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Development of ELISA Assays for Measurement of Can f 1 and Can f 3 in Dog Allergen Extracts
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Development of ELISA Assays for Measurement of Can f 1 and Can f 3 in Dog Allergen Extracts Taruna Khurana, PhD, CBER FDA, Philip Young, CBER/FDA, Jay E. Slater, MD; FDA/CBER/OVRR/DBPAP, Silver Spring, MD. RATIONALE: Dog allergen extract is a non-standardized extract with no potency measures. Understanding dog allergen extract potency will enhance the safety and efficacy of this product. Our goal is to develop monoclonal antibody-based sandwich ELISA assays for the measurement of Can f 1 and Can f 3. METHODS: Mouse monoclonal antibodies were generated against natural Can f 1 and dog serum albumin (Can f 3). Anti-Can f 1 and anti-Can f 3 hybridoma supernatants were screened against dog hair and dog epithelium extracts. Purified IgG antibodies were biotinylated for further pairwise screening and optimization. The defined concentrations of capture (nonbiotinylated) and primary (biotinylated) antibodies were used for initial testing of various commercially available dog allergen extracts. RESULTS: Screening of Can f 1 antibodies revealed consistent strong responses with natural protein and dog extracts during indirect ELISA. Three pairs were selected for Can f 1 measurement: 6G1 as a capture antibody (2.5 mg/mL) and 4C3, 7D12, or 9A9 as biotinylated primary antibody (each at 1.0 mg/mL). Anti Can f 3 antibodies exhibit greater variability and less affinity: 10-20 mg/mL of each capture and primary antibody are required for consistent measurement of Can f 3. Further studies are needed to optimize both the assays. CONCLUSIONS: sELISA assays have been developed for determining Can f 1 and Can f 3 contents of non-standardized dog allergen extracts.
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Comparison Between Intradermal Skin Testing and Serum Specific Immunoglobulin E in Detecting Allergic Sensitization in Patients with Negative Skin Prick Tests Denisa Ferastraoaru, M.D.1, Maria Shtessel, M.D.1, Golda Hudes, M.D. PhD2, Gabriele De Vos, M.D.3; 1Montefiore Medical Center, Bronx, NY, 2 Albert Einstein/ Montefiore Medical Center, New York, NY, 3Albert Einstein College of Medicine, Bronx, NY. RATIONALE: Studies show that both skin prick testing (SPT) and serum specific immunoglobulin E (ssIgE) complement each other to determine environmental sensitization. However, it is common practice to only use SPT and intradermal skin testing (IDST), assuming that IDSTwill complement SPT sufficiently. The aim of this study was to compare the capability of detecting allergic sensitization by IDST and ssIgE in patients with negative SPT. METHODS: We retrospectively analyzed 272 SPT and 197 IDST/ssIgE results for 11 environmental allergens done in 25 patients aged 10-65 years who presented to our clinic between January-June 2014 for evaluation of perennial or seasonal occulonasal symptoms. RESULTS: Of the 272 SPT performed, 45 were found positive. IDST and ssIgE testing was done for SPT negative allergens. Of the 197 ssIgE/IDST test pairs, 155 (78%) were IDST-/ssIgE-, 14 (7%) were IDST+/ssIgE+, 21 (11%) were IDST+/ssIgE-, and 7 (4%) were IDST-/ssIgE+. In contrast to ssIgE testing, IDST was stronger in detecting sensitizations to most pollen (tree, grass, ragweed) and cockroach. None of these sensitizations would have been detected using ssIgE testing alone. While ssIgE testing was overall weaker in detecting sensitizations than IDST, ssIgE testing was low positive (0.35-1kU/l) in a few cases despite negative IDST for mugwort pollen, dust mite, mouse and molds. CONCLUSIONS: While overall IDST was stronger than ssIgE testing in diagnosing allergic sensitization when SPT results are negative, for specific allergens ssIgE testing may be superior or equal to IDST. The correlation with clinical allergic symptoms in these patients has yet to be determined.
