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IgE Against Bed Bug (Cimex lectularius) Allergens Are Common Among Adults Bitten By Bed Bugs
Abstracts AB89
J ALLERGY CLIN IMMUNOL VOLUME 129, NUMBER 2
Allergen Specificity Of 3 Scfv Antibodies Developed For A Multiplex Assay Of Blattella Germanica Extract Potency T. Khurana1, M. Collison1, F. Chew2, K. Jeong3, J. Slater1; 1FDA, Bethesda, MD, 2National University of Singapore, Singapore, SINGAPORE, 3 Institute of Tropical Medicine, Seoul, KOREA, REPUBLIC OF DEMOCRATIC PEOPLE’S. RATIONALE: We are developing a multiplex antibody-based assay for allergen potency. In previous work, the overall allergenicity of German cockroach (GCr) allergen extracts did not correlate with content of any known specific allergens. We have developed 14 avian scFv antibodies to GCr. In this study we begin to identify their target proteins. METHODS: Targets of GCr-specific antibodies 2A1, 6A2 and 6A3, isolated by direct immunoprecipitation, were analyzed by SDS-PAGE and mass spectrometry (MS). The cDNA sequences of the target proteins of 2A1 and 6A2 were expressed in E. coli and purified using affinity chromatography. Allergenicity of these expressed proteins was determined by screening a GCr-allergic human serum pool (S1-Cr) for presence of specific IgE antibodies. Thermal stability of these proteins was determined by antibody binding following 1 min incubations at 40-808C. RESULTS: The target proteins of antibodies 2A1 and 6A2 have an apparent mass of ;33 kDa and ;78 kDa respectively. BLAST analyses of peptide sequences obtained from MS indicate the following closest matches: 2A1: Per a 3 homologue (isoform 2, GU086323); 6A2: Bla g 7 (AF260897); 6A3: vitellogenin (AJ0005115). Targets of the other 11 antibodies remain unidentified. ELISA using S1-Cr indicates the presence of specific IgE for Per a 3 homologue and Bla g 7. Per a 3 homologue appears to be the only heat stable protein in GCr extract. CONCLUSIONS: We have identified 3 specific target allergens in GCr: vitellogenin, Bla g 7, and a heat stable Per a 3 homologue. IgE Against Bed Bug (Cimex lectularius) Allergens Are Common Among Adults Bitten By Bed Bugs J. B. Price1, A. Divjan2, G. A. Freyer2, W. R. Montfort3, M. S. Perzanowski2; 1Department of Pediatrics, College of Physicians and Surgeons, Columbia University, New York, NY, 2Department of Environmental Health Sciences, Mailman School of Public Health, Columbia University, New York, NY, 3Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ. RATIONALE: Cimex lectularius, the bed bug, has had a resurgence in New York City (NYC). The potential health implications of this increase in exposure are unknown. Our goal was to develop assays for measuring IgE antibodies against crude C. lectularius extract and a salivary protein Cimex nitrophorin (cNP) in order to determine the prevalence of sensitization to bed bug allergens among adults with a report of bed bug bites. METHODS: We recruited 30 adult subjects in NYC who reported being bitten by bed bugs within the past year and having an itchy, raised bump in response. Serum drawn from qualified subjects was used in the development of an immunoassay using the ImmunoCAPÒ system. _0.1 IU/ml) RESULTS: Seventeen (57%) subjects had detectable IgE (> against the crude C. lectularius extract. Of those seventeen, nine subjects had detectable IgE against recombinant cNP. All subjects with IgE against cNP had measureable IgE against C. lectularius extract. Mean total IgE was higher among subjects with, as compared to subjects without IgE against C. lectularius extract (117 vs. 28.8 IU/ml, P 50.002) and cNP (125 vs. 47.6 IU/ml, P50.057). CONCLUSION: We developed assays for measuring IgE antibodies against C. lectularius extract and cNP. IgE antibodies to the crude extract and cNP were common among adults who reported being bitten by bed bugs. The dramatic rise in human exposure to bed bugs in NYC and the demonstration of an IgE response to allergens from C. lectularius potentially has important public health implications of bed bug exposure that should be further investigated.
