Protein-binding Membrane Research Articles

Differential expression of follicular fluid exosomal microRNA in women with diminished ovarian reserve.

Decreased ovarian reserve function is mainly characterized by female endocrine disorders and fertility decline. Follicular fluid (FF) exosomal microRNAs (miRNAs) have been shown to regulate the function of granulosa cells (GCs). The present study explored differentially expressed miRNAs (DEmiRNAs) in patients with diminished ovarian reserve (DOR). FF was collected from 12 DOR patients and 12 healthy controls. DEmiRNAs between the two groups were identified and analyzed using high-throughput sequencing technology and validated by real-time quantitative PCR (RT-qPCR). A total of 592 DEmiRNAs were identified using high-throughput miRNA sequencing, of which 213 were significantly upregulated and 379 were significantly downregulated. The sequencing results were further validated by RT-qPCR. These DEmiRNA target genes were mainly involved in the cancer pathway, phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway, regulation of actin cytoskeleton signaling pathway, and biological processes related to protein binding, nucleoplasm, cytoplasm, and cell membrane. FF exosomal miRNAs are significantly differentially expressed in DOR patients versus non-DOR patients, underscoring their crucial role in regulating the pathogenesis of DOR.

Why we should study protein binding to bicontinuous inverted cubic phases, to better understand membrane trafficking.

Many proteins bind to membranes in a curvature-sensitive manner. This includes some adaptors/effectors in the vesicle budding and fusion stages of membrane trafficking. Recently, others showed that some proteins bind preferentially to membranes with Gaussian curvature: they bind more strongly to spherical vesicles (with positive Gaussian curvature) than to tubules with the same mean curvature but zero local Gaussian curvature. Fusion and fission pores (which are intermediates in vesicle fusion & budding) have special curvature properties. Here I show that the surfaces of such pores have curvature that is qualitatively and quantitatively different than in the membrane systems conventionally used to assess curvature-sensitive protein binding (membrane vesicles and tubules). Hence, existing methods may miss proteins that have evolved to bind to pore-relevant curvatures. However, the curvature properties of bicontinuous inverted cubic (QII) phases are quite similar to those of fusion/fission pores. Specifically, both QII phases and fusion/fission pores have negative Gaussian curvature, which is true of neither vesicles nor tubules; have more negative mean curvature than many tubules; and gradients in mean curvature that are either absent or smaller in vesicles and tubules. QII phases can be equilibrated with peptide through the aqueous phase: it has been shown that proteins with molecular weights of several tens of kDa can enter the water channel networks and penetrate into aggregates of QII phase. Thus, we might find new adaptors/effectors of trafficking by studying peptide or protein binding to preparations of QII phase. Some features of QII phases make such experiments awkward. Only certain ranges of lipid compositions form QII phases, and, in phospholipids, temperature-cycling techniques or particular electrolyte compositions may be needed. Appropriate protocols will be proposed.

The Involvement of Insulin-Like Growth Factor 2 Messenger Ribonucleic Acid-Binding Protein 2 in the Regulation of the Expression of Breast Cancer-Related Genes.

AimThis study investigated the role and mechanism of insulin-like growth factor 2-IGF2BP2 in breast cancer.MethodsIGF2BP2 is overexpressed in MDA-MB-231 human breast cancer cells. Thus, RNA sequencing was used to analyze the differentially expressed genes, Cell Counting Kit-8 was used to detect cell proliferation, and a Transwell assay was used to assess cell invasion. Following on from the RNA sequencing results, Interferon-induced protein with tetratricopeptide repeats 2 (IFIT2), chemokine C-C motif ligand 20 (CCL20), chemokine C-C motif ligand 5 (CCL5), and chemokine C-X-C motif ligand 10 (CXCL10) regulated by IGF2BP2 were subjected to real-time reverse transcriptase-polymerase chain reaction verification.ResultsAfter IGF2BP2 overexpression, 67 genes were up-regulated, and 87 genes were down-regulated. The gene with the most significant up-regulation was homeobox protein 1 (PROX1), and the gene with the most significant down-regulation was Acidic β-crystallin 4 (CRYBA4). The most enriched gene ontology (GO) terms of up-regulated differentially expressed genes are protein binding and cell membrane and of down-regulated differentially expressed genes they are ion binding, cytoplasm, and response to virus. Kyoto Encyclopedia of Genes and Genomes analysis showed that the up-regulated differential genes were mainly enriched in protein processing, the endoplasmic reticulum, and the regulation of actin cytoskeleton, while down-regulated differential genes were mainly enriched in rheumatoid arthritis, chemokine signaling pathways, toll-like receptor signaling pathways, tumor necrosis factor signaling pathways, cytokine-cytokine receptor interaction, and Notch signaling pathways. IGF2BP2 overexpression significantly promoted the proliferation and invasion of breast cancer cells (P < 0.01). Compared with the control group, the IGF2BP2 overexpression group had significantly increased expressions of IFIT2, CCL20, and CXCL10 (P < 0.05).ConclusionIGF2BP2 may promote the invasion and proliferation of human breast cancer cells by up-regulating breast cancer-related genes, such as IFIT2, CCL20, and CXCL10.

