Direct on air sampling filter quantification of cat allergen

A direct on sampling filter in solution (DOSIS) method for quantification of airborne cat allergens has been developed. In this method, the allergens firmly adsorbed to a porous polytetrafluoroethylene filter are reacted with specific antibodies conjugated to alkaline phosphatase, generating a matrix-bound allergen-antibody-phosphatase complex. The treated filter is subsequently floated on a commercially available chemiluminescent phosphatase substrate solution. Aliquots of this solution are removed and analyzed luminometrically. The light intensity of the product is linearly related to the amount of allergen over a large mass range, 0-100 SQ units (1 SQ unit is about 146 pg of the allergenic protein Fel d 1). DOSIS demonstrated intra- and interassay precisions of 9% and 8% and 14% and 21% for the levels 4 and 20 SQ units per filter, respectively. The limit of quantification was estimated to 0.4 SQ units (58 pg Fel d 1) of cat allergen per filter. Application of DOSIS to analysis of cat allergen concentrations of indoor air in homes with and without cats revealed, on average, a six times higher concentration in the former (142 SQ units/m(3)) as compared to the latter (24 SQ units/m(3)). The recorded concentrations for airborne cat allergen in homes with cats are in accordance with previously reported figures. Allergen-specifically stained sampling filters revealed the particulate nature of airborne cat allergen which seemed predominantly to be carried by numerous large dust particles.     

Cat (Fel d 1) and dog (Can f 1) allergen levels in cars,dwellings and schools

Pets are an important source of indoor allergens. The aim of the study was to compare cat and dog allergen levels in cars, schools and homes. The study was carried out in 17 cars, 14 classrooms and 19 dwellings located in the highly industrialized and urbanized region of Poland. Dust and air samples were analyzed for Fel d 1 and Can f 1 using a double monoclonal ELISA assay. The highest amounts of cat and dog allergens (Fel d 1: 1169 μg/g; Can f 1: 277 μg/g) were found in dwellings with pets. Allergen concentrations were correlated with the number of animals kept at home. Although concentrations on automobile seats were lower, Fel d 1 levels exceeded 8 μg/g in 23.5 % of cars and high levels of Can f 1 (>10 μg/g) were found in 17.6 % of cars. The study revealed that cars of pet owners may be reservoirs of cat and dog allergens even when animals are not transported in them. In schools, concentrations of pet allergens did not reach high levels, but the moderate levels of Fel d 1 (≥1–8 μg/g) and Can f 1 (≥2–10 μg/g) were detected in 42.9 and 7.1 % of the investigated classrooms. Concentrations of cat and dog allergen in schools were higher than in homes without pets. While airborne Fel d 1 and Can f 1 levels were found low, residential allergen concentrations in settled dust and air were correlated. The study results suggest that classrooms and cars of pet owners may be important sites of exposure to cat and dog allergens, though the highest concentrations of Fel d 1 and Can f 1 are found in homes of pet owners.     

The mouse salivary androgen-binding protein (ABP) alpha subunit closely resembles chain 1 of the cat allergen Fel dI

Androgen-binding protein (ABP) is found in the salivas of a wide variety of rodents and it has been proposed that ABP functions in sex and/or subspecies recognition (Karn and Dlouhy,J. Hered. 82, 453, 1991). This is a report of significant identity between the alpha subunit of mouse salivary ABP and Chain 1 of cat allergen Fel dI (50% identity), as well as with two other proteins that share identity with Chain 1 of Fel dI, rabbit uteroglobin (27% identity with ABP alpha) and human lung Clara 10 (27% identity with ABP alpha). The secondary structure predicted for the mouse ABP alpha subunit is a very good fit with the secondary structure determined by X-ray crystallography for rabbit uteroglobin, a protein that shares with mouse ABP the capability of binding steroid. Fel dI is found in cat saliva, sebaceous glands, and pelt. Its function is not known but it has been proposed to be involved in protecting dry epithelia, a parallel to uteroglobin protecting wet epithelia. Since mice, like cats, lick themselves and each other extensively, coating their pelts with ABP may be part of this or another biological function.     

