tool in the diagnosis of allergic disorders. The optimal number and type of allergens used in different settings remains undefined. We aim to describe SPT in our clinical practice and propose the appropriateness of reducing the number of pollen allergens to three. The aim is to improve cost-effectiveness and reducing time spent on allergen testing, particularly in the community setting. Methods: Consecutive patients who attended the private rooms of 2 immunologists and the allergy/immunology clinic in a tertiary referral hospital who required skin prick testing were evaluated from June 2006 to November 2006. Statistical analysis was undertaken with the use of Pearson`s Chi-square and Fisher`s Exact test to assess for significance. Multivariate analysis was also performed. Results: There were a total of 273 skin prick test sets performed. There was no significant difference between the rates of SPT positivity with common pollen allergens between the two clinics. A positive SPT to Perennial Ryegass (Lolium perenne), Timothy (Phleum patense) or Bermuda grass (Cynodon dactylon) had a sensitivity of 100% to Bent or Orchard grass (Dactylis glomerate), with sensitivities of 97%, 96.3% and 94.8% to English Plantain (Plantago lanceolata), Bahia grass (Paspalum notatum)and Dock/Sorrel (Rumex sp) respectively. Use of Perennial Ryegrass, Timothy or Bermuda grass also detected tree pollen sensitivity with sensitivities in Birch mix (Betula sp) of 92.6%, Acacia (96.4%), Casuarina (89.1%), Platanus sp (92.3%) and Privet(Ligustrum sp) (88.6%). Conclusion: The use of 3 common grass pollen allergens in SPTs (Lolium perenne, Phleum pratense and Cynodon dactylon) detected 90% of atopic individuals with sensitivity to many pollen types. This information may be useful in defining the most appropriate allergens to determine pollen hypersensitivity in community settings.
Different allergenic activity of grass pollen allergens revealed by skin testing
European Journal of Clinical Investigation, 2008
ABSTRACTBackground Grass pollen is one of the most important allergen sources. The aim of this study was to compare the in vivo allergenic activity of two recently characterized major grass pollen allergens, Phl p 4 and Phl p 13, with three established major grass pollen allergens, Phl p 1, Phl p 2 and Phl p 5 as a basis for the formulation of a grass pollen allergy vaccine based on purified allergens.Material and methods Eighty‐two grass pollen allergic patients were skin prick tested with serial dilutions of approximately equimolar concentrations of the purified allergens in a double‐blind study.Results Phl p 4 and Phl p 13 were identified as major grass pollen allergens according to IgE binding frequency (Phl p 4: 85%; Phl p 13: 56%), but exhibited a five to nine‐fold lower allergenic skin reactivity compared to Phl p 1, Phl p 2 or Phl p 5.Conclusion Our results indicate that Phl p 4 and Phl p 13 are not essential components for a therapeutic grass pollen vaccine and underpin...
Journal of Allergy and Clinical Immunology, 1996
Background: Complementaly DNAs coding for the major timothy grass pollen (Phleum pratense) allergens Phl p 1, Phl p 2, and Phl p 5 and birch profilin were isolated, expressed as recombinant nonfusion proteins in Escherichia coli, and purified. Objective: In this study the in vitro IgE-binding capacity of recombinant Phl p 1, Phl p 2, Phl p 5, and birch profilin and their IgE recognition frequencies were investigated by using sera from different populations. Methods: One hundred eighty-three sera from patients allergic to grass pollen were obtained from different populations in Europe, Japan, and Canada. The sera were selected according to clinical criteria, skin testing, and RAST (CAP syswm: Pharmacia. Uppsala. Sweden ~ and then tested for IgE reactivity with natural and purified recombinant timothy grass pollen allergens by ELISA and Western blot. Results: Most (94.5%) of the patients allergic to grass pollen could be diagnosed with a combination of recombinant Phl p 1. Phl p 2, Phl p 5, and profilin by means of ELISA. Sera that did not react with the recombinant allergens contained low" levels of timothy grass pollenspecific IgE. Although considerable variability in IgE recognition frequency of the recombinant allergens was observed in certain populations, a good correlation was found between natural timothy CAP results and the combination of recombinant allergens in all 183 tested sera (r-0.87). Conclusions: Despite considerable variability in the IgE recognition frequency, purified recombinant timothy grass pollen allergens (Phl p 1. Phl p 2. Phl p 51 and profilin permitted successful in vitro diagnosis of grass pollen allergy in 94.5% of allergic individuals from different populations. The addition of other recombinant allergens (e.g., recombinant Phl p 4) would only slightly improve the in vitro test sensitivity.
Measurement of IgE to pollen allergen components is helpful in selecting patients for immunotherapy
Background: Pollen allergy still represents an important cause of allergic morbidity worldwide. Accurate diagnostic methods are important to determine the disease-causing allergen. Objective: To describe the sensitization patterns of patients with spring pollinosis and to make a real-life evaluation of the usefulness of a predetermined IgE molecular profile compared with other sensitization methods for choosing the composition of immunotherapy. Methods: One hundred seventy-five patients with a diagnosis of spring pollinosis completed a skin prick test study with Olea europaea, Phleum pratense, palm profilin, and peach peel and an in vitro study of the application of a specific recombinant IgE protocol (nOle e1, rPhl p1-5b, rPhl p12, rPhl p7, and rPru p3). Immunotherapy using the 2 methods was compared. Results: A high sensitization to nOle e1 and rPhl p1-5b was found. Profilin, polcalcin, and lipid transfer proteins seemed irrelevant for the differential diagnosis of olea and grass pollen sensitization in the most southern area of the Iberian Peninsula. Application of the component resolved the diagnosis, and the choice of immunotherapy was changed in more than 50% of patients. Conclusion: These results support the necessity of the habitual use of this kind of protocol in routine allergologic practice.
