Successful Standardization of Purified Natural and Recombinant Mite Allergens as part of the European Union (EU) CREATE Project.
LD Vailes, A Tsay, F Ferreira, R van Ree, MD Chapman. INDOOR Biotechnologies Inc, Charlottesville VA, University of Salzburg, Austria, Central Laboratory of Bloodtransfusion, Amsterdam, The Netherlands.
Rationale: To develop purified Group1 and Group2 mite allergens with verifiable allergen and protein content, as part of the EU CREATE program, which will serve as international standards for in vitro assays and for research use.
Methods: Natural allergens, or recombinant allergens from Ecoli or Ppastoris were purified by affinity chromatography or HPLC and analyzed by SDS-PAGE, protein assay, amino acid analysis, mass spectrometry and by ELISA. Purified allergens were compared by ELISA with three existing allergen standards and with six commercial Dpt or Df extracts.
Results: Amino acid analysis showed good agreement between the protein content, with recombinant:natural protein ratios of 2.4-2.7 and 0.51-1.4 for Group1 and Group2. Q-TOF MS showed major peaks at MW 14027-14096 for purified Group2 allergens. Multiple isoforms were detected in natural allergens. ELISA showed parallel dose response curves and consistent quantitative relationships between all five allergen standards with detection limits ranging from 4-13ng/ml (Group1) and 0.5-1ng/ml (Group 2). Inter-assay ELISA CV’s were 12-23% Group 1 (IBI), 37-45% Group 1 (ALK) and 26-36% Group 2. Allergen levels in Dpt or Df extracts ranged from 8-1000μg/vial (Group 1) and 3-170μg/vial (Group2).
Conclusions: Purified mite allergen standards have been developed and the quantitative relationship between current mite references and CREATE reference materials has been established. Immunoassays for both major mite allergens have been validated. The purified CREATE references are suitable for international comparisons of allergen levels by immunoassay and should have applications in standardizing allergenic products and environmental exposure measurements.
Allergen Immunotherapy Increases Suppressive Activity by CD4+CD25-, IL-10 Producing T cells, but does not Affect Suppression by CD4+CD25+ T cells.
EM Ling, M Calderon, D Nguyen, K Powrie, SR Durham, DS Robinson, Imperial College London and St Mary Hospital, London UK
Rationale: Allergen immunotherapy (AI) is associated with modulation of Th2-type T cell responses to allergen, and has previously been described as resulting in induction of IL-10 producing T cells. We have previously reported less suppression by CD4+CD25+ regulatory T cells in patients with allergic rhinitis than non-atopics. We asked whether AI- induced regulation of allergen-driven T cell responses resulted from increased activity of naturally occurring CD4+CD25+ or was due to a separate regulatory population of IL-10 producing T cells.
Methods: During the UK pollen season, peripheral blood CD4+CD25+ and CD4+CD25- T cells were isolated by immunomagnetic columns from patients who had received AI for severe summer hay fever for at least one year and from a control group of rhinitic patients who had not received AI. CD4+CD25- T cells were separated after overnight incubation into IL-10 producing cells and non-IL-10 T cells by a further immunomagnetic step. The ability of CD4+CD25+ or CD4+CD25- IL-10+ T cells to suppress grass pollen allergen-driven cultures of CD4+CD25- T cells was compared between groups.
Results: Suppression of grass pollen (whole allergen extract and PhlPV) driven proliferation was greater by CD4+CD25- IL-10 producing T cells from patients who had received AI when compared to those who had not (median percent suppression 75% vs 20%, p<0.01). There was no difference between groups in the ability of CD4+CD25+ T cells to suppress grass pollen stimulated T cells.
Conclusions: Allergen immunotherapy induces a population of IL-10 producing regulatory T cells, which are distinct from the naturally occurring CD4+CD25+ subset.
JACI 2004;113:S109
Chemically modified recombinant Fel d 1: a good alternative to hypoallergenic mutants.
SA Versteeg1, I Bulder1, WA van Leeuwen1, F Ferreira2, R van den Hout3, H van Schijndel3, R van Ree1. 1Sanquin Research, Amsterdam, The Netherlands 2University of Salzburg, Austria 3HAL Allergen Laboratories, Haarlem, The Netherlands.
Rationale: Hypoallergenic mutants of recombinant major allergens are proposed to make immunotherapy safer and more efficient. Their development, however, is laborious and requires detailed structural information. The aim of this study was to develop chemically-modified recombinant Fel d 1 as an alternative to a hypoallergenic mutant.