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Assessment of Allergenicity of GMO Maize Pollen H. Steinman; Food & Allergy Consulting & Testing Services, Milnerton, Cape Town, SOUTH AFRICA. RATIONALE: Farmers in a South African maize-growing area, Hartswater, Northern Cape Province, reported increased allergic symptoms as a result of contact with pollen of a specific GMO maize variety. METHODS: Forty five subjects previously assessed and diagnosed with allergic rhinitis, asthma and chronic sinusitis by two Hartswater physicians were recruited to the study. Detailed questionnaires were completed. Skin prick tests (SPT) for maize pollen from 3 GMO varieties and the 3 isolines, a positive and negative control were performed on all subjects. Serum IgE for maize pollen, a variety of grasses and moulds, olive tree, house dust mites, and CCD was evaluated. Patients serum was used to search for novel or up-regulated allergens by immunoblot. RESULTS: Thirty six patients (80%) were SPT positive for at least one variety of maize pollen. Nineteen subjects were SPT positive for all six maize varieties. Fifteen subjects were shown to have maize pollen specific IgE >0.35 kU/L, of which 6 >3.0 kU/L. SPT was significantly more positive for a specific GMO variety in 6 patients. SPT resulted in anaphylaxis in 4 subjects requiring reversal with intramuscular adrenaline. Immunoblot analysis is proceeding. CONCLUSIONS: Maize pollen is a common cause of hypersensitivity in the Hartswater region. Serum specific IgE determination for maize pollen does not appear to be useful in determining patients allergic to maize pollen suggesting that a maize pollen protein is not adequately represented in serum specific IgE assay. Some individuals appear to be more sensitised (reported history and SPT) to a specific GMO variety.
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Clinical Usefulness Of Diagnosis By Molecular Components In Mite Allergy F. Rodriguez Fernandez1, M. Lopez Hoyos2, P. Munoz Cacho3, C. Barbeitio1, G. Perdomo Gutierrez1, A. Galan Nieto4, M. Arina Sanchez4; 1 Hospital Marques de Valdecilla, Allergy Service, Santander, SPAIN, 2 Hospital Marques de Valdecilla, Immunology Service, Santander, SPAIN, 3Servicio Cntabro de Salud, Santander, SPAIN, 4ALK-Abello, S.A., Madrid, SPAIN. RATIONALE: In patients allergic to mites IgE antibody test with a natural allergen extract does not reveal the nature of the sensitizing molecules. Polisensitization to House Dust Mites (HDM) and Storage Mites (SM) is frequently observed. The aim of this study is to define whether determination of molecular patterns of sensitization is useful for optimizing the diagnosis of these patients. METHODS: 101 patients attending our outpatient clinic suffering rhinoconjunctivitis and/or asthma sensitized to mites were included. Skin prick test (SPT, ALK-Abello) and specific IgE (ImmunoCAP) to the HDM Dermatophaghoides pteronyssinus and to the SM Lepidoglyphus destructor were done. In parallel, IgE to a panel of allergen components including Der p1, Der p2, Der f1, Der f2, Lep d2 and Der p10 (mite tropomyosin) were performed in the Advia Centaur platform. RESULTS: All subjects showed positive SPT and 99% positive IgE to D. pteronyssinus. In these patients 81.2% sensitization to Der p1 and 93.1% to Der p2 was found. SPT and specific IgE to L. destructor was positive in 84.2% and 85.1% respectively, although only 42.6% had sensitization to Lep d2. Positivity to Derp 10 was found in only 2% of patients. CONCLUSIONS: In mite allergy the molecular diagnosis does not always coincide with the diagnosis performed with natural extracts, especially in SM. We consider important to know the molecular components responsible for the sensitization, in order to provide a more accurate diagnosis and etiological treatment to our patients.