Paper-based open microfluidic platform for protein electrophoresis and immunoprobing.

Protein electrophoresis and immunoblotting are indispensable analytical tools for the characterization of proteins and posttranslational modifications in complex sample matrices. Owing to the lack of automation, commonly employed slab-gel systems suffer from high time demand, significant sample/antibody consumption, and limited reproducibility. To overcome these limitations, we developed a paper-based open microfluidic platform for electrophoretic protein separation and subsequent transfer to protein-binding membranes for immunoprobing. Electrophoresis microstructures were digitally printed into cellulose acetate membranes that provide mechanical stability while maintaining full accessibility of the microstructures for consecutive immunological analysis. As a proof-of-concept, we demonstrate separation of fluorescently labeled marker proteins in a wide molecular weight range (15-120kDa) within only 15min, reducing the time demand for the entire workflow (from sample preparation to immunoassay) to approximately one hour. Sample consumption was reduced 10- to 150-fold compared to slab-gel systems, owing to system miniaturization. Moreover, we successfully applied the paper-based approach to complex samples such as crude bacterial cell extracts. We envisage that this platform will find its use in protein analysis workflows for scarce and precious samples, providing a unique opportunity to extract profound immunological information from limited sample amounts in a fast fashion with minimal hands-on time.

Network Analysis of Different Exogenous Hormones on the Regulation of Deep Sowing Tolerance in Maize Seedlings.

The planting method of deep sowing can make the seeds make full use of water in deep soil, which is considered to be an effective way to respond to drought stress. However, deep sowing will affect the growth and development of maize (Zea mays L.) at seedling stage. To better understand the response of maize to deep sowing stress and the mechanism of exogenous hormones [Gibberellin (GA3), Brassinolide (BR), Strigolactone (SL)] alleviates the damaging effects of deep-sowing stress, the physiological and transcriptome expression profiles of seedlings of deep sowing sensitive inbred line Zi330 and the deep-tolerant inbred line Qi319 were compared under deep sowing stress and the conditions of exogenous hormones alleviates stress. The results showed that mesocotyl elongated significantly after both deep sowing stress and application of exogenous hormones, and its elongation was mainly through elongation and expansion of cell volume. Hormone assays revealed no significant changes in zeatin (ZT) content of the mesocotyl after deep sowing and exogenous hormone application. The endogenous GA3 and auxin (IAA) contents in the mesocotyl of the two inbred lines increased significantly after the addition of exogenous GA3, BR, and SL under deep sowing stress compared to deep sowing stress, while BR and SL decreased significantly. Transcriptome analysis showed that the deep seeding stress was alleviated by GA3, BR, and SLs, the differentially expressed genes (DEGs) mainly included cellulose synthase, expansin and glucanase, oxidase, lignin biosynthesis genes and so on. We also found that protein phosphatase 2C and GA receptor GID1 enhanced the ability of resist deep seeding stress in maize by participating in the abscisic acid (ABA) and the GA signaling pathway, respectively. In addition, we identified two gene modules that were significantly related to mesocotyl elongation, and identified some hub genes that were significantly related to mesocotyl elongation by WGCNA analysis. These genes were mainly involved in transcription regulation, hydrolase activity, protein binding and plasma membrane. Our results from this study may provide theoretical basis for determining the maize deep seeding tolerance and the mechanism by which exogenous hormones regulates deep seeding tolerance.

Identification of key genes and pathways in benign prostatic hyperplasia.

