A novel multiplex method for the simultaneous detection and relative quantitation of pollen allergens

Standardization of pollen protein extracts is essential in order to ensure efficiency and safety in allergy diagnosis and immunotherapy. In this paper, we have optimized a multiplex Western blotting method for the simultaneous detection of four olive pollen allergens (Ole e 1, Ole e 2, Ole e 5, and Ole e 9) on a single blot using a monoclonal antibody from mouse and three polyclonal antibodies raised in rabbit. We utilized unconjugated Fab antibody fragments for blocking rabbit primary antibodies, and fluorescence-based detection. These changes allowed an accurate and reliable comparative quantitation of these allergens among pollen-protein samples from six olive cultivars. In addition, we also tested the IgE-binding capacity of these pollen extracts by reprobing the same blot with a pool of sera from eight patients allergic to olive and detection with enzyme conjugated antibodies. A noticeable variability regarding allergen content and IgE-reactivity was found among the olive cultivars analyzed. Moreover, we could easily confirm the identity of some of the IgE-binding proteins by simply overlapping both fluorescence and chemiluminescence images. This method is versatile since it can be applied to other allergogenic plant species and extended to other allergens.     

Chip‐based capillary electrophoresis profiling of olive pollen extracts used for allergy diagnosis and immunotherapy

Standardization of protein extracts for clinical purposes represents an important task in order to maintain adequate reactivity, presence of the relevant allergens, and safety among other factors. The main objective of this work was to explore the potential use of a chip‐based automated CE system commercially available to analyze several of the most common forms of allergenic extracts from olive pollen used in allergy clinics. These include experimental extracts prepared from olive pollens, in‐house reference extracts, extracts designed for skin prick test assays, and a panel of vaccine variants aimed to specific immunotherapy. As a major conclusion of the study, chip‐based CE allowed in all cases to determine accurate protein profiles with different degrees of sensitivity, where several allergens (particularly the major olive pollen allergen Ole e 1) were easily recognized. Moreover, several purified allergens were also analyzed by this method, and proposed as specific standards for different purposes. In the present condition, the method can only provide the protein profile of the extracts with respect to a preestablished standard extract, but not allergen identification. However, these and other future developments and applications are discussed.     

Proteomics of cypress pollen allergens using double and triple one-dimensional electrophoresis

Italian cypress (Cupressus sempervirens, Cups) pollen causes allergic diseases in inhabitants of many of the cities surrounding the Mediterranean basin. However, allergens of Cups pollen are still poorly known. We introduce here a novel proteomic approach based on double one‐dimensional gel electrophoresis (D1‐DE) as an alternative to the 2‐DE immunoblot, for the specific IgE screening of allergenic proteins from pollen extracts. The sequential one‐dimensional combination of IEF and SDS‐PAGE associated with IgE immunoblotting allows a versatile multiplexed immunochemical analysis of selected groups of allergens by converting a single protein spot into an extended protein band. Moreover, the method appears to be valuable for MS/MS identification, without protein purification, of a new Cups pollen allergen at 43 kDa. D1‐DE immunoblotting revealed that the prevalence of IgE sensitization to this allergen belonging to the polygalacturonase (PG) family was 70% in tested French allergic patients. In subsequent triple one‐dimensional gel electrophoresis, the Cups pollen PG was shown to promote lectin‐based protein‐protein interactions. Therefore, D1‐DE could be used in routine work as a convenient alternative to 2‐DE immunoblotting for the simultaneous screening of allergenic components under identical experimental conditions, thereby saving considerable amounts of sera and allergen extracts.     