Methods: Fel d 1 was cloned and expressed in Pichia pastoris as a hybrid molecule of both chains of the allergen. The resulting recombinant (r)Fel d 1 was compared to purified natural (n)Fel d 1 by CD and mass spectroscopy, RAST(-inhibition), competitive RIA, ELISA, immunoblot, basophil histamine release (BHR) and T-cell proliferation assays. Chemical modifocation was carried out using glutaraldehyde. Hypoallergenicity was assessed by RAST-inhibition (RI) and BHR.
Results: rFel d 1 was expressed at ~500 mg/L and was shown to be glycosylated by the yeast. Physico-chemical and immunological comparison with nFel d 1 showed that the recombinant molecule is well-folded and that the immune-reactivity of both molecules is very similar. Deglycosylation did not influence immune-reactivity. Chemical modification resulted in at least a 200-fold decrease in IgE reactivity (RI) and a 1000-fold decrease in biological activity (BHR).
Conclusions: Chemically modification of recombinant major allergens is a good and easy alternative to the development of hypoallergenic mutants.
JACI 2004;113:S137
Ligand Binding: an Intrinsic Property of Cockroach Allergen Bla g 2
S. Wuenschmann1, M. Li2, A. Gustchina2, M. D. Chapman1, A. Pom s1;
1INDOOR Biotechnologies, Inc., Charlottesville, VA, 2Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, MD.
Rationale: Bla g 2 is an inactive aspartic protease similar to pregnancy-associated glycoproteins. We investigated the effects of amino acid substitutions in the catalytic site and the "flap" region of Blag2 on enzymatic activity and binding to pepstatin, a specific aspartic protease inhibitor.
Methods: Recombinant Blag2 mutant (Mut1) was expressed in Pichia pastoris after site-directed mutagenesis of the wild type (Wt) allergen. De-glycosylated mutant (N93Q) was produced for crystallography. The Wt and Mut1 were assayed for enzymatic activity (milk-clotting-MCA- or hemoglobin Assay-HA-) or for binding to pepstatin.
Results: The Wt and Mut1 allergens did not show aspartic protease activity at 10microg/ml and 80microg/ml for MCA and HA, respectively, while pepsin was active at <0.3microg/ml and <10microg/ml. Approximately 70% of pepsin bound to pepstatin-agarose, and was fully recovered by elution. rBlag2 Wt and Mut1 needed more stringent conditions for elution, indicating a stronger binding to pepstatin than pepsin. 2-5 fold more rBlag2 Mut1 than Wt was eluted, suggesting that the inhibitor binds stronger when the resemblance to the active site of aspartic proteases is higher. Crystals were obtained with the N93Q mutant, and diffraction data were collected at 2.2A resolution. Structure determination is in progress.
Conclusions: Since engineering of rBlag2 did not convert the allergen into an active aspartic protease, we conclude that pepstatin binding to Blag2 indicates that this allergen may function as a ligand binding molecule, rather than as a protease. Determination of the crystal structure of Blag2 will help in clarifying this matter and in the search for natural ligands of Blag2.
Animal House Exposure to Endotoxin and Rodent Allergens
JA Platts-Mills, N Custis, A Kenney, MD Chapman, A Tsay, TA Platts-Mills.
Rationale: A consistent method for measuring airborne allergens and endotoxin in animal houses is essential for evaluating the risk of sensitization and the impact of measures taken to reduce exposure.
Methods: Levels of airborne mouse and rat allergens in animal rooms were measured using a recently described method for collecting airborne allergens employing an ion charging device (The Sharper Image Ionic Breeze Quadra) running at 1.7m3/min for 24 hours, along with a two-site monoclonal assay for rat and a monoclonal primary/polyclonal secondary assay for mouse (Indoor Biotechnologies). Airborne endotoxin was assayed in parallel using the FDA-standardized Limulus Amoebocyte Lysate test QCL 1000 (Bio-Whittaker).
Results: Airborne mouse and rat was present in relevant animal rooms, however there was a very wide range of results for both allergens (<.004ng/m3 to >6ng/m3) and endotoxin (<2.4pg/m3 to >1000pg/m3). The primary factor influencing airborne allergen and endotoxin was the presence of filter tops on individual animal cages for mouse (allergen p=.003, endotoxin p=<.01) and rat (allergen p=.013, endotoxin p =.001). Each of the values were reduced by >98%.
