Quercetin Alleviates Asthma-Induced Airway Inflammation and Remodeling through Downregulating Periostin via Blocking TGF-ß1/Smad Pathway.

INTRODUCTION: The aim of the study was to discuss whether the anti-asthmatic effect of quercetin is related to periostin and the downstream molecular pathway of quercetin's anti-asthmatic effect. METHODS: We constructed asthmatic mice, sensitized by ovalbumin, and administrated different treatments into mice according to the experimental design. In this study, we mainly observed the inflammatory response, airway fibrosis, and airway hyperresponsiveness in asthmatic mice. Pathological stains (H&E, PAS, and Masson) were performed. We also detected the inflammation factors and fibrosis-related cytokines by enzyme-linked immunosorbent serologic assay. In addition, we also explored the level of periostin by enzyme-linked immunosorbent serologic assay and Western blot. At the same time, TGF-ß1/Smad pathway was also determined by Western blot. RESULTS: A high expression of periostin was found in asthmatic mice, and quercetin decreases periostin content in bronchoalveolar lavage fluid. Quercetin and OC-20 inhibit airway inflammation response, airway fibrosis, and airway hyperreactivity. Quercetin downregulated TGF-ß1/Smad pathway in the lung tissues of asthmatic mice. Anti-asthma role of quercetin is related to periostin. Then deeper mechanical study revealed that inhibiting TGF-ß1 could improve asthmatic symptoms, and quercetin exerted the protective effect on asthmatic mice through inhibition of TGF-ß1/Smad pathway. CONCLUSION: Quercetin provided a protective role against asthma via periostin, manifested by mild inflammatory infiltration, reduced goblet cell proliferation, and reduced airway fibrosis. TGF-ß1/Smad pathway is an important transduction system, participating in the protective effect of quercetin on asthma.

Progress in affinity ligand-functionalized bacterial magnetosome nanoparticles for bio-immunomagnetic separation of HBsAg protein.

Efficient Bio-immunomagnetic separation (BIMS) of recombinant hepatitis B surface antigen (rHBsAg) with high binding capacity was studied using affinity ligand immobilized bacterial magnetosome nanoparticles (Magnetospirillum gryphiswaldense strain MSR-1 bacteria) as an immunomagnetic sorbent. Our results showed immunomagnetic adsorption, acted by affinity interactions with the immobilized monoclonal antibody, offered higher antigen adsorption and desorption capacities as compared with the commercially available immunoaffinity sorbents. Four different ligand densities of the Hep-1 monoclonal antibody were examined during covalent immobilization on Pyridyl Disulfide-functionalized magnetosome nanoparticles for HBsAg immunomagnetic separation. The average of adsorption capacity was measured as 3 mg/ml in optimized immunomagnetic sorbent (1.056 mg rHBsAg/ml immunomagneticsorbent/5.5 mg of total purified protein) and 5mg/ml in immunoaffinity sorbent (0.876 mg rHBsAg/ml immunosorbent/5.5 mg total purified protein during 8 runs. Immunomagnetic sorbent demonstrated ligand leakage levels below 3 ng Mab/Ag rHBsAg during 12 consecutive cycles of immunomagnetic separation (IMS). The results suggest that an immunomagnetic sorbent with a lower ligand density (LD = 3 mg Mab/ml matrix) could be the best substitute for the immunosorbent used in affinity purification of r-HBsAg there are significant differences in the ligand density (98.59% (p-value = 0.0182)), adsorption capacity (97.051% (p-value = 0.01834)), desorption capacity (96.06% (p-value = 0.036)) and recovery (98.97% (p-value = 0.0231)). This study indicates that the immunosorbent approach reduces the cost of purification of Hep-1 protein up to 50% as compared with 5 mg Mab/ml immunoaffinity sorbent, which is currently used in large-scale production. As well, these results demonstrate that bacterial magnetosome nanoparticles (BMs) represent a promising alternative product for the economical and efficient immobilization of proteins and the immunomagnetic separation of Biomolecules, promoting innovation in downstream processing.

Development of immunosorbents coupled on-line to immobilized pepsin reactor and micro liquid chromatography-tandem mass spectrometry for analysis of butyrylcholinesterase in human plasma.