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A Comparative Analysis of Skin Prick Testing, Specific IgE Levels and Total Nasal Symptom Scores in the Environmental Exposure Unit (EEU) Daniel Adams, BSc1, Lisa Steacy, BSc1, Terry J. Walker, BA1, Barnaby Hobsbawn1, Anne Ellis, MD, MSc, FAAAAI1,2; 1Allergy Research Unit, Kingston General Hospital, Kingston, ON, Canada, 2Departments of Medicine and Biomedical & Molecular Science, Queen’s University, Kingston, ON, Canada. RATIONALE: Skin prick testing(SPT), allergen-specific IgE(sIgE) and Total Nasal Symptom Scores(TNSS) are all useful measurements to help diagnose and assess allergic rhinitis(AR). In addition to diagnosis, these are often used for inclusion criteria in clinical trials of AR. A comparative analysis of these three measurements was performed to evaluate for predictive trends. METHODS: Participants had a history of seasonal AR with a SPT to rye _3mm than negative control. TNSS data were collected during four grass > consecutive 3hr exposures to rye grass pollen in the EEU. Rye grass sIgE measurements were completed at study conclusion. Data were analyzed descriptively. RESULTS: There was a clear association between SPT wheal size and IgE levels. Only 10.0% of participants with a wheal size of 3 or 4mm (n520) _0.35 IgE(kU/L) and 5.0% of these participants had IgE had IgE levels > _10mm _0.70(kU/L). 94.3% of participants with SPT wheal > levels > _0.35 IgE(kU/L) and 89.4% of these (n5141) also had IgE levels > _0.70(kU/L). Those participants with specific individuals had IgE levels > IgE levels of <0.35(kU/L)(n556) developed lower TNSS scores following _10.0(kU/ 2hr of pollen exposure than individuals with sIgE levels of > L)(n573)(Average TNSS scores of 6.5 and 7.9, respectively). Notably, 38.9% of participants with SPT53mm or 4mm and sIgE <0.35 (n518) still achieved a TNSS of 6 within 2hr, however. CONCLUSIONS: SPT may be predictive of an individual’s sIgE. sIgE levels may have some predictive ability for TNSS, but any given SPT or sIgE cut-off will not fully exclude a participant capable of developing a robust TNSS response.
SATURDAY
Abstracts AB57
J ALLERGY CLIN IMMUNOL VOLUME 135, NUMBER 2
Development of ELISA Assays for Measurement of Can f 1 and Can f 3 in Dog Allergen Extracts Taruna Khurana, PhD, CBER FDA, Philip Young, CBER/FDA, Jay E. Slater, MD; FDA/CBER/OVRR/DBPAP, Silver Spring, MD. RATIONALE: Dog allergen extract is a non-standardized extract with no potency measures. Understanding dog allergen extract potency will enhance the safety and efficacy of this product. Our goal is to develop monoclonal antibody-based sandwich ELISA assays for the measurement of Can f 1 and Can f 3. METHODS: Mouse monoclonal antibodies were generated against natural Can f 1 and dog serum albumin (Can f 3). Anti-Can f 1 and anti-Can f 3 hybridoma supernatants were screened against dog hair and dog epithelium extracts. Purified IgG antibodies were biotinylated for further pairwise screening and optimization. The defined concentrations of capture (nonbiotinylated) and primary (biotinylated) antibodies were used for initial testing of various commercially available dog allergen extracts. RESULTS: Screening of Can f 1 antibodies revealed consistent strong responses with natural protein and dog extracts during indirect ELISA. Three pairs were selected for Can f 1 measurement: 6G1 as a capture antibody (2.5 mg/mL) and 4C3, 7D12, or 9A9 as biotinylated primary antibody (each at 1.0 mg/mL). Anti Can f 3 antibodies exhibit greater variability and less affinity: 10-20 mg/mL of each capture and primary antibody are required for consistent measurement of Can f 3. Further studies are needed to optimize both the assays. CONCLUSIONS: sELISA assays have been developed for determining Can f 1 and Can f 3 contents of non-standardized dog allergen extracts.