Structural functionality, catalytic mechanism modeling and molecular allergenicity of phenylcoumaran benzylic ether reductase, an olive pollen (Ole e 12) allergen

Isoflavone reductase-like proteins (IRLs) are enzymes with key roles in the metabolism of diverse flavonoids. Last identified olive pollen allergen (Ole e 12) is an IRL relevant for allergy amelioration, since it exhibits high prevalence among atopic patients. The goals of this study are the characterization of (A) the structural-functionality of Ole e 12 with a focus in its catalytic mechanism, and (B) its molecular allergenicity by extensive analysis using different molecular computer-aided approaches covering (1) physicochemical properties and functional-regulatory motifs, (2) sequence analysis, 2-D and 3D structural homology modeling comparative study and molecular docking, (3) conservational and evolutionary analysis, (4) catalytic mechanism modeling, and (5) sequence, structure-docking based B-cell epitopes prediction, while T-cell epitopes were predicted by inhibitory concentration and binding score methods. Structural-based detailed features, phylogenetic and sequences analysis have identified Ole e 12 as phenylcoumaran benzylic ether reductase. A catalytic mechanism has been proposed for Ole e 12 which display Lys133 as one of the conserved residues of the IRLs catalytic tetrad (Asn-Ser-Tyr-Lys). Structure characterization revealed a conserved protein folding among plants IRLs. However, sequence polymorphism significantly affected residues involved in the catalytic pocket structure and environment (cofactor and substrate interaction-recognition). It might also be responsible for IRLs isoforms functionality and regulation, since micro-heterogeneities affected physicochemical and posttranslational motifs. This polymorphism might have large implications for molecular differences in B- and T-cells epitopes of Ole e 12, and its identification may help designing strategies to improve the component-resolving diagnosis and immunotherapy of pollen and food allergy through development of molecular tools.     

Capillary electrophoresis in allergen preparation research and in production control

A CE method has been developed for the analysis of wasp venoms and pollen extracts of Phleum pratense and Betula verrucosa. Various electrolyte systems can be used but the best separation and reproducibility are attained in 0.15 mol/l phosphoric acid, pH 1.8. UV photometric detection at 190 nm is sufficiently sensitive for determination of the polypeptides and glycoproteins contained in these materials (the detection limit for phospholipase A₂ is 0.4 ng). The allergens from wasp venoms and pollen extracts yield characteristic electropherograms which can be used for control of the allergen production. Modification of capillary wall with poly(ethylhydroxy methacrylate) causes a decrease in the migration times and the separation efficiency is retained.     

Cloning and Expression of Che a 1, the Major Allergen of <Emphasis Type="Italic">Chenopodium album</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Chenopodium album is a weedy annual plant in the genus Chenopodium. C. album pollen represents a predominant allergen source in Iran. The main C. album pollen allergens have been described as Che a 1, Che a 2, and Che a 3. The aim of this work was to clone the Che a 1 in Escherichia coli to establish a system for overproduction of the recombinant Che a 1 (rChe a 1). In order to clone this allergen, the pollens were subjected to RNA extraction. A full-length fragment encoding Che a 1 was prepared by polymerase chain reaction amplification of the first-strand cDNA synthesized from extracted RNA. Cloning was carried out by inserting the cDNA into the pET21b (+) vector, thereafter the construct was transformed into E. coli Top10 cells and expression of the protein was induced by IPTG. The rChe a 1 was purified using histidine tag in recombinant protein by means of Ni–NTA affinity chromatography. IgE immunoblotting, ELISA, and inhibition ELISA were done to evaluate IgE binding of the purified protein. In conclusion, the cDNA for the major allergen of the C. album pollen, Che a 1, was successfully cloned and rChe a 1 was purified. Inhibition assays demonstrated allergic subjects sera reacted with rChe a 1 similar to natural Che a 1 in crude extract of C. album pollen. This study is the first report of using the E. coli as a prokaryotic system for Che a 1 cloning and production of rChe a 1.     