Benign prostatic hyperplasia (BPH) is one of the most common causes of lower urinary tract symptoms (LUTS) in elderly man. However, the underlying molecular mechanisms of BPH have not been completely elucidated. We identified the key genes and pathways by using analysis of Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were identified using edgeR. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed for the DEGs by Database for Annotation, Visualization and Integrated Discovery (DAVID) database and ConsensusPathDB, respectively. Then, protein-protein interaction (PPI) networks were established by the Search Tool for the Retrieval of Interacting Genes (STRING) database and visualized by Cytoscape software. Finally, we identified 660 DEGs ultimately including 268 upregulated genes and 392 downregulated genes. GO analysis revealed that DEGs were mainly enriched in extracellular exosome, identical protein binding, mitochondrial adenosine triphosphate (ATP) synthesis coupled proton transport, extracelluar matrix, focal adhesion, cytosol, Golgi apparatus, cytoplasm, protein binding, and Golgi membrane. Focal adhesion pathway, FoxO signaling pathway, and autophagy pathway were selected. Ubiquitin-conjugating enzyme E2 C (UBE2C), serine/threonine kinase (AKT1), mitogen-activated protein kinase 1 (MAPK1), cyclin B1 (CCNB1), polo-like kinase 1 (PLK1) were filtrated as the hub genes according to the degree of connectivity from the PPI network. The five hub genes including UBE2C, AKT1, MAPK1, CCNB1, PLK1 may play key roles in the pathogenesis of benign prostatic hyperplasia (BPH). Focal adhesion pathway, FoxO signaling pathway, and autophagy pathway may be crucial for the progression of BPH.

Phencynonate S-isomer as a eutomer is a novel central anticholinergic drug for anti-motion sickness

To compare and evaluate the differences of stereoselective activity, the binding affinity, metabolism, transport and molecular docking of phencynonate isomers to muscarinic acetylcholine receptor (mAChR) were investigated in this study. The rotation stimulation and locomotor experiments were used to evaluate anti-motion sickness effects. The competitive affinity with [3H]-QNB and molecular docking were used for studying the interactions between the two isomers and mAChR. The stereoselective mechanism of isomers was investigated by incubation with rat liver microsomes, a protein binding assay and membrane permeability assay across a Caco-2 cell monolayer using a chiral column HPLC method. The results indicated that S-isomer was more effective against motion sickness and had not anxiogenic action at therapeutic doses. S-isomer has the higher affinity and activity for mAChR in cerebral cortex and acted as a competitive mAChR antagonist. The stereoselective elimination of S-isomer was primarily affected by CYP1B1 and 17A1 enzymes, resulting in a higher metabolic stability and slower elimination. Phencynonate S isomer, as a eutomer and central anticholinergic chiral drug, is a novel anti-motion sickness drug with higher efficacy and lower central side effect. Our data assisted the development of a novel drug and eventual use of S-isomer in clinical practice.

Transition from curvature sensing to generation in a vesicle driven by protein binding strength and membrane tension.

The ability of proteins to sense and/or generate membrane curvature is crucial for many biological processes inside the cell. We introduce a model for the binding and unbinding of curvature inducing proteins on vesicles using Dynamic Triangulation Monte Carlo (DTMC) simulations. In our study, the interaction between membrane curvature and protein binding is characterised by the binding affinity parameter μ, which indicates the interaction strength. We demonstrate that both sensing and generation of curvature can be observed in the same system as a function of the protein binding affinity on the membrane. Our results show that at low μ values, proteins only sense membrane curvature, whereas at high μ values, they induce curvature. The transition between sensing and generation regimes is marked by a sharp change in the μ-dependence of the protein bound fraction. We present ways to quantitatively characterise these two regimes. We also observe that imposing tension on the membrane (through internal excess pressure for liposomes) extends the region of curvature sensing in the parameter space.