SUNDAY
336
J ALLERGY CLIN IMMUNOL VOLUME 129, NUMBER 2
Allergen Specificity Of 3 Scfv Antibodies Developed For A Multiplex Assay Of Blattella Germanica Extract Potency T. Khurana1, M. Collison1, F. Chew2, K. Jeong3, J. Slater1; 1FDA, Bethesda, MD, 2National University of Singapore, Singapore, SINGAPORE, 3 Institute of Tropical Medicine, Seoul, KOREA, REPUBLIC OF DEMOCRATIC PEOPLE’S. RATIONALE: We are developing a multiplex antibody-based assay for allergen potency. In previous work, the overall allergenicity of German cockroach (GCr) allergen extracts did not correlate with content of any known specific allergens. We have developed 14 avian scFv antibodies to GCr. In this study we begin to identify their target proteins. METHODS: Targets of GCr-specific antibodies 2A1, 6A2 and 6A3, isolated by direct immunoprecipitation, were analyzed by SDS-PAGE and mass spectrometry (MS). The cDNA sequences of the target proteins of 2A1 and 6A2 were expressed in E. coli and purified using affinity chromatography. Allergenicity of these expressed proteins was determined by screening a GCr-allergic human serum pool (S1-Cr) for presence of specific IgE antibodies. Thermal stability of these proteins was determined by antibody binding following 1 min incubations at 40-808C. RESULTS: The target proteins of antibodies 2A1 and 6A2 have an apparent mass of ;33 kDa and ;78 kDa respectively. BLAST analyses of peptide sequences obtained from MS indicate the following closest matches: 2A1: Per a 3 homologue (isoform 2, GU086323); 6A2: Bla g 7 (AF260897); 6A3: vitellogenin (AJ0005115). Targets of the other 11 antibodies remain unidentified. ELISA using S1-Cr indicates the presence of specific IgE for Per a 3 homologue and Bla g 7. Per a 3 homologue appears to be the only heat stable protein in GCr extract. CONCLUSIONS: We have identified 3 specific target allergens in GCr: vitellogenin, Bla g 7, and a heat stable Per a 3 homologue. IgE Against Bed Bug (Cimex lectularius) Allergens Are Common Among Adults Bitten By Bed Bugs J. B. Price1, A. Divjan2, G. A. Freyer2, W. R. Montfort3, M. S. Perzanowski2; 1Department of Pediatrics, College of Physicians and Surgeons, Columbia University, New York, NY, 2Department of Environmental Health Sciences, Mailman School of Public Health, Columbia University, New York, NY, 3Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ. RATIONALE: Cimex lectularius, the bed bug, has had a resurgence in New York City (NYC). The potential health implications of this increase in exposure are unknown. Our goal was to develop assays for measuring IgE antibodies against crude C. lectularius extract and a salivary protein Cimex nitrophorin (cNP) in order to determine the prevalence of sensitization to bed bug allergens among adults with a report of bed bug bites. METHODS: We recruited 30 adult subjects in NYC who reported being bitten by bed bugs within the past year and having an itchy, raised bump in response. Serum drawn from qualified subjects was used in the development of an immunoassay using the ImmunoCAPÒ system. _0.1 IU/ml) RESULTS: Seventeen (57%) subjects had detectable IgE (> against the crude C. lectularius extract. Of those seventeen, nine subjects had detectable IgE against recombinant cNP. All subjects with IgE against cNP had measureable IgE against C. lectularius extract. Mean total IgE was higher among subjects with, as compared to subjects without IgE against C. lectularius extract (117 vs. 28.8 IU/ml, P 50.002) and cNP (125 vs. 47.6 IU/ml, P50.057). CONCLUSION: We developed assays for measuring IgE antibodies against C. lectularius extract and cNP. IgE antibodies to the crude extract and cNP were common among adults who reported being bitten by bed bugs. The dramatic rise in human exposure to bed bugs in NYC and the demonstration of an IgE response to allergens from C. lectularius potentially has important public health implications of bed bug exposure that should be further investigated.