Human butyrylcholinesterase (HuBuChE) has been widely used as a biomarker of exposure to organophosphorus (OPs) warfare agents. Indeed, intoxication by OPs can be proven by LC-MS/MS analysis of a specific HuBuChE nonapeptide on which OPs covalently bind. Therefore, we developed a fast, selective and sensitive on-line set-up for the analysis of HuBuChE from plasma that combines immunoextraction by anti-HuBuChE antibodies, pepsin digestion on Immobilized Enzyme Reactors (IMER) and microLC-MS/MS analysis of the target nonapeptide, FGESAGAAS. Two pepsin-based IMERs were prepared and characterized in terms of grafting and digestion yields and were coupled on-line to microLC-MS/MS analysis. In addition, immunosorbents were prepared by covalent grafting of three anti-HuBuChE antibodies on CNBr-sepharose and epoxy-polymethacrylate supports and packed in precolumns. The best antibody grafting yields were obtained with sepharose-based supports, with grafting yields up to 98%. B2 18-5 monoclonal antibody grafted on sepharose led to the best immunosorbent, with HuBuChE recovery close to 100%. The immunosorbent was introduced upstream of the on-line digestion set-up and immunoextraction of HuBuChE was achieved in 14min while digestion was performed in 20min, allowing detection of the target nonapeptide in less than 1h. The global recovery of the nonapeptide was higher than 42% using the best immunosorbent with a RSD value lower than 7% (n=3). Finally, the limit of quantification evaluated in plasma sample was 2fmol of nonapeptide. This value, corresponding to 0.5fmol of HuBuChE tetramer, is well below the average amount of HuBuChE tetramer in 50µL of plasma (590fmol).

Protein-Grafted Polymers Prepared Through a Site-Specific Conjugation by Microbial Transglutaminase for an Immunosorbent Assay.

Protein-polymer conjugates have been developed in many fields. Most hybrids are composed of one protein attached to one or several polymer chains. The other form of hybrid involves the construction of multiple proteins on one polymer chain, thereby facilitating protein assemblies that provide multivalent effects. Unfortunately, synthetic methods for production of these types of hybrids are limited and challenging because precise control of the conjugation sites is needed. Herein, a novel synthetic polymer that can enzymatically assemble multiple proteins was developed. Polyacrylamide grafted with multiple microbial transglutaminase (MTG)-recognizable peptide derivatives was synthesized, and MTG-catalyzed site-specific conjugation of proteins with the polymer was achieved. The application for immunological biosensing was demonstrated using the assembly of a fusion protein composed of antibody-binding and enzyme moieties. This enzymatic method to synthesize a one-dimensional protein assembly on a synthetic polymer is versatile and can be expanded to a wide range of applications.

Metal-linked Immunosorbent Assay (MeLISA): the Enzyme-Free Alternative to ELISA for Biomarker Detection in Serum.

Determination of disease biomarkers in clinical samples is of crucial significance for disease monitoring and public health. The dominating format is enzyme-linked immunosorbent assay (ELISA), which subtly exploits both the antigen-antibody reaction and biocatalytic property of enzymes. Although enzymes play an important role in this platform, they generally suffer from inferior stability and less tolerant of temperature, pH condition compared with general chemical product. Here, we demonstrate a metal-linked immunosorbent assay (MeLISA) based on a robust signal amplification mechanism that faithfully replaces the essential element of the enzyme. As an enzyme-free alternative to ELISA, this methodology works by the detection of α-fetoprotein (AFP), prostatic specific antigen (PSA) and C-reactive protein (CRP) at concentrations of 0.1 ng mL(-1), 0.1 ng mL(-1) and 1 ng mL(-1) respectively. It exhibits approximately two magnitudes higher sensitivity and is 4 times faster for chromogenic reaction than ELISA. The detection of AFP and PSA was further confirmed by over a hundred serum samples from hepatocellular carcinoma (HCC) and prostate cancer patients respectively.

Accurate Quantification of Disease Markers in Human Serum Using Iron Oxide Nanoparticle-linked Immunosorbent Assay.