P024 Automated solid-phase HLA antibody assay using the BioTek ELx50 microplate washer

Aim Our laboratory currently uses the manual method to perform the wash steps of the Luminex-based single antigen bead (SAB) assay for HLA antibody analysis. Automation of the wash steps using an automated microplate washer with a vacuum filtration module could be beneficial by reducing tech-to-tech variability and hands-on time. The goal of this study was to investigate the use of this instrument toward the automation of the SAB assay. Methods 5 well-characterized samples with antibody profiles containing both HLA Class I and Class II specificities were analyzed in duplicate within run and on 3 separate runs by means of the LABScreen SAB assay (One Lambda, Inc.). Tests were performed with the BioTek ELx50 microplate washer (BioTek Instruments, Inc.) with a MultiScreen 96-well filter plate with a 1.2 μm low protein-binding Durapore membrane (EMD Millipore Corp.), which performed all wash buffer dispensations, aspirations and incubations. The acquisition was performed on the same Luminex 100 instrument. The results obtained with the microplate washer were compared to results obtained using the manual method. Calculations of % coefficient of variation (CV%) were adapted from the NCCLS global consensus guidelines. Results The BioTek ELx50 microplate washer showed consistent results in comparison with the manual method. The MFI CV% values for each sample within run and between run was calculated. No well failures were observed and antibody strength specificities assignments were comparable in all samples. In addition, antibody specificities (MFI > 2000) showed similar MFI and CV% values between the manual and BioTek ELx50-washed samples. Conclusions We have demonstrated the ability of the BioTek ELx50 microplate washer to automate the LABScreen SAB assay. Automation using this microplate washer speeds processing, improves washing efficiency, increases sensitivity and improves accuracy by eliminating tech to tech variability.

Generalized Born and Explicit Solvent Models for Free Energy Calculations in Organic Solvents: Cyclodextrin Dimerization.

Evaluation of solvation (binding) free energies with implicit solvent models in different dielectric environments for biological simulations as well as high throughput ligand screening remain challenging endeavors. In order to address how well implicit solvent models approximate explicit ones we examined four generalized Born models (GB(Still), GB(HCT), GB(OBC)I, and GB(OBC)II) for determining the dimerization free energy (ΔG(0)) of β-cyclodextrin monomers in 17 implicit solvents with dielectric constants (D) ranging from 5 to 80 and compared the results to previous free energy calculations with explicit solvents ( Zhang et al. J. Phys. Chem. B 2012 , 116 , 12684 - 12693 ). The comparison indicates that neglecting the environmental dependence of Born radii appears acceptable for such calculations involving cyclodextrin and that the GB(Still) and GB(OBC)I models yield a reasonable estimation of ΔG(0), although the details of binding are quite different from explicit solvents. Large discrepancies between implicit and explicit solvent models occur in high-dielectric media with strong hydrogen bond (HB) interruption properties. ΔG(0) with the GB models is shown to correlate strongly to 2(D-1)/(2D+1) (R(2) ∼ 0.90) in line with the Onsager reaction field ( Onsager J. Am. Chem. Soc. 1936 , 58 , 1486 - 1493 ) but to be very sensitive to D (D < 10) as well. Both high-dielectric environments where hydrogen bonds are of interest and low-dielectric media such as protein binding pockets and membrane interiors therefore need to be considered with caution in GB-based calculations. Finally, a literature analysis of Gibbs energy of solvation of small molecules in organic liquids shows that the Onsager relation does not hold for real molecules since the correlation between ΔG(0) and 2(D-1)/(2D+1) is low for most solutes. Interestingly, explicit solvent calculations of the solvation free energy ( Zhang et al. J. Chem. Inf. Model . 2015 , 55 , 1192 - 1201 ) reproduce the weak experimental correlations with 2(D-1)/(2D+1) very well.

A Filter Binding Assay to Quantify the Association of Cyclic di-GMP to Proteins.

Cyclic di-GMP (c-di-GMP) is a ubiquitous second messenger that regulates many processes in bacteria including biofilm formation, motility, and virulence (Hengge, 2009). Analysis of c-di-GMP binding properties of bacterial proteins is an important step to characterize c-di-GMP signaling pathways. C-di-GMP binds numerous proteins such as transcription factors, enzymes, and multimeric protein complexes (Hickman and Harwood, 2008, Ryjenkov et al., 2006, Weinhouse et al., 1997). The c-di-GMP binding assay described here is a relatively simple and cost effective method to characterize c-di-GMP binding to a protein using [32P]-labeled c-di-GMP. Radiolabeled c-di-GMP is readily synthesized with a purified GGDEF enzyme [such as WspR from Pseudomonas aeruginosa (P. aeruginosa)] and [a-32P]-GTP (Srivastava et al., 2013). After incubation of the labeled c-di-GMP with the protein of interest in solution, the resulting mixture is filtered through a nitrocellulose protein binding membrane. The amount of labeled c-di-GMP that is retained on the membrane indicates the interaction between the signal and protein. The specificity of c-di-GMP binding can be tested by competing with unlabeled c-di-GMP or other nucleotides such as GTP in the reaction. By examining binding of a fixed protein concentration to increasing concentrations of c-di-GMP, this method is able to determine the dissociation constant of c-di-GMP-protein interaction.