Comparison of airborne spore concentrations and fungal allergen content

The exposure to spores causing health effects is usually assessed by determining the concentration of viable spores per cubic meter of air (CFU/m³).Since allergens might also be present in dead spores or smaller particles, the objective of this study was to investigate the correlation between the viable spores of Alternaria and Cladosporium at different indoor and outdoor sites and the corresponding allergen concentration detected with a specially developed ELISA (Enzyme Linked Immunosorbent Assay). In outdoor air, the results show a strong correlation between the different sampling techniques applied for viable spores (Slit-Sampler and Multistage Liquid Impinger) and between the viable spores and the allergen concentrations detected in the liquid samples of the impingers. Indoors, the number of viable spores and the allergen concentration do not correlate and the allergen load is underestimated if colony counting methods are used. This revised version was published online in June 2006 with corrections to the Cover Date.     

Formation of disulfide bonds and homodimers of the major cat allergen Fel d 1 equivalent to the natural allergen by expression in Escherichia coli

Dander from the domestic cat (Felis domesticus) is one of the most common causes of IgE-mediated allergy. Attempts to produce tetrameric folded major allergen Fel d 1 by recombinant methods with structural features similar to the natural allergen have been only partially successful. In this study, a recombinant folded Fel d 1 with molecular and biological properties similar to the natural counterpart was produced. A synthetic gene coding for direct fusion of the Fel d 1 chain 2 N-terminally to chain 1 was constructed by overlapping oligonucleotides in PCR. Escherichia coli expression resulted in a non-covalently associated homodimer with an apparent molecular mass of 30 kDa defined by size exclusion chromatography. Furthermore, each 19,177-Da subunit displayed a disulfide pattern identical to that found in the natural Fel d 1, i.e. Cys3(1) Cys73(2), Cys44(1)-Cys48(2), Cys70(1)-Cys7(2), as determined by electrospray mass spectrometry after tryptic digestion. Circular dichroism analysis showed identical folds of natural and recombinant Fel d 1. Furthermore, recombinant Fel d l reacted specifically with serum IgE, inducing expression of CD203c on basophils and lymphoproliferative responses in cat-allergic patients. The results show that the overall fold and immunological properties of the recombinant Fel d 1 are very similar to those of natural Fel d 1. Moreover, the recombinant Fel d 1 construct provides a tool for defining the three-dimensional structure of Fel d 1 and represents a reagent for diagnosis and allergen-specific immunotherapy of cat allergy.     

Structural characterization of the tetrameric form of the major cat allergen Fel d 1

Felis domesticus allergen 1(Fel d 1) is a 35 kDa tetrameric glycoprotein formed by two heterodimers which elicits IgE responses in 95% of patients with allergy to cat. We have previously established in vitro conditions for the appropriate folding of recombinant Fel d 1 using a direct linkage of chain 1 to chain 2 (construct Fel d 1 (1+2)) and chain 2 to chain 1 (construct Fel d 1 (2+1)). Although the crystal structure of Fel d 1 (2+1) revealed a striking structural similarity to that of uteroglobin, a steroid-inducible cytokine-like molecule with anti-inflammatory and immunomodulatory properties, no functional tetrameric form of Fel d 1 could be identified. Here we present the crystal structure of the Fel d 1 (1+2) tetramer at 1.6 A resolution. Interestingly, the crystal structure of tetrameric Fel d 1 reveals two different calcium-binding sites. Symmetrically positioned on each side of the Fel d 1 tetramer, the external Ca(2+)-binding sites correspond to a putative Ca(2+)-binding site previously suggested for uteroglobin. The second Ca(2+)-binding site lies within the dimerization interface, stabilizing the formation of the Fel d 1 tetramer, and inducing important local conformational changes that directly govern the shape of two water-filled cavities. The crystal structure suggests a potential portal for an unknown ligand. Alternatively, the two cavities could be used by the allergen as a conditional inner space allowing for the spatial rearrangement of centrally localized side-chains, such as Asp130, without altering the overall fold of the molecule. The striking structural similarity of the major cat allergen to uteroglobin, coupled to the identification in the present study of a common Ca(2+)-binding site, let us speculate that Fel d 1 could provoke an allergic response through the modulation of phospholipase A2, by sequestering Ca ions in a similar manner as previously suggested for uteroglobin.     