Accurate and reliable quantification of biomarkers in the blood is essential in disease screening and diagnosis. Here we describe an iron oxide nanoparticle (IONP)-linked immunosorbent assay (ILISA) for detecting biomolecules in human serum. Sandwich ILISA was optimized for the detection of four important serological markers, IgA, IgG, IgM, and C-reactive protein (CRP), and assessed with normal sera, simulated disease-state sera and the serum samples from patients infected with West Nile virus (WNV) or human herpes virus (HHV). Our study shows that using the detection assay formulated with 18.8 nm wüstite nanocrystals, ILISA can achieve sub-picomolar detection sensitivity, and all four markers can be accurately quantified over a large dynamic range. In addition, ILISA is not susceptible to variations in operating procedures and shows better linearity and higher stability compared with ELISA, which facilitates its integration into detection methods suitable for point of care. Our results demonstrate that ILISA is a simple and versatile nanoplatform for highly sensitive and reliable detection of serological biomarkers in biomedical research and clinical applications.

High background in Luminex® assay for HLA antibody screening: Interest of Adsorb Out™.

INTRODUCTION: The Luminex® technology has become an integral component of clinical decision-making and diagnosis of transplanted organ rejection. Despite the superior sensibility of this technology, it is not completely problem free. We have observed in these bead-based assays that sera of some patients give a high negative control bead (NC) value which makes assessing HLA antibodies difficult. Treatment of sera by the Adsorb Out™ reagent may reduce the high background. In this study, we want to evaluate the effect of the Adsorb Out™ on the NC's MFI value by comparing treated and untreated patients' sera. METHODS: HLA antibody screening was performed on 3011 sera. These sera came from patients awaiting and undergoing renal transplant from different Moroccan hospitals. The sera were analyzed using the standard protocol for Luminex® antibody screening. Sera with high NC's value has been pre-incubated by the Adsorb Out™, and analyzed on Luminex®. RESULTS: 3% of studied samples have high NC's value. The Adsorb Out™ decreases the NC's value and brings it back to a normal range in 62.2% treated sera. It has no effect in 12.3%. The Adsorb Out™ effect depends only of NC's value, independently to age, storage date, sex and immunization. CONCLUSION: The Adsorb Out™ reagent has an important effect in decreasing NC value of sera. However, it has no effect in some patient's sera. In these cases we could try another treatment, as EDTA, DTT. The non-specific binding may be caused by multiple patient-specific factors, it would be important to search correlation between them and NC's values.

Isolation of antibodies against different protein conformations using immunoaffinity chromatography.

A polyclonal antiserum obtained after the immunization of a rabbit with recombinant human sperm-specific glyceraldehyde-3-phosphate dehydrogenase lacking in 68 N-terminal amino acid residues (dN-GAPDS) was purified using different immunosorbents with immobilized dN-GAPDS in the native or denatured states. The procedure resulted in isolation of two types of polyclonal antibodies. The first type interacted with native recombinant dN-GAPDS as well as with native human sperm-specific glyceraldehyde-3-phosphate dehydrogenase, not cross-reacting with muscle glyceraldehyde-3-phosphate dehydrogenase (GAPD). The second type interacted with both native and denatured forms of the sperm-specific proteins, exhibiting some cross-reaction with GAPD. Thus, the suggested approach allows isolation of the antibodies against conformational or linear epitopes from the same polyclonal serum.

Optimization of a nonradioactive immunosorbent assay for p38alpha mitogen-activated protein kinase activity.

Here we describe an optimization of a nonradioactive immunosorbent p38alpha mitogen-activated protein kinase (MAPK) activity assay to determine inhibitory potency of small molecule inhibitors. The assay omits the secondary antibody and, therefore, is shorter, more accurate, and easier to handle (total assay time of 3 h). This direct assay uses a new monoclonal anti-phospho-ATF-2 (Thr69/71) peroxidase-conjugated antibody, increasing specificity and eliminating problems with cross-reactivities of secondary antibodies.

A sensitive immunosorbent bio-barcode assay combining PCR with icELISA for detection of gonyautoxin 2/3.