Food allergen protein families and their structural characteristics and application in component-resolved diagnosis: new data from the EuroPrevall project

A large number of food allergens able to induce allergic symptoms in predisposed individuals, including severe, even life-threatening reactions, have been identified and characterized. However, proteins able to cause such IgE-mediated reactions can be assigned to only a limited number of protein families. Detailed knowledge about the characteristics of food allergens, their 3D structures, biological activity and stability, will help to improve diagnosis of food allergy, avoid unnecessary exclusion diets and assess the risk of cross-reactive allergies to other food sources. This review is dedicated to summarizing current knowledge about the most important food allergen protein families and to presenting data from the EuroPrevall allergen library, a proof-of-concept collection of highly purified, characterized and authenticated food allergens from animal and plant food sources to facilitate improved diagnosis of food allergies. Relevant food allergen sources     

Structural characterization of the ligstroside aglycone isoforms in virgin olive oils by liquid chromatography–high‐resolution Fourier‐transform mass spectrometry and H/Dexchange

A systematic structural characterization of the isomeric forms related to ligstroside aglycone (LA), one of the most relevant secoiridoids contained in virgin olive oils, was performed using reverse phase liquid chromatography with electrospray ionization Fourier‐transform single and tandem mass spectrometry, operated in negative ion mode (RPLC‐ESI(?)‐FTMS and FTMS/MS). The high mass resolution and accuracy provided by the adopted orbital trap mass analyzer enabled the recognition of more than 10 different isomeric forms of LA in virgin olive oil extracts. They were related to four different types of molecular structure, two of which including a dihydropyranic ring bearing one or two aldehydic groups, whereas the others corresponded to dialdehydic open‐structure forms, differing just for the position of a C═C bond. The contemporary presence of enolic or dienolic tautomers associated to most of these compounds, stable at room temperature (23°C), was also assessed through RPLC‐ESI‐FTMS analyses operated under H/D exchange conditions, ie, by using D₂O instead of H₂O as co‐solvent of acetonitrile in the RPLC mobile phase. As discussed in the paper, the results obtained for LA indicated a remarkable structural similarity with oleuropein aglycone (OA), the most abundant secoiridoid of olive oil, whose isoforms had been previously characterized using the same analytical approach.     

Analysis of contact allergenic compounds in oxidizedd-limonene

Summary A detailed chemical analysis and quantification of oxygen-containing monoterpenes in auto-oxidizedd-limonene was performed. Some of these, e.g. the cis- and trans-isomers of limonene-2-hydroperoxide, have previously been shown to exhibit strong contact allergenic properties. GC-MS with chemical ionization in negative ion mode was shown to be a successful method for the identification and determination of the molecular weight of chemically unstable limonene hydroperoxides. An HPLC method for isolation of individual compounds in auto-oxidizedd-limonene is presented. Two different stationary phases were used in normal phase mode, cyanopropyl- and diamino-modified silica. The method described can be used for the isolation of individual contact allergens in sensitization experiment vehicles, such as petrolatum and olive oil. This makes it possible to study whether test compounds, such as hydroperoxides, are chemically stable during a sensitization experiment.     

A 2‐D‐HPLC‐CE platform coupled to ESI‐TOF‐MS to characterize the phenolic fraction in olive oil

A 2‐D‐HPLC/CE method was developed to separate and characterize more in depth the phenolic fraction of olive oil samples. The method involves the use of semi‐preparative HPLC (C18 column 250×10 mm, 5 μm) as a first dimension of separation to isolate phenolic fractions from commercial extra‐virgin olive oils and CE coupled to TOF‐MS (CE‐TOF‐MS) as a second dimension, to analyze the composition of the isolated fractions. Using this method, a large number of compounds were tentatively identified, some of them by first time, based on the information concerning high mass accuracy and the isotopic pattern provided by TOF‐MS analyzer together with the chemical knowledge and the behavior of the compounds in HPLC and CE. From these results it can be concluded that 2‐D‐HPLC‐CE‐MS provides enough resolving power to separate hundreds of compounds from highly complex samples, such as olive oil. Furthermore, in this paper, the isolated phenolic fractions have been used for two specific applications: quantification of some components of extra‐virgin olive oil samples in terms of pure fractions, and in vitro studies of its anti‐carcinogenic capacity.     