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Comparison Between Intradermal Skin Testing and Serum Specific Immunoglobulin E in Detecting Allergic Sensitization in Patients with Negative Skin Prick Tests Denisa Ferastraoaru, M.D.1, Maria Shtessel, M.D.1, Golda Hudes, M.D. PhD2, Gabriele De Vos, M.D.3; 1Montefiore Medical Center, Bronx, NY, 2 Albert Einstein/ Montefiore Medical Center, New York, NY, 3Albert Einstein College of Medicine, Bronx, NY. RATIONALE: Studies show that both skin prick testing (SPT) and serum specific immunoglobulin E (ssIgE) complement each other to determine environmental sensitization. However, it is common practice to only use SPT and intradermal skin testing (IDST), assuming that IDSTwill complement SPT sufficiently. The aim of this study was to compare the capability of detecting allergic sensitization by IDST and ssIgE in patients with negative SPT. METHODS: We retrospectively analyzed 272 SPT and 197 IDST/ssIgE results for 11 environmental allergens done in 25 patients aged 10-65 years who presented to our clinic between January-June 2014 for evaluation of perennial or seasonal occulonasal symptoms. RESULTS: Of the 272 SPT performed, 45 were found positive. IDST and ssIgE testing was done for SPT negative allergens. Of the 197 ssIgE/IDST test pairs, 155 (78%) were IDST-/ssIgE-, 14 (7%) were IDST+/ssIgE+, 21 (11%) were IDST+/ssIgE-, and 7 (4%) were IDST-/ssIgE+. In contrast to ssIgE testing, IDST was stronger in detecting sensitizations to most pollen (tree, grass, ragweed) and cockroach. None of these sensitizations would have been detected using ssIgE testing alone. While ssIgE testing was overall weaker in detecting sensitizations than IDST, ssIgE testing was low positive (0.35-1kU/l) in a few cases despite negative IDST for mugwort pollen, dust mite, mouse and molds. CONCLUSIONS: While overall IDST was stronger than ssIgE testing in diagnosing allergic sensitization when SPT results are negative, for specific allergens ssIgE testing may be superior or equal to IDST. The correlation with clinical allergic symptoms in these patients has yet to be determined.
183
A Comparative Analysis of Skin Prick Testing, Specific IgE Levels and Total Nasal Symptom Scores in the Environmental Exposure Unit (EEU) Daniel Adams, BSc1, Lisa Steacy, BSc1, Terry J. Walker, BA1, Barnaby Hobsbawn1, Anne Ellis, MD, MSc, FAAAAI1,2; 1Allergy Research Unit, Kingston General Hospital, Kingston, ON, Canada, 2Departments of Medicine and Biomedical & Molecular Science, Queen’s University, Kingston, ON, Canada. RATIONALE: Skin prick testing(SPT), allergen-specific IgE(sIgE) and Total Nasal Symptom Scores(TNSS) are all useful measurements to help diagnose and assess allergic rhinitis(AR). In addition to diagnosis, these are often used for inclusion criteria in clinical trials of AR. A comparative analysis of these three measurements was performed to evaluate for predictive trends. METHODS: Participants had a history of seasonal AR with a SPT to rye _3mm than negative control. TNSS data were collected during four grass > consecutive 3hr exposures to rye grass pollen in the EEU. Rye grass sIgE measurements were completed at study conclusion. Data were analyzed descriptively. RESULTS: There was a clear association between SPT wheal size and IgE levels. Only 10.0% of participants with a wheal size of 3 or 4mm (n520) _0.35 IgE(kU/L) and 5.0% of these participants had IgE had IgE levels > _10mm _0.70(kU/L). 94.3% of participants with SPT wheal > levels > _0.35 IgE(kU/L) and 89.4% of these (n5141) also had IgE levels > _0.70(kU/L). Those participants with specific individuals had IgE levels > IgE levels of <0.35(kU/L)(n556) developed lower TNSS scores following _10.0(kU/ 2hr of pollen exposure than individuals with sIgE levels of > L)(n573)(Average TNSS scores of 6.5 and 7.9, respectively). Notably, 38.9% of participants with SPT53mm or 4mm and sIgE <0.35 (n518) still achieved a TNSS of 6 within 2hr, however. CONCLUSIONS: SPT may be predictive of an individual’s sIgE. sIgE levels may have some predictive ability for TNSS, but any given SPT or sIgE cut-off will not fully exclude a participant capable of developing a robust TNSS response.
SATURDAY
Abstracts AB57
J ALLERGY CLIN IMMUNOL VOLUME 135, NUMBER 2