Monoclonal antibodies to the major feline allergen Fel d I. II. Single step affinity purification of Fel d I, N-terminal sequence analysis, and development of a sensitive two-site immunoassay to assess Fel d I exposure

Two mAb were used to develop new techniques for the purification and quantitation of the major feline salivary allergen, Felis domesticus allergen I (Fel d I). The allergen was purified from aqueous house dust extract with a high Fel d I content by affinity chromatography over a monoclonal immunosorbent and elution with 4 mM HCl, pH 2.5. This single step procedure gave 40 to 50% recovery of 90% pure allergen which, following final purification by size exclusion HPLC, showed a single line on immunodiffusion and crossed immunoelectrophoresis against monospecific anti-Fel d I and polyclonal anti-cat dander antibodies. The m.w. of native Fel d I was 39,000 on size exclusion HPLC, and 17,000 under nonreducing conditions on gel electrophoresis. The N-terminal amino acid sequence (33 residues) showed no homology with other known protein sequences. The combination of the SDS-PAGE and N-terminal sequence data suggests that Fel d I is a non-covalently linked homodimer. A two-site RIA was developed using mAb directed against different epitopes on Fel d I. This assay was species-specific, highly sensitive (0.0004 U/ml), and showed an excellent correlation with a polyclonal inhibition RIA (n = 27, r = 0.93, p less than 0.001). Cat allergen extracts used for immediate skin tests showed marked differences in Fel d I content (from 0.1 to 30 U/ml). Consistently high Fel d I levels were found at monthly intervals in six dust samples from four houses with cats (10 to 100 U/g of dust). Comparisons of Fel d I and mite and pollen allergen levels showed that house dust can contain greater than 100 micrograms/g of either of these allergens and is a potent source of foreign environmental antigens. Monoclonal affinity chromatography provides a major breakthrough in the purification of Fel d I, from a source material that would otherwise have been considered impossible (house dust). The mAb assay for Fel d I is both more sensitive and more easily standardized than existing techniques. These techniques will allow full structural and antigenic analysis of Fel d I and more detailed studies on the relationship between cat antigen exposure and the development of asthma.     

Recognition of the major cat allergen Fel d 1 through the cysteine-rich domain of the mannose receptor determines its allergenicity

Dendritic cells are professional antigen-presenting cells that are specialized in antigen uptake and presentation. Allergy to cat has increased substantially in recent years and has been shown to be positively associated with asthma. We have recently shown that the mannose receptor (MR), a C-type lectin expressed by dendritic cells, recognizes various glycoallergens from diverse sources and is involved in promoting allergic responses to a major house dust mite allergen in vitro. Here we investigated the potential role of MR in allergic responses to Fel d 1, a major cat allergen. Fel d 1 binding to MR was confirmed by ELISA. Using blocking, gene silencing (siRNA) experiments, and MR knock-out (MR(-/-)) cells, we have demonstrated that MR plays a major role in internalization of Fel d 1 by human and mouse antigen-presenting cells. Intriguingly, unlike other glycoallergens, recognition of Fel d 1 by MR is mediated by the cysteine-rich domain, which correlates with the presence of sulfated carbohydrates in natural Fel d 1. WT and MR(-/-) mice were used to study the role of MR in allergic sensitization to Fel d 1 in vivo. MR(-/-) mice sensitized with cat dander extract and Fel d 1 produced significantly lower levels of total IgE, Fel d 1-specific-IgE and IgG1, the hallmarks of allergic response, compared with WT mice. Our data show for the first time that Fel d 1 is a novel ligand of the cysteine-rich domain of MR and that MR is likely to play a pivotal role in allergic sensitization to airborne allergens in vivo.     

