Multiplex Array for Detection of Airborne Allergen Levels

S230 Abstracts

887

Der f 22: A Novel Allergen From Dermatophagoides farinae That Is Encoded By A Gene Paralogous To Der f 2 F. T. Chew, K. Reginald, C. L. Tan, T. C. Ong, K. N. Wong, Y. S. Tiong, L. Y. Yit, H. S. Shang; National Unversity of Singapore, Singapore, Singapore. RATIONALE: We isolated a cDNA clone from a Dermatophagoides farinae cDNA library which showed 32% amino acid sequence identity to Der f 2. METHODS: To confirm the presence of a gene encoding this protein in the mite genome, we isolated its genomic DNA and performed a Southern blot to compare its loci and copy number relative to the gene encoding for Der f 2. The allergenicity and biochemical property of these two allergens were also crossed compared. RESULTS: The novel allergen was named Der f 22. The full length cDNA sequence coded for 155 amino acids, with a 20 amino acid signal peptide, and 6 cysteine residues. Genomic DNA analysis showed that the gene encoding Der f 22 had one intron flanking two exons, sharing the same organization as the Der f 2 gene. Nevertheless, the position of their introns varied, and both single copy genes were found to be on different loci of the genome. Sera from 50% of dust mite sensitized individuals showed IgE binding to Der f 22 but no IgE cross-reactivity to Der f 2. Both allergens however were localized to the gut region of Dermatophagoides farinae sections, and showed similar dose response binding to cholesterol. CONCLUSIONS: The low sequence identity but potential structural and functional similarities between Der f 22 and Der f 2 suggest that the genes encoding these allergens may be paralogous. The antigens were both allergenic but not cross-reactive.

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TUESDAY

Purification of Serine-protease Free nDer p 1 by Affinity Chromatography S. Wuenschmann1, M. Himly2, L. D. Vailes1, R. Maltais1, M. D. Chapman1; 1INDOOR Biotechnologies Inc., Charlottesville, VA, 2Dept. of Molecular Biology, University of Salzburg, Salzburg, Austria. RATIONALE: Natural Der p 1 purified from Dermatophagoides pteronyssinus by mAb affinity chromatography has been reported to contain trace amounts of serine proteases as detected by proteolytic assays. Our goal was to produce a serine-protease free nDer p 1 with high cysteine protease activity and to characterize D. pteronyssinus serine proteases. METHODS: Natural Der p 1 was purified from extracts of D. pteronyssinus house dust mites by mAb affinity chromatography and by a combination of Benzamidine and mAb affinity chromatography. Proteolytic activity was assessed in both nDer p 1 samples and in Benzamidine-bound proteins. Benzamidine-eluted proteins were further characterized by SDSPAGE and identified by mass spectrometry. RESULTS: Proteolytic assays showed that serine protease activity, susceptible to inhibition with AEBSF (serine protease inhibitor), is present in mAb-affinity purified natural Der p 1 and in Benzamidine-bound proteins. The addition of Benzamidine chromatography in the purification process removed the serine-protease activity, producing natural Der p1 exhibiting cysteine-protease activity only. SDS PAGE of Benzamidinebound proteins showed a major band at 30kD, which was identified as Der p 3 by MS-peptide mapping and sequencing. CONCLUSIONS: Trace amounts of serine proteases in affinity-purified nDer p1, attributable to nDer p3, were successfully removed by an additional purification step using a Benzamidine pre-column. This improved nDer p 1 contains cysteine-protease activity and is serine-protease free.

J ALLERGY CLIN IMMUNOL FEBRUARY 2009

889

The Effects of Mailing on In vivo and In vitro Potencies of Standardized Timothy Grass Extract M. Moore1, M. Tucker2, T. Grier3, D. LeFevre3, J. Quinn1; 1Wilford Hall Medical Center, Lackland AFB, TX, 2Naval Medical Center, San Diego, CA, 3Greer Laboratories, Lenoir, NC. RATIONALE: Allergen extracts can degrade when exposed to temperatures significantly beyond the optimum storage recommendation of 48 C. Many allergen extracts are mailed to their final destination, especially within the context of the U.S. Military. We evaluated the effect of summer mailing on Timothy grass extract concentration and in vivo potency. METHODS:1:1 v/v and 1:10 v/v dilutions of 100,000 BAU/ml standardized Timothy grass extract were mailed round-trip between San Antonio, Texas and Phoenix, Arizona during August 2007. The in-transit temperatures were recorded with a portable temperature logger. Following mailing of the extracts, we performed in vitro (ELISA inhibition) and in vivo potency analyses (IDEAL50 on 3 Timothy grass sensitive patients). RESULTS: Extract exposure temperatures were measured above 208 C for 11 days and 308 C for 6 hours during the standard mailing with weather temperatures exceeding 388 C. ELISA inhibition results for the control samples, 1:1 v/v and 1:10 v/v, were 97,900 and 10,580 BAU/ml respectively. The mailed extracts, 1:1 v/v and 1:10 v/v, measured 96,800 and 7,830 BAU/ml respectively. These measurements fell within the current FDA lot release limits (76-132%) and stability limits (50-200%) relative to the standardized reference. IDEAL50 of the control and mailed extracts were 12.98 vs.12.28, 12.66 vs. 12.32, and 11.97 vs. 11.7 for the 3 patients. These differences were not statistically significant. CONCLUSIONS: Mailing of Timothy grass extract produced no significant reductions of in vitro relative potencies or in vivo skin test reactivity in 3 sensitive patients.

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Multiplex Array for Detection of Airborne Allergen Levels A. Heisler, F. Pacheco, J. Portnoy, C. Barnes; Children’s Mercy Hospital, Kansas City, MO. RATIONALE: Since current techniques for detecting allergen levels are relatively time consuming and labor intensive, we examined the practicality of developing a multiplex array to measure multiple outdoor allergen levels simultaneously. This was done by developing the ragweed portion of the array to examine its efficiency and accuracy compared to standard methods of allergen detection. The multiplex array could be developed because of a platform technology manufactured by the Luminex Corporation which allows for the detection of up to 100 different analytes simultaneously. METHODS: Carboxylated fluorescent microspheres purchased from the Luminex Corporation were coupled to rabbit anti ragweed antibodies. These antibody coupled microspheres, along with air samples collected using an Omni 3000, were added to wells of a 96 well filter plate. After ragweed from the air samples attached to the antibody coated microspheres, the relative amount of ragweed bound to the microspheres was measured on a detection system manufactured by the Luminex Corporation. A standard curve was developed to determine the concentration of ragweed in the air sample. RESULTS: Ragweed concentrations for 45 different days were measured. Concentrations determined from the Luminex analyzer were consistent with results obtained from visual enumeration of airborne ragweed pollen grains. Additionally, the results displayed early high airborne allergen levels not shown in the traditional method of allergen detection. This is consistent with previous reports of similar findings. CONCLUSIONS: Development of the ragweed portion of the array showed that a multiplex array for detection of airborne allergen levels would be more efficient, and possibly more accurate, than current methods.