Conclusions: Though previous studies have attributed reduced allergen levels to filter tops, this study shows the same effect on endotoxin levels. The levels of allergen and endotoxin measured in animal rooms with filter tops is similar to the levels of endotoxin and common indoor airborne allergens in domestic studies and thus is in itself a sufficient prevention against exposure for animal technicians.
Recombinant allergens as diagnostics: correlation with IgE antibody to natural allergens.
LD Vailes, K Ichikawa, A Pom?/font>s, E Best, M McDermott,A Jacquet, and MD Chapman.
U. Virginia, Charlottesville, VA, USA; U. Tsukuba,Ibaraki, Japan; Heska Corporation, Fort Collins, CO, USA; and U. Libre deBruxelles, Gosselies, Belgium.
Recombinant forms of cat, mite, and cockroach allergens were compared to the purified natural allergens for IgE ab reactivity using a chimeric ELISA. Recombinant Bla g 1, Bla g 2, and Pro Der f 1 were produced in Pichia pastoris; rFel d 1 in Baculovirus; and Pro Der p 1 in CHO cells. Allergens were purified using ion exchange, or mAb affinity chromatography, followed by gel filtration HPLC. Natural allergens were purified from extracts of house dust, spent mite culture or cockroach fras. With the exception of rBla g 1, the recombinant allergens were glycosylated showing diffuse banding at a higher molecular weight than the natural allergen on SDS-PAGE. Allergen specific IgE ab to natural or recombinant allergens was measured in large panels of sera by ELISA. Results were quantified using a chimeric mouse anti Der p 2 / human IgE ab to form a control curve. The correlation between IgE ab binding to recombinant versus natural allergen were as follows:Bla g 1: r = 0.92, n = 34 sera; Bla g 2: r = 0.93, n = 26; Der f 1: r =0.96, n = 98; Der p 1: r = 0.92 , n = 33; Fel d 1 r = 0.95, n = 34. The results showed an excellent quantitative correlation between IgE ab to natural and recombinant allergens (p < 0.001). Glycosylation had no effect on IgE ab binding. Recombinant allergens provide new tools for allergy diagnosis.
Validation of recombinant allergens: ELISA reactivity, IgE antibody binding, and skin test reactivity.
LD Vailes, K Ichikawa, A Pom?/font>s, A Smith, E Best, M McDermott, A Jacquet, and MD Chapman. U. Tsukuba, Ibaraki, Japan; Heska Corporation, Fort Collins, CO, USA; U. Libre de Bruxelles, Gosselies, Belgium; and U. Virginia, Charlottesville, VA, USA.
In order for recombinant allergens to be useful as diagnostics and in therapy, they must react with mAb and with IgE antibodies from allergic individuals to a comparable degree as natural allergenic products. In this study, two site mAb ELISA were used to compare natural and recombinant allergens. A chimeric ELISA was used to correlate allergen specific IgE ab by linear regression analysis in >250 sera from allergic patients. Recombinant forms of cat, mite and cockroach proteins, many of which have been difficult to express in the past, were successfully produced in four different systems. Recombinant Bla g 1, Bla g 2, and ProDer f 1 were produced in Pichia pastoris; rFel d 1 in Baculovirus and Pichia; ProDer p 1 in CHO cells and Pichia; and rDer p 2 in E. coli. Allergens were purified using ion exchange, or mAb affinity chromatography, and gel filtration HPLC. Natural allergens were purified from extracts of house dust, spent mite culture or cockroach frass.
All recombinant allergens showed parallel binding curves and had comparable levels of sensitivity to natural allergens in mAb ELISA. Allergens were then used at 0.5μg/ml in a serum based ELISA to correlate IgE antibody binding to natural versus recombinant protein. Results were quantified (ng/ml IgE) using a chimeric mouse anti Der p 2 standard curve.
| Allergen |
System |
Sera |
r value |
p value |
| Bla g 2 |
P.past. |
54 |
0.96 |
< 0.001 |
| Bla g 1 |
P.past. |
34 |
0.92 |
< 0.001 |
| Fel d 1 |
Bacul |
68 |
0.98 |
< 0.001 |
| Fel d 1 |
P.past. |
57 |
0.91 |
< 0.001 |
| Der p 1 |
P.past, |
33 |
0.93 |
< 0.001 |
| Der p 1 |
CHO |
33 |
0.92 |
< 0.001 |
| Der f 1 |
P.past. |
98 |
0.96 |
< 0.001 |
| Der p 2 |
E. coli |
50 |
0.92 |
< 0.001 |
A significant correlation (p > 0.001) was also obtained between IgE ab binding to recombinant Fel d 1 and Der f 1 and Pharmacia CAP score for cat epithelium and D. farinae respectively. Recombinant Derp 2 and Bla g 2 gave skin test reactivity from 10-6 to 10-2μg/ml respectively. With the exception of rBla g 1 and rDer p 2, the recombinant allergens were glycosylated, showing diffuse banding at a higher molecular weight than the natural allergen on SDS-PAGE. However, glycosylation had no effect on IgE ab binding. The results show that the immunoreactivity of recombinant allergens can be successfully validated using a combination of in vitro tests (mAb and IgE) and in vivo skin tests. These validated reagents will provide new tools for allergy diagnosis and treatment.