Clinical & Experimental Allergy, 2011
Background Grass pollens are major triggers of allergic rhinitis and asthma, but the immunological relationships between pollen allergens of the subtropical Bahia grass, Paspalum notatum, and temperate grasses are unresolved. Objective To assess serum IgE cross-reactivity between subtropical P. notatum and temperate Lolium perenne (Ryegrass) pollen allergens. Methods Serum IgE reactivities of grass pollen-allergic patients with P. notatum, L. perenne and Cynodon dactylon (Bermuda grass) pollen extracts and their respective purified group 1 allergens, Pas n 1, Lol p 1 and Cyn d 1, were compared by immunoblotting, ELISA and basophil activation. Results In a cohort of 51 patients from a temperate region, a high frequency of IgE reactivity with each grass pollen was detected, but reactivity with L. perenne pollen was substantially greater than with P. notatum and C. dactylon pollen. Similarly, serum IgE reactivity with Lol p 1 was greater than with Pas n 1 or Cyn d 1. For seven of eight sera studied in detail, asymmetric serum IgE cross-reactivity was observed; L. perenne pollen inhibited IgE reactivity with P. notatum pollen but not the converse, and IgE reactivity with Pas n 1 was inhibited by Lol p 1 but IgE reactivity with Lol p 1 was not inhibited by Pas n 1 or Cyn d 1. Importantly, P. notatum pollen and Pas n 1 activated basophils in grass pollen-allergic patients from a temperate region, although stimulation was greater by pollen of L. perenne than P. notatum or C. dactylon, and by Lol p 1 than Pas n 1 or Cyn d 1. In contrast, a cohort of 47 patients from a subtropical region showed similar IgE reactivity with P. notatum and L. perenne pollen, and reciprocal cross-inhibition of IgE reactivity between L. perenne and P. notatum. Conclusions Pollen allergens of the subtropical P. notatum, including Pas n 1, show clinically relevant IgE cross-reactivity with pollen allergens of L. perenne but also species-specific IgE reactivity.
Environmental Research, 2009
Trees are considered producers of allergenic pollen. The aims of this work were to characterize the aerobiology of the Platanus, Acer, Salix, Quercus, Betula and Populus pollen, linking it with monthly emergency hospital admissions and to identify the different reactivity levels in sensitized patients. This information would be of great importance to evaluate the convenience of changing the inventory of pollen producer trees related to the risk of allergenic reactions. The study was conducted in Porto, Portugal, from 2005 to 2007. Airborne pollen was sampled using a Hirst-type volumetric trap. The antigenic and allergenic properties of Acer negundo, Betula pendula, Platanus occidentalis, Populus hybrida, Quercus robur and Salix babylonica pollen, collected in public gardens or sidewalks, were investigated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunological techniques using polysensitized-patient sera. Monthly hospital admissions of asthma or dyspnea related with respiratory diseases were obtained from the Emergency Room database of Hospital Geral de Santo António. Tree pollen and hospital admissions were positively correlated. Tree pollen peaked in March which coincides with the hospital admissions maximum. The highest binding affinity was observed with A. negundo, S. babylonica and P. occidentalis pollen extracts and the lowest with P. hybrida. Consistently, Acer and Platanus maximum airborne pollen concentrations were observed during March attaining levels considered moderate to high risk for allergenic reactions. Prominent bands with approximately 71, 35, 31, 22, 19, 16, 14, 13 and 11 kDa were revealed. A 52 kDa band was shared by all analyzed sera. High levels of airborne pollen and emergency hospital admissions were related. High binding affinity of specific IgE to pollen extracts of the most abundant tree pollen present in the atmosphere was observed. Patient sera revealed multiple similar allergenic bands shared by the different extracts. This multidisciplinary approach is useful in day-to-day medical practice to help in diagnostic, therapeutic and allergy alerting system adjusting.
Biological Chemistry Hoppe-Seyler, 1993
The identication and characterization of allergenic components is a vital step towards improving diagnosis and therapy. Members of the grass family (Poaceae) reveal a high cross-reactivity among each other caused by the close phylogenetical relationship. In order to investigate the variability between allergenic components, we studied the allergen grass group V, one of the major allergens. Pollen extracts of 4 different tribes (timothy grass (Phlewn pratense)-Agrostidae, perennial rye grass (Lolium perenne)-Festuceae, meadow velvet (Holcus lanatus)-Aveneae, and rye (Secale cereale)-Triticeae) of the Festucoideae subfamily were separated by 2D PAGE and investigated by immunoblotting using patients' poolserum and monoclonal antibodies (raised against group V allergens of timothy grass pollen). The antibodies identify different allergens in the four grass species.The components vary from 30-50 kDa and pi 4.8-7.0.The eight NH 2-terminal amino acids were determined and indicated high similarities between the different components.These results cast doubt on the suitability of classifying allergens into groups based only on their molecular mass, isoelectric point and N-terminal sequence analysis. It suggests to classify allergens according to their IgE-reactive epi topes.