Demonstration of aeroallergenicity of fungal hyphae and hyphal fragments among allergic rhinitis patients using a novel immunostaining technique

Abstract More than 80 species of fungi are suspected of inducing immunoglobulin E (IgE)-mediated hypersensitivity. Exposure to airborne fungal conidia has been linked to the respiratory symptoms in individuals with fungal allergy; however, the contribution of airborne fungal hyphae and hyphal fragments to allergic diseases is poorly understood. We sought to investigate the expression of allergens from airborne fungal hyphae and hyphal fragments using the halogen immunoassay, which uses patients’ serum IgE to immunostain immobilized allergens extracted from individual fungal particles. Airborne fungi were collected from the nasal cavities of 25 patients and 10 controls using the refined nasal wash technique, fixed on mixed cellulose ester protein-binding membranes, incubated overnight in a humid chamber to promote the germination of conidia, and immunostained with the participants own serum IgE. The samples were examined by means of light microscopy, and positively immunostained fungal particles were classified and counted. All samples contained fungal particles that expressed soluble allergens and were significantly higher in concentration than counts of conidia of individual well-characterized allergenic genera (P < 0.05). Resultant immunostaining of fungal hyphae was heterogeneous, and ∼27% of all hyphae expressed detectable allergens compared with nonstained hyphae (P < 0.05). This study conclusively demonstrates that fungal hyphae and fragments are underestimated sources of aeroallergens because positively immunostained hyphal fragments were detected in all samples and the number of the detected fungal hyphae in any of the individual protein-binding membranes was significantly higher than the conidial counts in any of the commonly recognized aeroallergenic species.

Surveillance of fungal allergic sensitization using the fluorescent halogen immunoassay

OBJECTIVE: Conidia derived from a small number of common fungal genera are widely accepted as the etiological agents responsible for fungal allergic sensitization. The contribution of fungal conidia, spores, airborne hyphae, and subcellular fragments from other uncharacterized fungal genera remains unclear. In this proof-of-concept study, we examined the composition of mycoaerosols that atopic women were exposed and sensitized to in their own indoor environment using the fluorescent halogen immunoassay (fHIA). PATIENTS AND METHODS: Mycoaerosols were collected onto mixed cellulose ester protein binding membranes (PBMs) for 30 minutes with volumetric air sampling pumps. The PBMs were laminated with an adhesive cover slip and indirectly immunostained with individual patient serum IgE using the fHIA. Samples were examined using confocal laser scanning microscopy and immunostained particles were expressed as a percentage of total particles. RESULTS: All air samples contained a broad spectrum of fungal spores, conidia, hyphae, and other fungal particulates. Airborne concentrations varied between individual study participant environments. Positively immunostained conidia belonging to moniliaceous amerospores, Cladosporium, Alternaria, and many unknown species were observed in the majority of air samples. Other fungal genera including Bipolaris, Curvularia, Pithomyces, and Stachybotrys, in addition to, ascospore genera and dematiaceous hyphal fragments released detectable allergen. Twelve percent of all fHIA haloes quantified in the analysis were directed towards fungal particles. No immunostaining was detected to conidia belonging to Epicoccum, Fusarium, and Spegazzinia species. CONCLUSION: In addition to characterized fungal aeroallergens, we observed a wider composition of fungi that bound human IgE. Field surveillance studies that utilize immunodiagnostic techniques such as the fHIA will provide further insight into the diversity of fungi that function as aeroallergen sources in individual study participant environments.

High-Capacity, Protein-Binding Membranes Based on Polymer Brushes Grown in Porous Substrates

The use of atom transfer radical polymerization to grow poly(2-hydroxyethyl methacrylate) (PHEMA) brushes in porous alumina followed by functionalization of the PHEMA with nitrilotriacetate−Cu2+ complexes yields membranes that adsorb proteins via coordination of Cu2+ to histidine residues. Adsorption isotherms show that these membranes have binding capacities as high as 0.9 mg of bovine serum albumin (BSA)/cm2 of external membrane surface area (150 mg/cm3 of membrane), and breakthrough curves indicate that saturation of the membranes with BSA or myoglobin occurs in less than 15 min. The efficiency of protein elution with ethylenediaminetetraacetic acid (EDTA) solutions is essentially 100%, and the membranes show no detectable decrease in capacity over nine cycles of binding, elution, and regeneration with Cu2+. The unusually high capacity of these membranes for rapid protein binding makes them attractive for applications such as purification of His-tag proteins.