Validation of the Rapid Test for Monitoring Mite Allergen Exposure in the Home
A Tsay, C Roman, EL Chapman, L. Williams, MD Chapman.
Indoor Biotechnologies, Inc. Charlottesville, VA. Allergen avoidance is recommended as the first step in the treatment of asthma and allergic diseases. Exposure to indoor allergens is usually assessed by ELISA analysis of dust samples, however, ELISA is not suitable for home use. The rapid test (RT) uses gold labeled antibodies, develops within 10 minutes and has a sensitivity of ~ 0.1 mg/g Group 2 allergen. The use of the RT with a dust-sampling device (MITEST) was compared to the standard allergen assay (ELISA). Dust samples (n=228) were collected and extracted from 0.25m2 bedding, carpet, or soft furnishings using the MITEST dust collector and assayed for mite Group 2 allergen by ELISA and RT. Samples were collected from Virginia, Australia, Sweden and France. The evaluation of Der p 2 ELISA with RT showed a good correlation, with 209/228 samples (92%) showing a good to excellent results when compared on a + to +++ visual scale. The RT was used to compare dust mite levels in air ducts with other sites (bed, floor, and sofa) in 26 homes from Virginia.The results showed that only 26% of air duct samples contained detectable mite allergens by RT or ELISA. We have been able to validate the use of RT together with MITEST collector for assessing mite exposure. The results also show that the test is sensitive and can be used to monitor allergen levels at different sites within the home.
Rapid Test for Mite Allergen Detection in the Home: Correlation with ELISA.
A. Tsay* BS, L. Williams PhD, J. Chandler PhD, M.D. Chapman PhD.
Cardiff, U.K., and Charlottesville, VA.
(J Allergy Clin Immunol 1999; 103:S235)
Allergen avoidance is the first line of treatment for the management of patients with allergic rhinitis and asthma. Exposure to indoor allergensis usually assessed by ELISA analysis of dust samples, however, ELISA is not suitable for home use. We compared Der p 1 and Der p 2 ELISA on dust samples collected in the UK, Brazil and New Zealand. The Der p 2 ELISA results were compared with a semi-quantitative lateral flow rapid test for mite Group 2 allergen. The rapid test uses gold labelled antibodies, develops within 10 minutes and has a sensitivity of ~10ng/ml Group 2 allergen. The use of the rapid test with a dust sampling device (MITEST) was also evaluated. Der p 1 and Der p 2 ELISA results in dust samples were highly correlated (n=168, r=0.70, p<0.001). Semi-quantitative evaluation of Der p 2 ELISA with the rapid test showed a good correlation, with 108/130 samples (83%) showing good to excellent results when compared on a + to +++ visual scale. Dust samples (n=37) were collected and extracted from 0.25m2 bedding, carpet, or soft furnishings using the MITEST dust collector and assayed for mite Group 2 allergen by ELISA and rapid test. Mean dust weight collected by MITEST was 140mg (bed); 747mg (carpet) and 87mg (furn). Group 2 allergen levels (ELISA) ranged from 10-140ng/ml. Twenty two samples contained no detectable Group 2 by ELISA and were negative on the rapid test. There were two false negative samples on the rapid test, but otherwise the results correlated with ELISA. The results suggest that the rapid test for Group2 is suitable for assessing mite allergen exposure in the home and will enable patients to monitor current exposure and the effects of allergen of avoidance procedures.
Performance Evaluation Of A Rapid Test Method For Dust Mite Allergen Detection in the Home
Guyton S, Tsay A, Chapman M, Eggleston P Johns Hopkins University Baltimore MD, Indoor Biotechnologies, Inc. Charlottesville, VA.