Sampling dust from human dwellings to estimate the prevalence ofDermatophagoides mite and cat allergens

Summary Quantification of specific allergens in household dust samples may provide important information for selecting appropriate allergen control methods, and monitoring efficacy and compliance. The purpose of this study was to investigate the source of variation in mite and cat allergen measurements associated with dust sample collection. Discrete and composite dust samples were collected on a filter using a special vacuum sampling device. Aqueous extracts of the dust samples were prepared thenDer p I,Der f I, andFel d I were quantitated by enzyme immunoassays (EIA). Mite and cat allergens were frequently detected in dust samples from human dwellings, and the amounts of these allergens varied significantly (p<0.01) among dwellings. The differences of allergen measurements among duplicate samples taken immediately and up to three weeks later appear to be much smaller than differences among houses and between rooms. Variation among dust samples taken from living rooms and bedrooms of the same dwelling suggest differences in allergen reservoirs. Composite samples formed by sampling specific objects within a room may provide a reliable estimate of allergen exposure in that room. Dust samples from discrete objects are useful to find and monitor specific reservoirs of mite and cat allergens.     

Direct effects of atmospheric carbon dioxide concentration on whole canopy dark respiration of rice

The purpose of this study was to test for direct inhibition of rice canopy apparent respiration by elevated atmospheric carbon dioxide concentration ([CO₂]) across a range of short‐term air temperature treatments. Rice (cv. IR‐72) was grown in eight naturally sunlit, semiclosed, plant growth chambers at daytime [CO₂] treatments of 350 and 700 μmol mol^?1. Short‐term night‐time air temperature treatments ranged from 21 to 40 °C. Whole canopy respiration, expressed on a ground area basis (R_d), was measured at night by periodically venting the chambers with ambient air. This night‐time chamber venting and resealing procedure produced a range of increasing chamber [CO₂] which we used to test for potential inhibitory effects of rising [CO₂] on R_d. A nitrous oxide leak detection system was used to correct R_d measurements for chamber leakage rate (L) and also to determine if apparent reductions in night‐time R_d with rising [CO₂] could be completely accounted for by L. The L was affected by both CO₂ concentration gradient between the chamber and ambient air and the inherent leakiness of each individual chamber. Nevertheless, after correcting R_d for L, we detected a rapid and reversible, direct inhibition of R_d with rising chamber [CO₂] for air temperatures above 21 °C. This effect was larger for the 350 compared with the 700 μmol mol^?1 daytime [CO₂] treatment and was also increased with increasing short‐term air temperature treatments. However, little difference in R_d was found between the two daytime [CO₂] treatments when night‐time [CO₂] was at the respective daytime [CO₂]. These results suggest that naturally occurring diurnal changes in both ambient [CO₂] and air temperature can affect R_d. Because naturally occurring diurnal changes in both [CO₂] and air temperature can be expected in a future higher CO₂ world, short‐term direct effects of these environmental variables on rice R_d can also be expected.     

The crystal structure of the major cat allergen Fel d 1, a member of the secretoglobin family

The domestic cat (Felis domesticus) is one of the most important causes of allergic asthma worldwide. The dominating cat allergen, Fel d 1, is composed of two heterodimers. Recently, it has been shown that recombinant Fel d 1, consisting of chain 2 and chain 1 fused together without additional linker, has immunological properties indistinguishable from the natural heterodimeric protein. Herein, we report the crystal structure of recombinant monomeric Fel d 1 at 1.85-A resolution, determined by multi-wavelength anomalous diffraction using selenomethionine substituted protein. Fel d 1 is an all-helical protein and consists of eight helices. The two halves of the recombinant Fel d 1 molecule, corresponding to the wild-type Fel d 1 chains, are very similar in three-dimensional structure, despite the lack of significant sequence identity. The structure of the Fel d 1 presents a striking similarity to that of uteroglobin, a steroid-inducible cytokine-like molecule with anti-inflammatory and immunomodulatory properties. An internal, asymmetric cavity is formed in the Fel d 1 that could bind an endogenous ligand. The distribution of residues lining this cavity suggests that such a ligand must be amphipathic. The structure of Fel d 1 displays the localization of three previously defined Fel d 1 IgE epitopes on the surface of the protein. The three-dimensional structure provides a framework for rational design of hypoallergenic mutants aimed for treatment of cat allergy.     