337
338
Assessment of Allergenicity of GMO Maize Pollen H. Steinman; Food & Allergy Consulting & Testing Services, Milnerton, Cape Town, SOUTH AFRICA. RATIONALE: Farmers in a South African maize-growing area, Hartswater, Northern Cape Province, reported increased allergic symptoms as a result of contact with pollen of a specific GMO maize variety. METHODS: Forty five subjects previously assessed and diagnosed with allergic rhinitis, asthma and chronic sinusitis by two Hartswater physicians were recruited to the study. Detailed questionnaires were completed. Skin prick tests (SPT) for maize pollen from 3 GMO varieties and the 3 isolines, a positive and negative control were performed on all subjects. Serum IgE for maize pollen, a variety of grasses and moulds, olive tree, house dust mites, and CCD was evaluated. Patients serum was used to search for novel or up-regulated allergens by immunoblot. RESULTS: Thirty six patients (80%) were SPT positive for at least one variety of maize pollen. Nineteen subjects were SPT positive for all six maize varieties. Fifteen subjects were shown to have maize pollen specific IgE >0.35 kU/L, of which 6 >3.0 kU/L. SPT was significantly more positive for a specific GMO variety in 6 patients. SPT resulted in anaphylaxis in 4 subjects requiring reversal with intramuscular adrenaline. Immunoblot analysis is proceeding. CONCLUSIONS: Maize pollen is a common cause of hypersensitivity in the Hartswater region. Serum specific IgE determination for maize pollen does not appear to be useful in determining patients allergic to maize pollen suggesting that a maize pollen protein is not adequately represented in serum specific IgE assay. Some individuals appear to be more sensitised (reported history and SPT) to a specific GMO variety.
339
Clinical Usefulness Of Diagnosis By Molecular Components In Mite Allergy F. Rodriguez Fernandez1, M. Lopez Hoyos2, P. Munoz Cacho3, C. Barbeitio1, G. Perdomo Gutierrez1, A. Galan Nieto4, M. Arina Sanchez4; 1 Hospital Marques de Valdecilla, Allergy Service, Santander, SPAIN, 2 Hospital Marques de Valdecilla, Immunology Service, Santander, SPAIN, 3Servicio Cntabro de Salud, Santander, SPAIN, 4ALK-Abello, S.A., Madrid, SPAIN. RATIONALE: In patients allergic to mites IgE antibody test with a natural allergen extract does not reveal the nature of the sensitizing molecules. Polisensitization to House Dust Mites (HDM) and Storage Mites (SM) is frequently observed. The aim of this study is to define whether determination of molecular patterns of sensitization is useful for optimizing the diagnosis of these patients. METHODS: 101 patients attending our outpatient clinic suffering rhinoconjunctivitis and/or asthma sensitized to mites were included. Skin prick test (SPT, ALK-Abello) and specific IgE (ImmunoCAP) to the HDM Dermatophaghoides pteronyssinus and to the SM Lepidoglyphus destructor were done. In parallel, IgE to a panel of allergen components including Der p1, Der p2, Der f1, Der f2, Lep d2 and Der p10 (mite tropomyosin) were performed in the Advia Centaur platform. RESULTS: All subjects showed positive SPT and 99% positive IgE to D. pteronyssinus. In these patients 81.2% sensitization to Der p1 and 93.1% to Der p2 was found. SPT and specific IgE to L. destructor was positive in 84.2% and 85.1% respectively, although only 42.6% had sensitization to Lep d2. Positivity to Derp 10 was found in only 2% of patients. CONCLUSIONS: In mite allergy the molecular diagnosis does not always coincide with the diagnosis performed with natural extracts, especially in SM. We consider important to know the molecular components responsible for the sensitization, in order to provide a more accurate diagnosis and etiological treatment to our patients.
SUNDAY
336