In the current study, we developed a nanosphere bio-barcode technology to detect trace gonyautoxin 2/3 (GTX 2/3). GTX 2/3-glucose oxidase (GOX) conjugates were first prepared as the coating antigen in a periodate reaction. Subsequently, gold nanoparticles (NP) dual-labeled with anti-GTX 2/3 monoclonal antibodies (Mab) and DNA oligonucleotides were synthesized via a one-step preparation method. Combining PCR with indirect competitive ELISA (icELISA), a novel immunosorbent bio-barcode assay was established utilizing the Mab-NP-dsDNA complex to convert enzymatic signals to DNA signals. Importantly, the limit of detection of the method was lower than 0.74 microg mL(-1). Thus, the immunosorbent bio-barcode assay is a rapid and high-throughput screening tool to detect GTX 2/3 in aquatic products.

A novel affinity ligand for polystyrene surface from a phage display random library and its application in anti-HIV-1 ELISA system.

In order to develop an affinity ligand for site-directed immobilization of target proteins on polystyrene (PS) surface, a linear 12-mer peptide phage display random library was screened. Phage clones that specifically bound to PS plate were sequenced after three rounds of biopanning. The obtained DNA sequences revealed that there were several aromatic and basic amino acid residues, which may be critical to binding. One of the selected dodecapeptides, named Lig1 (FKFWLYEHVIRG), was genetically fused to the N/C-terminus of recombinant antigen ENV which could be recognized by specific antibodies against human immunodeficiency virus type 1 (HIV-1), to investigate its performance as an affinity ligand. The ligand-fused ENVs overexpressed in Escherichia coli were compared to the original one in terms of the immobilization characteristics on PS plate in enzyme-linked immunosorbent assay (ELISA). The results indicated that the ligand-fused proteins showed a considerably improved affinity to PS surface, and were preferentially adsorbed on PS plate suffering only scarcely from interference by coexisting protein molecules. Anti-HIV-1 ELISA system, which employed Lig1-ENV (Lig1 fused to ENV N-terminus) as immobilization antigen also exhibited sufficiently high sensitivity and specificity in serodiagnosis tests.

Immunoadsorption in pemphigus.

The principle of extracorporal immunoadsorption (IA) is based on affinity adsorption of pathogenic (auto-)antibodies and circulating immune complexes (CIC) which reversibly bind to an immobilized ligand of the adsorber. In pemphigus, a blistering autoimmune disease affecting skin and mucous membranes, autoantibodies, mainly of the IgG subclass are directed against desmosomal adhesion molecules and other non-desmosomal antigens on the surface of epidermal keratinocytes, such as acetylcholine receptors. The pathogenicity of these autoantibodies has been shown in various in vitro and in vivo systems. Recently, IA was applied in severe pemphigus demonstrating that a rapid and dramatic decline in desmoglein (Dsg)-reactive autoantibodies is accompanied by clinical remission of mucocutaneous blisters and erosions. As an adjuvant treatment, IA was combined with systemic immunosuppressive medication and current protocols initially apply treatment cycles of 3-4 IAs on consecutive days followed by immunoapheresis once a week or repeating the initial cycle in 4 week intervals depending on the disease activity. IA in pemphigus is generally safe and well tolerated.

Soluble epidermal growth factor receptor acridinium-linked immunosorbent assay.

This chapter provides basic information for quantifying soluble epidermal growth factor receptor (sEGFR) isoforms in human sera using an acridinium-linked immunosorbent assay (ALISA) developed by Baron and coworkers. This ALISA has been shown to be specific for epidermal growth factor receptor (EGFR) and the isoforms of EGFR encoded by alternate transcripts (i.e., sEGFRs); it, therefore, does not detect other EGFR/ErbB receptor family members, i.e., ErbB2, ErbB3, or ErbB4. In addition, this ALISA recognizes EGFR and sEGFR isoforms that contain subdomains I-IV of the extracellular domain, but it does not recognize EGFR isoforms lacking subdomain IV. The ALISA described here also may be useful for determining EGFR and sEGFR concentrations in lysates of cultured cells, conditioned culture medium, or tissue/tumor specimens, as well as in other human body fluids such as serum, plasma, ascites, urine, saliva, and cystic fluids.

Preparation of an immunoadsorbent coupled with a recombinant antigen to remove anti-acetylcholine receptor antibodies in abnormal serum.