Mouse allergen, lung function, and atopy in Puerto Rican children

Objective

To examine the relation between mouse allergen exposure and asthma in Puerto Rican children.

Methods

Mus m 1, Der p 1, Bla g 2, and Fel d 1 allergens were measured in dust samples from homes of Puerto Rican children with (cases) and without (controls) asthma in Hartford, CT (n = 449) and San Juan (SJ), Puerto Rico (n = 678). Linear or logistic regression was used for the multivariate analysis of mouse allergen (Mus m 1) and lung function (FEV₁ and FEV₁/FVC) and allergy (total IgE and skin test reactivity (STR) to ≥1 allergen) measures.

Results

Homes in SJ had lower mouse allergen levels than those in Hartford. In multivariate analyses, mouse allergen was associated with higher FEV₁ in cases in Hartford (+70.6 ml, 95% confidence interval (CI) = 8.6–132.7 ml, P = 0.03) and SJ (+45.1 ml, 95% CI =  −0.5 to 90.6 ml, P = 0.05). In multivariate analyses of controls, mouse allergen was inversely associated with STR to ≥1 allergen in non-sensitized children (odds ratio [OR] for each log-unit increment in Mus m 1 = 0.7, 95% CI = 0.5–0.9, P<0.01). In a multivariate analysis including all children at both study sites, each log-increment in mouse allergen was positively associated with FEV₁ (+28.3 ml, 95% CI = 1.4–55.2 ml, P = 0.04) and inversely associated with STR to ≥1 allergen (OR for each log-unit increment in Mus m 1 = 0.8, 95% CI = 0.6–0.9, P<0.01).

Conclusions

Mouse allergen is associated with a higher FEV₁ and lower odds of STR to ≥1 allergen in Puerto Rican children. This may be explained by the allergen itself or correlated microbial exposures.     

Molecular characterization of major cat allergen Fel d 1: expression of heterodimer by use of a baculovirus expression system

Fel d 1 is a major cat allergen inducing allergic rhinitis and asthma in sensitized individuals. It has a more complex structure when compared with other allergens and therefore expression of recombinant Fel d 1 has been considered a challenge. The present study shows for the first time that a Baculovirus expression system is able to produce an intact rFel d 1 molecule that is glycosylated and structurally equivalent to the natural cat allergen, nFel d 1. Enzymatic digestion of rFel d 1 and further analysis by use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) resulted in a complete coverage of the amino acid sequence of rFel d 1. In addition, the three disulfide bridges at the positions alpha70-beta7, alpha44-beta48, and alpha3-beta73 were verified. The N-glycan structure of rFel d 1 was investigated by a combination of MALDI-TOF MS and monosaccharide analysis by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAC). The N-glycosylation analyses of rFel d 1 refer to a pattern of glycoforms including core alpha1.3-fucosylation that is different from nFel d 1. Further characterization by use of human serum IgE, histamine release, and lymphocyte proliferation assays demonstrated that the immunological characteristics of rFel d 1 are similar to those of nFel d 1. Detailed characterization of both natural and recombinant allergens provides tools to explore immunological mechanisms associated with allergen sensitization and desensitization.     

The combination of airborne pollen and allergen quantification to reliably assess the real pollinosis risk in different bioclimatic areas