化妆品中24种过敏原的气相色谱-质谱法测定

建立了气相色谱-质谱(GC-MS)法同时测定化妆品中24种过敏原的分析方法。样品经甲醇超声提取,以选择离子模式(SIM)进行定性及定量分析。24种过敏原在质量浓度0.2～100 mg/L范围内呈良好的线性关系,相关系数r2﹥0.995,检出限为2～10 mg/kg,平均回收率为80%～104%,相对标准偏差为1.8%～9.3%。该方法快速、准确、灵敏度高。通过对77名患者使用的150份可疑致敏化妆品测试发现,24种过敏物质的阳性检出率为78%,其中至少有1种过敏物质含量大于10 mg/kg的样本占阳性样本的81.2%。71名过敏患者(92.2%)使用的化妆品中检出致敏物;其中至少有一种过敏物质的检出值大于10 mg/kg的占90.1%。结果显示,24种过敏物质是化妆品接触性皮炎的主要致敏原。     

Online preconcentration and two‐dimensional separation of cationic compounds via hyphenation of capillary zone electrophoresis with cyclodextrin‐modified micellar electrokinetic capillary chromatography

A novel preconcentration/separation approach, which online combined CZE with CD‐modified MEKC, was developed for simultaneous enhancing resolving power and detection sensitivity. CZE with cation‐selective exhaustive injection and transient ITP preconcentration was used as the first dimension, from which the effluent fractions were further analyzed by CD‐modified MEKC acting as the second dimension. As the key to successful integration of CZE with MEKC, a new interface was designed and electroaccumulation focusing strategy was employed to avoid analyte band diffusion at the interface. The comprehensive 2‐D system was successfully established with only one high voltage and four electrodes. The grouping of two orthogonal separation techniques, together with analytes preconcentration techniques, significantly enhanced resolution and sensitivity for 2‐D separation of cationic compounds. The resulting electrophoregram was quite different from that of either single CZE or MEKC. Up to 14 000‐ to 35 000‐fold improvement in sensitivity was obtained relative to conventional electrokinetic injection method. The limits of detection (S/N=3) were in the range of 0.03–0.1 μg/L. The number of theoretical plates was in the range of 103 000–184 000. This method was successfully applied to the analysis of trace cationic cardiovascular drugs in wastewater.     

2D Crystal Engineering of Nanosheets Assembled from Helical Peptide Building Blocks

The successful integration of 2D nanomaterials into functional devices hinges on developing fabrication methods that afford hierarchical control across length scales of the entire assembly. We demonstrate structural control over a class of crystalline 2D nanosheets assembled from collagen triple helices. By lengthening the triple helix unit through sequential additions of Pro‐Hyp‐Gly triads, we achieved sub‐angstrom tuning over the 2D lattice. These subtle changes influence the overall nanosheet size, which can be adjusted across the mesoscale size regime. The internal structure was observed by cryo‐TEM with direct electron detection, which provides real‐space high‐resolution images, in which individual triple helices comprising the lattice can be clearly discerned. These results establish a general strategy for tuning the structural hierarchy of 2D nanomaterials that employ rigid, cylindrical structural units.     