An immunoadsorbent that removes anti-acetylcholine receptor antibodies (AChRAb) in abnormal serum of myasthenia gravis (MG) patient was efficiently prepared by an expression product, the functional fragment of AChR(alpha205) fused with maltose binding protein (MBP). The ligand can then covalently bind to amylose resin through MBP fusion protein. It was shown from the result of this study with anti-AChR mice sera that the removal rate of AChRAb on this immunoadsorbent reached 87+/-10% (mean value of 10 mice) and the maximally binding capacity of AChRAb was approximately 260 microg/g immunoadsorbent (wet weight). Moreover, the immunoadsorption test of sera in two MG patients indicated that about 90% and 96% of abnormal AChRAb could be eliminated, while other serum components such as albumin, IgG, IgM and IgA only dropped 18%, 35%, 22%, 15% and 24%, 27%, 15%, 12%, respectively, for two MG patient sera. It is anticipated from this study that the immunoadsorbent reported here could, with further development, find its clinical application for removal of AChRAb from patient serum.

A novel immunoadsorbent for rheumatoid arthritis therapy--preparation and efficacy evaluation.

AIM: To develop a novel immunoadsorbent for rheumatoid arthritis (RA) therapy. METHODS: A RA immunoadsorbent was developed by binding heat-aggregated human IgG(HAHIgG) to porous agar gel beads. Its adsorption capacity for rheumatoid factors (RFs), storage stability and blood compatibility were evaluated. RESULTS: The coupling yield of HAHIgG on the carrier was 6.0 mg/g wet gel. Saturation adsorption capacity of the adsorbent for IgMRF, IgGRF and IgARF were 3400, 2240 and 2400 IU/g, respectively. The adsorbent can be stored at 4 degrees C for three months without significant variance in its activity. Its fine permeability and hemocompatibility were demonstrated by extracorporeal hemoperfusion on rabbits. CONCLUSION: HAHIgG/agar gel is a safe and effective immunoadsorbent for RA therapy, its potential clinical use is promising in the future.

Molecular heterogeneity of free PSA in sera of patients with benign and malignant prostate tumors.

OBJECTIVE: To analyze free prostate-specific antigen (f-PSA) in sera from patients with prostate cancer (PCa) and benign prostatic hyperplasia (BPH), and to detect possible differences in subtypes as potential diagnostic parameters. MATERIALS AND METHODS: PSA was purified from sera by an immunoaffinity procedure developed on the basis of oriented antibody immobilization, and subjected to size exclusion chromatography (SEC), Western blotting, and N-terminal amino acid sequencing. RESULTS: The novel procedure allowed the purification of PSA with high yield from sera containing PSA <10 ng/ml. SEC under nonreducing conditions as well as Western blots demonstrated the presence of several molecular forms of f-PSA. Three of the smaller polypeptides exhibited the N-terminal sequence of PSA while one represented the C-terminal fragment Lys(146)-Pro(237). Shortening of some polypeptides by the N-terminal amino acid Ile(1) suggestive of aminopeptidase action was also observed. No propeptide sequence could be detected, and none of the bands from patient sera reacted with antibodies raised against propeptide antigens. BPH sera expressed higher proportions of smaller PSA fragments per unit p33, and contained significant amounts of fragments <14,000 which appeared to be very low or absent from most PCa sera. CONCLUSIONS: f-PSA as obtained from BPH and PCa sera represents a heterogeneous fraction. The major component (p33) is not in the nicked form and does not contain proPSA. Diagnostic potential could arise from the quantitative differences of the smaller PSA derivatives seen between PCa and BPH sera.

The SH2-containing 5'-inositol phosphatase (SHIP) is tyrosine phosphorylated after Fc gamma receptor clustering in monocytes.