Exposure to allergens represents a key factor among the environmental determinants of asthma. The most common information available for pollinosis patients is the concentration of pollen grains in the bioaerosol and their temporal distribution. However, in recent years, discordance between pollen concentrations and allergic symptoms has been detected. The purpose of this research is to evaluate the relationship between pollen counts and the atmospheric aeroallergen concentrations in different Spanish bioclimatic areas. For the monitoring of allergen content in the air, a quantitative antigen–antibody technique combined with the Cyclone sampling methodology was used. The study was conducted during 2007 by considering some of the most common allergens that induce pollinosis in each area: Platanus and Urticaceae in Ourense and Cartagena, and Poaceae in Ourense and León. In Ourense, pollen counts and aeroallergen concentrations coincided for the three pollen types studied, and the pollen and allergen data associated with the meteorological factors were highly significant for the pollen counts. In Cartagena (for Platanus and Urticaceae) and León (for Poaceae), the low correlations between pollen counts and allergen concentrations obtained could be due to the specific bioclimatic conditions. In contrast, the higher allergen concentrations found in the atmosphere in Cartagena and León compared to Ourense could be related to the existing pollutant levels there, inducing a higher expression of plant pathogenesis-related proteins in the plants of polluted cities. The combination of pollen counts and allergen quantification must be assessed to reliably estimate exposure of allergic people to allergens in different bioclimatic areas.     

A role for IL-10-mediated HLA-DR7-restricted T cell-dependent events in development of the modified Th2 response to cat allergen

Although high dose exposure to inhaled cat allergen (Fel d 1) can cause a form of tolerance (modified Th2 response), the T cell mechanism for this phenomenon has not been studied. T cell responses to Fel d 1 were characterized in both allergic (IgE(pos)) and modified Th2 (IgE(neg)IgG(pos)) responders as well as serum Ab-negative controls (IgE(neg)IgG(neg)). Fel d 1 stimulated high levels of IL-10 in PBMC cultures from all individuals, with evidence of Th2 and Th1 cytokine skewing in allergic and control subjects, respectively. Using overlapping peptides, epitopes at the N terminus of Fel d 1 chain 2 were shown to stimulate strong T cell proliferation and to preferentially induce IL-10 (peptide 2:1 (P2:1)) or IFN-gamma (P2:2) regardless of the allergic status of the donor. Injection of cat extract during conventional immunotherapy stimulated expansion of IL-10- and IFN-gamma-producing chain 2 epitope-specific T cells along with increased Fel d 1-specific serum IgG and IgG4 Ab. Six of 12 modified responders expressed the major HLA-DRB1 allele, *0701, and both P2:1 and P2:2 were predicted ligands for this allele. Cultures from DR7-positive modified responders produced the highest levels of IL-10 to P2:1 in addition to other major and minor epitopes within chains 1 and 2. In the presence of anti-IL-10 mAb, both T cell proliferation and IFN-gamma production were enhanced in a Fel d 1- and epitope-specific manner. We conclude that IL-10-producing T cells specific for chain 2 epitopes are relevant to tolerance induction, and that DR7-restricted recognition of these epitopes favors a modified Th2 response.     

Separation of bronchoconstriction from increased ventilatory drive in a nonhuman primate model of chronic allergic asthma.

The relationship between allergen-induced ventilatory drive and bronchoconstriction was investigated in dust mite-sensitive cynomolgus macaques periodically exposed to low doses of aerosolized antigen for up to 5.5 yr. Initially, the animals responded to aerosolized dust mite allergen at a concentration of 350 arbitrary units (AU)/ml with simultaneous increases in lung resistance (RL) and respiratory rate (RR). With time, RL and RR became differentially sensitive to allergen provocation. At the end of the study period, aerosolized allergen at a concentration of 15 AU/ml doubled RR without increasing RL. When mechanically ventilated to maintain tidal volume, higher concentrations of allergen could be delivered, and RL increased. Inhaled disodium cromoglycate and intravenous diphenhydramine attenuated the increase in RR, indicating that allergen-induced release of histamine and activation of H(1) receptors mediated the response. Inhaled beta-adrenergic agonists attenuated the RR response to dust mite and to direct histamine provocation. These results demonstrate that chronic periodic allergen challenge increases the allergic sensitivity of histamine-dependent reflexes controlling ventilatory drive. Activation of these reflexes is independent of overt bronchoconstriction, but can be inhibited by beta-adrenergic agonists, indicating that beta-adrenergic agonists exert their effect independent of bronchodilation.     