Halogen Immunoassay, a New Method for the Detection of Sensitization to Fungal Allergens; Comparisons with Conventional Techniques

Accurate diagnosis of allergy to specific fungal species is confounded by the variability in allergens occurring with different diagnostic systems. We compared the halogen immunoassay (HIA), which uses allergens expressed by freshly germinated spores that are bound to protein binding membranes (PBM), with the commercial Pharmacia UniCap assay (CAP) and with skin prick tests (SPT). Serum from 60 subjects was used; 30 were SPT positive and sensitized to at least one of Alternaria alternata or Aspergillus fumigatus and the other 30 were SPT negative to these fungi but known to be sensitized to non-fungal allergens. All sera were analyzed by CAP against A. alternata, A. fumigatus, Cladosporium herbarum and Epicoccum purpurascens. For HIA, spores from reference cultures belonging to these four species were germinated on PBM, laminated and then probed with each serum. Two independent observers using an ordinal ranking system quantified the intensity and occurrence of the resultant immunoglobulin E (IgE) immunostained haloes around spores and this was statistically compared with the results of the two conventional immunodiagnostic techniques. Germinated conidia of each species expressed detectable allergen in the HIA. The agreement between the ordinal rank scores assigned by the pair of observers was very good (k >or= 0.8) and only differed for A. fumigatus (k = 0.66) . Between 3% and 7% of SPT negative sera was identified by HIA to have specific IgE towards A. fumigatus and A. alternata. For all four species tested there were strong correlations between HIA and CAP (P < 0.0001). However the correlation of both HIA and CAP to SPT was weaker for A. alternata (r(s) = 0.44, P < 0.0153) and absent for A. fumigatus. Overall, the HIA is a new immunodiagnostic technique for the detection of sensitization to fungal allergens that correlates significantly with CAP and to a lesser extent with SPT. This may be due to extract variability and system differences. The significance of this derives from the unique ability of the HIA to measure IgE antibodies to the undegraded allergens that are actively secreted by germinating conidia and hyphae. These are the natural agents of exposure to fungi, and as such, are most likely to be relevant to clinical disease.

Fungal fragments and undocumented conidia function as new aeroallergen sources

More than 100 genera of fungal conidia are currently recognized as sources of allergens. The contribution of other fungal genera plus airborne fungal hyphae and fragmented conidia to allergic diseases is poorly understood. We sought to investigate the expression of allergens from airborne wild-type fungi using the Halogen immunoassay, which uses allergic serum IgE to immunostain immobilized allergens extracted from individual fungal particles. Airborne fungi were collected onto mixed cellulose ester protein-binding membranes for 2.5 hours with volumetric air pumps. Collected fungi were incubated overnight in a humid chamber to promote the germination of conidia. The membranes were laminated with an adhesive cover slip and immunostained with an Alternaria species-sensitive serum IgE pool. The samples were examined by means of light microscopy, and positively immunostained fungal particles were classified and counted. All air samples contained fungal hyphae that expressed soluble allergens and were significantly higher in concentration than counts of conidia of individual well-characterized allergenic genera (P < .05). Resultant immunostaining of fungal hyphae was heterogeneous, and approximately 25% of all hyphae expressed detectable allergen compared with nonstained hyphae (P < .05). Fungal conidia of 10 genera that were previously uncharacterized as allergen sources were shown to demonstrate IgE binding to expressed antigens and accounted for 8% of the total airborne conidia count. Our analysis of wild-type fungi collected indoors presents a new paradigm of natural fungal exposure, which, in addition to commonly recognized species, implicates airborne hyphae, fragmented conidia, and the conidia of a much more diverse range of genera as airborne allergens.