Current models of Fc gamma R signal transduction in monocytes describe a molecular cascade that begins upon clustering of Fc gamma R with the phosphorylation of critical tyrosine residues in the cytoplasmic domains of Fc gamma RIIa or the gamma-chain subunit of Fc gamma RI and Fc gamma RIIIa. The cascade engages several other tyrosine-phosphorylated molecules, either enzymes or adapters, to manifest ultimately an array of biological responses, including phagocytosis, cell killing, secretion of a variety of inflammatory mediators, and activation. Continuing to assess systematically the molecules participating in the cascade, we have found that the SH2-containing 5'-inositol phosphatase (SHIP) is phosphorylated on tyrosine early and transiently after Fc gamma R clustering. This molecule in other systems, such as B cells and mast cells, mediates an inhibitory signal. We find that clustering of either Fc gamma RIIa or Fc gamma RI is effective in inducing SHIP phosphorylation, that SHIP binds in vitro to a phosphorylated immunoreceptor tyrosine-based activation motif, peptide from the cytoplasmic domain of Fc gamma RIIa in activation-independent fashion, although SHIP binding increases upon cell activation, and that Fc gamma RIIb and Fc gamma RIIc are not responsible for the observed SHIP phosphorylation. These findings prompt us to propose that SHIP inhibits Fc gamma R-mediated signal transduction by engaging immunoreceptor tyrosine-based activation motif-containing cytoplasmic domains of Fc gamma RIIa and Fc gamma RI-associated gamma-chain.

Preparation of DNA-immobilized immunoadsorbent for treatment of systemic lupus erythematosus.

A new DNA-immobilized immunoadsorbent was prepared to remove the pathogenic anti-DNA antibody from the serum of systemic lupus erythematosus (SLE) patients. A non-woven poly(ethylene terephthalate) (PET) fabric made of 3.5-microm diameter fibers was used as the support of the immunoadsorbent. A cationic monomer, N,N-dimethylaminoethyl methacrylate (DAM), was graft polymerized onto the PET fiber surface by UV irradiation or with a chemical initiator. Polyion complexation between the cationic groups of the graft chains and DNA molecules was employed to immobilize DNA onto the fiber surface. No DNA leaching was observed when the DNA-immobilized fabrics were placed in 0.9 and 2.0 wt% NaCl solution at 37 degrees C overnight. In vitro evaluation of this DNA-immobilized immunoadsorbent demonstrated that this adsorbent could selectively adsorb anti-DNA antibody from the serum of SLE patients.

Simultaneous extraction of selected benzodiazepines and benzodiazepine-glucuronides from urine by immunoadsorption.

A rapid and selective cleanup procedure based on immunoadsorption is described for the simultaneous extraction of diazepam and its free or glucuronidated metabolites nordiazepam, temazepam, and oxazepam from urine. The method can also be used for the extraction of lorazepam and lorazepam-glucuronide. Because the samples do not have to be hydrolyzed before extraction, valuable information is preserved. With the exception of lorazepam-glucuronide, recoveries between 86 and 100% were obtained at spiking levels up to 200 ng of benzodiazepine or glucuronide per milliliter of urine. Using methanol/water (90:10, v/v) as an eluent, the immunoadsorber could be used at least 20 times. High-performance liquid chromatograms of urine samples from patients receiving low therapeutic dosages of diazepam or lorazepam are shown to demonstrate the high purity of the extracts.

Quantitation of IgG and IgM human anti-mouse antibodies (HAMA) interference in CA 125 measurements using affinity chromatography.

Currently no available immunoassay system offers complete protection against spuriously elevated or lowered results due to interference by Human Anti-Mouse Antibodies (HAMA). Although routine use of chromatography procedures is not an acceptable option because of the extra cost and workload involved, such a procedure would be highly desirable to ensure accurate immunoassay results. The present report describes a relatively simple affinity chromatography procedure using a HiTrap Protein G column to isolate immunoglobulin G (IgG) HAMA, followed by a HiTrap N-hydroxy-succinimide (NHS)-activated column coupled to goat-anti human immunoglobulin M (IgM) to bind IgM HAMA. To examine the usefulness of this purification procedure we determined CA 125 in forty serum samples prior to and following chromatography. Pre- and post-injection samples were obtained from 20 patients injected with 1 mg of 111In-labelled murine OC 125 F(ab')2 fragments in an immunoscintigraphy study. It is shown that this analytical procedure provides a technique to determine the extent and the nature of the existing HAMA interference in samples of patients after in vivo use of monoclonal antibodies for diagnostic or therapeutic purposes. The procedure can also contribute to the clarification of clinically discordant CA 125 results. Finally, the availability of such a procedure in the clinical laboratory provides an opportunity to test the robustness of newly developed immunoassay systems towards HAMA interference.