Evaluation of extraction buffers using the current approach of detecting multiple allergenic and nonallergenic proteins in food

The detection of food allergens has been a challenge because of the increasing need to ensure the absence of undeclared allergens in foods. The current trend in the detection of some food allergens, like peanuts, is based on the detection of multiple allergenic and nonallergenic proteins, and this is the approach that kit manufacturers have adopted. Because commercial kits differ in their ability to detect allergens, regulatory agencies, the food industry, and kit manufacturers are working together to standardize the detection methods. Three kits for the detection of peanuts have been evaluated for performance by the AOAC Research Institute. For this evaluation, a peanut butter suspension was used as a reference material. Several kit components contribute to between-kit analytical variation, even when the same sample is used. One component of commercial kits, which may be contributing to this variability, is the sample extraction buffer. In this study, differences in extractability of 3 allergenic foods were evaluated by using 4 different extraction buffers. The conclusion is that optimum allergen extractability was buffer-dependent, and no single buffer is appropriate for use as a universal extraction solution for all allergenic foods. Therefore, a thorough evaluation of sample preparation buffers needs to be performed for every individual allergenic food. In light of the results obtained, the current approach used for detection of peanut allergens based on the detection of multiple allergenic and nonallergenic proteins is being analyzed.     

Two‐Dimensional Semiconductors Grown by Chemical Vapor Transport

Developing controlled approaches for synthesizing high‐quality two‐dimensional (2D) semiconductors is essential for their practical applications in novel electronics. The application of chemical vapor transport (CVT), an old single‐crystal growth technique, has been extended from growing 3D crystals to synthesizing 2D atomic layers by tuning the growth kinetics. Both single crystalline individual flakes and continuous films of 1 L MoS₂ were successfully obtained with CVT approach at low growth temperatures of 300–600 °C. The obtained 1 L MoS₂ exhibits high crystallinity and comparable mobility to mechanically exfoliated samples, as confirmed by both atomic resolution microscopic imaging and electrical transport measurements. Besides MoS₂, this method was also used in the growth of 2D WS₂, MoSe₂, Mo_x W_1−x S₂ alloys, and ReS₂, thus opening up a new way for the controlled synthesis of various 2D semiconductors.     

HPLC enantioseparation of α,α‐diphenyl‐2‐pyrrolidinemethanol and methylphenidate using a chiral fluorescent derivatization reagent and its application to the analysis of rat plasma

Enantioseparation of α,α‐diphenyl‐2‐pyrrolidinemethanol (D2PM) and methylphenidate (MPH; Ritalin^®) using (R)‐(?)‐4‐(N,N‐dimethylaminosulfonyl)‐7‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐2,1,3‐benzoxadiazole as the chiral derivatization reagent has been achieved for the first time, and a simple, reliable detection method using HPLC with fluorescence detection has been developed. D2PM and MPH have been derivatized with (R)‐(?)‐4‐(N,N‐dimethylaminosulfonyl)‐7‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐2,1,3‐benzoxadiazole at 55°C for 15 min. The derivatives of D2PM and MPH have been separated, completely and rapidly, using a reversed‐phase system within 16 min (resolution factor (R_s)=1.60 and 2.53, respectively). The detection limits of (R)‐ and (S)‐D2PM were found to be 6.8 and 13 ng/mL, respectively, and those of D ‐ and L ‐threo‐MPH were 61 and 66 ng/mL, respectively (S/N=3). The proposed method was successfully applied to the analysis of rat plasma, where the rats were separately dosed with D2PM and MPH (Ritalin).     

Analytical and preparative native polyacrylamide gel electrophoresis: Investigation of the recombinant and natural major grass pollen allergen Phl p 2

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot are amongst the most popular methods for allergen characterization, such as comparison of recombinant allergens with their natural counterparts. Native PAGE was evaluated as a possible robust and simple method offering high-resolution capacity for characterization of the major grass pollen allergen Phl p 2. Analytical separation of recombinant Phl p 2 provided a superior quality control in terms of homogeneity and, after Western blotting, immunoglobulin E (IgE) reactivity. Separation of natural Phl p 2 identified two major isoforms which were shown to have different N-terminal sequences and IgE-binding properties. After isolation using preparative native PAGE in combination with electrodialysis, both isoforms were investigated by specific proteolysis and reversed-phase high-performance liquid chromatography (RP-HPLC). The results demonstrate differences in the primary structures and that the recombinant counterpart corresponds exactly to one isoform. Analytical and preparative native PAGE thus proved to be powerful tools for the investigation of allergen isoforms and quality control of recombinant counterparts.     