Immunological identification of types I and III collagen in bovine lens epithelium and its anterior lens capsule

To define the molecular structure of bovine lens epithelium and its anterior lens capsule, we investigated the composition of lens capsule basement membrane proteins. Immunofluorescence and immunogold techniques were used to demonstrate the presence of type I and type III collagen in the lens capsule and in primary explant epithelial cultures grown on protein-binding membranes. Immunofluorescence staining with specific antibodies indicated that type I and type III collagen were constituents of lens basement membrane. We observed that deposition of type III collagen was more than type I collagen. The synthesis of fibrillar collagen by lens epithelium and its deposition in the lens capsule was established by localization of fibrillar collagen by transmission immunoelectron microscopy. These results demonstrate for the first time that normal lens epithelium synthesize fibrillar collagen which is an intrinsic component of the anterior lens capsule basement membrane.

Competitive Immunoassay for Microliter Protein Samples with Magnetic Beads and Near-Infrared Fluorescence Detection

A competitive immunoassay with near-infrared (NIR) fluorescence detection to analyze microliter biological samples with an amol limit of detection (LOD) is described. An important feature about this technique is that the immunoreaction and fluorescence detection are separated into two distinct steps, allowing for independent optimization. In the immunoreaction step, NIR fluorescence-labeled antigen (Ag) competes with the unlabeled analyte (Ag) for antibodies (Ab) immobilized on the surface of paramagnetic beads. A magnet is then used to separate the bound antigen from the free in the supernatant. As the amount of Ag in the sample increases, there is less binding between Ag and immobilized Ab; therefore, the amount of Ag in the supernatant is proportionally related to the amount of Ag in the sample. In the fluorescence detection step, aliquots of the supernatant are concentrated onto a protein binding membrane by a capillary blotting technique with an optimized 33 nL/min flow rate. The fluorescence of the blotted spots is detected with a NIR sensitive photon counting system that is optimized to an instrumental LOD of 30 000 fluorophore molecules. This competitive assay demonstrates a sample LOD of 400 pg/mL of unlabeled rabbit immunoglobulin G spiked into bovine serum. This design features low sample volumes and reagent consumption.

Diagnosis of fungal allergy: Comparison of detection of allergens from germinating fungi (Halogen method) with Pharmacia UniCap assay and skin prick tests

Rationale Accurate diagnosis of allergy to specific fungal species is confounded by the variability in allergens occurring with different diagnostic systems. We compared the Halogen immunoassay (HIA), where allergens from germinating fungal spores are directly bound to protein binding membranes (PBM), with the Pharmacia UniCap assay (CAP) and with skin prick tests (SPT). Methods Serum from 60 subjects was used; 30 were SPT positive to at least one of Alternaria alternata or Aspergillus fumigatus and another 30 subjects were SPT negative to fungi. All were analyzed by CAP against A. alternata, A. fumigatus, Cladosporium herbarum and Epicoccum nigrum. For HIA, spores from reference cultures belonging to these species were germinated on PBM, laminated and then probed with each serum. The intensity and occurrence of the resultant IgE immunostaining haloes around spores were quantified by two independent examiners using an ordinal ranking system. Results In HIA, the between-examiner ordinal ranking scores only differed for C. herbarum (Wilcoxon non-parametric analysis), p=0.03. Two SPT negative sera were identified by HIA and CAP to be positive to A. alternata. For the SPT positive sera, significant non-parametric correlations (p<0.0001) were found between HIA and CAP scores as follows: A. alternata (r s=0.75), A. fumigatus (r s=0.56), E. nigrum (r s=0.63) and C. herbarum (r s=0.51). Correlations between HIA and SPT were significant for A. alternata (r s=0.62, p<0.0001) but not for A. fumigatus (r s=0.17, p<0.1790). Conclusions Overall, HIA is a new immunodiagnostic technique that correlates significantly with CAP and to a lesser extent with SPT, which may be due to extract variability and system differences.

Protein arrays from cDNA expression libraries.

This chapter describes the production of a cDNA expression library from human fetal brain, the construction of a high-density protein array from such a library, and two applications to screen the array for binding proteins. After producing the library and decollating the expression clones, one can pick thousands of expression clones with a laboratory robot and can deposit them into microtiter plates in an ordered manner. Such ordered clone libraries are the starting material for the construction of a high-density protein array. This array is constructed by spotting the expression clones onto a protein-binding membrane. Following cell growth and induction of protein expression on the membrane, the cell spots are lysed and their recombinant protein immobilized on the membrane. The so-constructed array carries thousands of proteins without the need to clone, express, and spot individual proteins. Such arrays allow one to screen for numerous protein functions in a high-throughput manner.