Lack of development of phagocytic inhibitory activity in serum of cats immunized withEnterobacter cloacae lipopolysaccharide

Sequentially collected sera from cats immunized withEnterobacter cloacae lipopolysaccharide (LPS) were assessed for their effect on phagocytosis by incubating alveolar marcophage monolayers in the presence of³H-labeled bacteria and 5% serum from control or immunized animals. Unlike serum fromPseudomonas aeruginosa LPS-immunized orP. aeruginosa-infected cats in previous studies, which contained phagocytic inhibitory activity specific forP. aeruginosa, serum from 12 of 13 cats immunized withE. cloacae LPS did not contain phagocytic inhibitory activity forE. cloacae orP. aeruginosa. An enzyme-linked immunosorbent assay (elisa) demonstrated a significant rise inE. cloacae LPS specific IgG (peak titer 1:10,240) by week 13 after immunization. This study suggests that long-term immunization withE. cloacae LPS does not result in the induction of macrophage phagocytic inhibitory activity as previously demonstrated withP. aeruginosa LPS immunization.     

An automated microfluidic-based immunoassay cartridge for allergen screening and other multiplexed assays

A microfluidic cartridge and system for multiplexed immunoassays is described. The passive microfluidic cartridge was composed of three layers of injection molded plastic sealed together using a thermal staking technique. Using this platform technology, a specific immunoglobulin E (IgE) panel assay was constructed. Allergen extract targets, positive and negative controls, and IgE calibration standards were immobilized within the cartridge as a microarray. A computer-controlled solenoid array provided the necessary actuation force for pumping reagents within the cartridge to perform an automated, chemiluminescent indirect immunoassay. A 20-target allergen extract panel was demonstrated on the device with a total analysis time of 27 min. Allergen screening results showed 84% agreement for 3 house dust mites (N = 300) compared with a commercial test and 80% agreement overall (N = 978). Average coefficients of variation (N = 80) were measured as 20.5% for low/medium levels and 20.4% for medium/high levels. The average limit of detection (N = 160) was measured at 0.535 AU, and cutoff levels of 1.0 AU were estimated at less than 1 IU/ml (2.4 ng/ml). Such a system has potential applications in decentralized allergen screening as well as in other near-patient diagnostic immunoassays where multiplexed analysis, ease of use, and short analysis time are critical.     

A comparison of airborne and dust-borne allergens and toxins collected from home,office and outdoor environments both in New Haven,United States and Nanjing,China

In this study, the airborne and dust-borne concentrations of endotoxin, (1,3)-β-d-glucan and five house dust allergens were measured in office, home, and outdoor environments both in New Haven, United States and Nanjing, China. Air samples were collected using a BioSampler at a flow rate of 12.5 l/min for 30 min. Dust samples were simultaneously collected using a surface sampler. Dust samples went through extraction and dilution before analysis, while air samples were analyzed directly. Limulus Amoebocyte Lysate (LAL) Pyrochrome and Glucatell assays were used to quantify endotoxin and (1,3)-β-d-glucan concentration levels, respectively. Enzyme-linked sorbent assay was used to measure the dust mites, cat, dog, and cockroach allergens. The experimental results indicated that endotoxin, (1,3)-β-d-glucan and allergen concentrations vary greatly both with samples and environments. In all tested environments, endotoxin concentration ranged from 0.8 to 83.7 ng/m³ for air, and 7.8 to 14.3 ng/mg for dust. (1,3)-β-d-glucan concentration ranged from 0.1 to 9.8 ng/m³ for air, and 6.6 to 110 ng/mg for dust. Cockroach allergens were detected only in New Haven office and outdoor environments, and other allergens ranged from 0.1 to 90 ng/mg for dust samples, and from 1.5 to 1,282 ng/m³ for air samples. In general, similar profiles of allergens and toxins were observed in New Haven and Nanjing environments. Linear regression analysis showed that there were better endotoxin and (1,3)-β-d-glucan linear correlations (R ² = 0.78, 0.87, respectively) between the dust and air samples compared to those of the allergens Der f 1 and Der p 1 (R ² = 0.5, 0.7, respectively). This research contributes to the development of robust biological exposure assessment and the elaboration of airborne and dust-borne bio-mass in the living environments.