(J Allergy Clin Immunol 2000;105:S289)
The performance of devices to allow home evaluation of allergen exposure, the MITEST collector and the rapid test for mite group2 allergens (RAPID), were compared to standard allergen assays. In twelve homes in Maryland and Pennsylvania, dust was collected from the bedroom floor, living room floor, bed, and soft furnishings using 4 separate collectors. Approximately 0.25 m2 of surface was vacuumed for 2 min at each site; then the collectors was sealed with Parafilm and transported in plastic bags to the laboratory for extraction. In the laboratory the bottom of each collectorwas sealed with collector caps provided with the kit and the collector was filled with 10 ml of 1% BSA/PBS /0.5%Tween20. The collector was sealed, shaken by hand for 2 min and allowed to sit for 5 min and a 150 ml aliquot was placed in the testing well of the RAPID testing device where it developed for 10 minutes. Group 2 mite concentrations were estimated by comparing the color intensity of a line with the color of 3 standard lines according to the following scale: 0, + (low), ++ (medium), +++ (medium-high), ++++(high). The remaining extract was removed centrifuged for 10 minutes and stored at 4°C until assayed for Der f1, Der P1, and Der p 2 using 2-site ELISA assays.
The MITEST collectors functioned well during the collection step, and facilitated the dust sampling. However during the extraction 26 of the collectors leaked at the caps. In 11 cases, the dust sample completely filled the device chamber, making extraction difficult. The test presented some difficulties in interpretation in that it was difficult to match the color of the test line with that of the standard lines on the device and that the lines were blurred in 14 tests. Despite these concerns with test interpretation, the results of the RAPID agreed with the ELISA results. For the 12 samples with a "0" value on the RAPID, Der p 2 ELISA values were all below the detection level (BD). For the 15 samples with a "low" result, ELISA values ranged from 0 to 264 ng/ml with an geometric mean of 20 ng/ml and 93% under 50ng/ml, the given value for a low indication from the RAPID test. For RAPID values of "medium", the ELISA results ranged from BD to 651 ng/ml (geo mean 195 ng/ml),which is lower than the given value of 250 ng/ml. In the 8 samples where the RAPID test was "medium-high", the Der p 2 ELISA results ranged from 235 to 794 ng/ml (geo mean 445 ng/ml). No samples were ranked as "high" by the RAPID test. We concluded that the MITEST and the RAPID worked well in this field test and would be a useful tool for a patient to estimate mite allergen levels in their home.
Abstract on Latex Assays presented at "first world congress on work-related and environmental allergy" - Fourth International Symposium on Irritant Contact Dermatitis
Seventh International NIVA Course on Work-Related Respiratory Hypersensitivity. 9-12 July 2003.
P59
SIGNIFICANT DECREASE IN LATEX ALLERGENS Hev b 1, 3, 5, and 6.02 IN MEDICAL GLOVES MARKETED IN FINLAND BETWEEN 1995-2001
Palosuo T*, Karkkainen T, Frisk K, Reinikka-Railo H, Qjajavi J, Alenius H, Reunala T, Turjanmaa K
National Public Health Institute, FIT Biotech Ltd., Natinal Agency of Medicines, Finnish Institute of Occupational Health, Tampere University Hospital, Helsinki and Tampere, Finland
Objective. Latex allergy continues to be an important occupational health problem. Methods that can reliably measure specific allergens in gloves and other latex devices, instead of nonspecific total protein, are needed.
Methods. A new immunoassay, making use of monoclonal-antibodies and recombinant allergens was used to quantify clinically relevant latex allergens Hev b 6.02, 5, 3 and 1, all known to retain their IgE-binding ability during rubber manufacturing. Finnish National Agency of Medicines has arranged on a biennial basis nationwide market surveys on medical gloves. We report here results for the new latex allergen-specific assay in surveys in 1995 (22 gloves) and 2001 (71 gloves).
Results. In 1995 Hev b6.02 was detected in 73%, Hev b5 in 73%, Hev b3 in 45% and Hev b1 in 14% of medical gloves. In 2001, Hev b6.02 was detected in 53%, Hev b5 in 24%, Hev b3 in 22% and Hev b1 in 0%. When the sum of the 4 allergens in gloves exceeded 1g/g, most latex-allergic patients showed positive skin prick test reactions against extracts of such gloves. In 1995 41% of the gloves revealed more than 1 g/g of latex allergens, while in 2001 only 14% exceeded this value.
Conclusion. A marked decrease in the concentrations 4 clinically relevant latex allergens in medical gloves was noted between years 1995 and 2001. Quantifying selected latex allergens in manufactured products offers means to reliably monitor their allergen content. This methodology could eventually be used to assess and set up meaningful safety limits for latex devices.
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