Mapping of Malus domestica allergens by 2-D electrophoresis and IgE-reactivity

The importance of apple allergens has been repeatedly emphasized, and their presence has been confirmed both in pollen and in fruits. In the present study, a combination of proteomic tools have been used to build a complete allergen map of apple. The water-soluble fraction of an apple extract was precipitated using a phenol-based procedure and separated by 2-DE. Initially four previously classified allergens, Mal d 1, Mal d 2, Mal d 3 and Mal d 4, could be identified in Western blots with polyclonal rabbit antibodies directed to the four respective allergens, and subsequently matched to the bands recognized by several patient sera. Further, all four known apple allergens were localized on a 2-DE map and they were matched with spots recognized by sera of patients with different allergic patterns. Moreover, a new, putative allergen could be identified using MS. We evaluated the influence of post-translational modifications and the immunoreactivity under different analytical conditions. The comparison of different visualization methods for 2-DE gels and blots revealed that even very low concentrations of the intact epitopes are detectable by IgEs of patients, and therefore might be sufficient to trigger allergic symptoms in sensitized individuals.     

Identification, quantitation and method validation for the analysis of suspected allergens in fragrances by comprehensive two-dimensional gas chromatography coupled with quadrupole mass spectrometry and with flame ionization detection

The first part of this study first aims at validating a method to identify suspected allergens limited by EU regulations in fragrances by comprehensive two-dimensional gas chromatography (GC x GC) coupled with rapid scanning quadrupole MS (GC x GC-qMS). The effectiveness of the quadrupole MS operating at different scanning speed (1000 and 11,111 amu/s) was evaluated in identifying (full scan mode acquisition--TIC) and in quantifying (single ion monitoring--SIM) the target analytes in complex mixtures. In full scan acquisition mode the mass range was reduced to 40-240 amu to increase the scan acquisition rate while in SIM mode the influence of different dwell-times (40, 10 and 5 ms) was tested. The number of scans for each single modulated chromatographic GC x GC peak and the total number of scans for the 2D peak, together with half height peak width (referred to apex) of each allergen in the standard mixture in both TIC and SIM modes were determined. Moreover, the match quality of the spectra obtained by GC/MS at 1000 and 11,111 amu/s and by GC x GC-MS at 11,111 amu/s were compared and the occurrence of spectral skewing verified. In the second part of this work quantitative methods by GC x GC-SIM/qMS and GC x GC-FID were validated on the basis of Eurachem/CITAC protocols through which the following performance parameters were determined: confirmation of identity, selectivity and specificity, limit of detection (LOD), limit of quantitation (LOQ), linearity (working and linear range), precision and accuracy and uncertainty. Suspected allergens were spiked in a concentration range between 2 and 25 ppm (microg/mL) on a Test fragrance taken as a reference, while 1,4 dibromo-benzene and 4,4'-dibromodiphenyl were used as internal standards.     

Fast multidimensional localized parallel NMR spectroscopy for the analysis of samples

A parallel localized spectroscopy (PALSY) method is presented to speed up the acquisition of multidimensional NMR (nD) spectra. The sample is virtually divided into a discrete number of nonoverlapping slices that relax independently during consecutive scans of the experiment, affording a substantial reduction in the interscan relaxation delay and the total experiment time. PALSY was tested for the acquisition of three experiments 2D COSY, 2D DQF‐COSY and 2D TQF‐COSY in parallel, affording a time‐saving factor of 3–4. Some unique advantages are that the achievable resolution in any dimension is not compromised in any way: it uses conventional NMR data processing, it is not prone to generate spectral artifacts, and once calibrated, it can be used routinely with these and other combinations of NMR spectra. Copyright © 2010 John Wiley & Sons, Ltd.