Curing viruses in Pleurotus ostreatus by growth on a limited nutrient medium containing cAMP and rifamycin

Oyster mushroom spherical virus (OMSV) and oyster mushroom isometric virus (OMIV) are the causative agents of a fruiting body deformation disease in the edible mushroom Pleurotus ostreatus. The curing of these mycoviruses was facilitated by a serial transfer of infected mycelia onto a limited nutrient medium containing 1mM of cAMP and 75 μg/ml of rifamycin (cAMP-rifamycin plate). The mycelia were grown on cAMP-rifamycin plates for 5 successive passages. ELISA and RT-PCR showed that the amount of mycoviruses inside the mycelia decreased significantly with increasing numbers of passages. The mycelia became free of viruses after 5 successive passages. Cultivation of the virus-cured mycelia on a mushroom compost medium produced a normal harvest, whereas the spawn infected with viruses failed to produce any fruiting bodies.     

Penicillin-specific antibodies: Monoclonals versus polyclonals in ELISA and in an optical biosensor

Two penicillin-specific monoclonal antibodies mAb 19C9 and mAb 9H3 and the penicillin-specific polyclonal antibodies pAb K2 were evaluated for their use in a competitive ELISA and in the BIAcore™ optical biosensor. In the ELISA, an ampicillin-protein conjugate was used as a coating molecule. For the biosensor assay, ampicillin was immobilized on a CM5 chip. With both monoclonal antibodies and in both test systems, ampicillin, amoxicillin and benzylpenicillin were better recognized than oxacillin, cloxacillin and dicloxacillin. Because the reproducibility was better in the biosensor (CV = 1.6%) than in the ELISA (CV = 8.9%), the limit of detection for ampicillin in buffer solution using mAb 19C9 was lower in the biosensor (46 ng ml^-1) as compared to the ELISA (356 ng ml^-1). Ampicillin can thus be detected below the MRL (50 ng ml^-1) in the biosensor assay but not in the ELISA. Both the ELISA and biosensor assay using the polyclonal antibodies pAb K2 were more sensitive as compared to the assays with the monoclonals. The ELISA using pAb K2 allowed the detection of all tested penicillins below the MRL. In the biosensor assay, ampicillin was also detected below the MRL (IC50 = 10 ng ml^-1). In contrast to the binding of the monoclonals, no spontaneous dissociation was observed after injection of the polyclonal antibodies in the biosensor. Whereas the monoclonals were completely removed from the sensor surface using ampicillin in the buffer solution as regeneration solution, stronger conditions were necessary for the pAb binding.     

Localized surface plasmon resonance biosensor integrated with microfluidic chip

A sensitive and low-cost microfluidic integrated biosensor is developed based on the localized surface plasmon resonance (LSPR) properties of gold nanoparticles, which allows label-free monitoring of biomolecular interactions in real-time. A novel quadrant detection scheme is introduced which continuously measures the change of the light transmitted through the nanoparticle-coated sensor surface. Using a green light emitting diode (LED) as a light source in combination with the quadrant detection scheme, a resolution of 10⁻⁴ in refractive index units (RIU) is determined. This performance is comparable to conventional LSPR-based biosensors. The biological sensing is demonstrated using an antigen/antibody (biotin/anti-biotin) system with an optimized gold nanoparticle film. The immobilization of biotin on a thiol-based self-assembled monolayer (SAM) and the subsequent affinity binding of anti-biotin are quantitatively detected by the microfluidic integrated biosensor and a detection limit of 270 ng/mL of anti-biotin was achieved. The microfluidic chip is capable of transporting a precise amount of biological samples to the detection areas to achieve highly sensitive and specific biosensing with decreased reaction time and less reagent consumption. The obtained results are compared with those measured by a surface plasmon resonance (SPR)-based Biacore system for the same binding event. This study demonstrates the feasibility of the integration of LSPR-based biosensing with microfluidic technologies, resulting in a low-cost and portable biosensor candidate compared to the larger and more expensive commercial instruments.     

Direct Versus Competitive Biosensor Immunoassays for the Detection of (Dihydro)Streptomycin Residues in Milk

A monoclonal antibody (MAb) against dihydrostreptomycin (4G8) was developed and its performance compared with a previously developed MAb against streptomycin (4E2) in biosensor immunoassays (BIAs) using a surface plasmon resonance (SPR)-based biosensor (BIACORE 3000). Direct BIAs for the detection of dihydrostreptomycin (DHS; 583 Da) and streptomycin (STREP; 581 Da) were developed by immobilising the MAbs on the sensor chip (CM5). These direct BIAs were compared with competitive inhibition BIAs, using a STREP- protein conjugate immobilized on the chip. The sensitivities of the direct and competitive BIAs for both drugs in buffer were comparable (10-20 ng ml- 1 at 50% binding or inhibition). With milk, interferences, probably due to the nonspecific binding of proteins to the sensor chips, were observed in both BIAs. These interferences could be largely reduced using ultra filtration (UF) as sample pre-treatment. Another option was the use of a reference flow channel to correct for nonspecific binding. Using this option with five times diluted milk, MAb 4G8 was found to be suited for the direct BIA of both drugs with a limit of detection (LOD) of 20 ng ml- 1 and both MAbs could be applied in the competitive BIA format with similar LODs.     

Novel assay format permitting the prolonged use of regeneration-based sensor chip technology

A polyclonal antibody raised against morphine-3-glucuronide (M3G, the main metabolite of heroin and morphine) was used in the development of a novel assay format using a surface plasmon resonance (SPR)-based biosensor. Previously developed assays have generated calibration curves based on differences in the quantity of response units binding to the surface of a chip coated with the analyte. The novel assay described here was based on the development of a standard curve using the slope of a series of consecutive binding interactions. Using this format, regeneration between each assay cycle was no longer required. This increased the useable life span of the chip surface and, as a result, decreased the cost associated with the assay. Thus, at least 15 binding interactions could be carried out before the saturation of antibody on the surface of the chip caused the response to deviate significantly from linearity. After 15 nonregenerated binding interactions, the slope still remained within 1.5% of the slope after a single binding event. Analysis time, and the sample volumes required were also markedly decreased while sensitivity was enhanced. The inhibition assay developed had a detection range of 270 to 17,500 pg ml(-1).     

Solid state cultivation--an efficient method to use toxic agro-industrial residues

Studies were carried out to screen twelve strains of Lentinus edodes for their efficiency to grow on toxic agro-industrial residues of coffee industry in solid state cultivation (SSC). Based on best mycelial growth (7.57 mm/day) and biomass production (48.78 mg/plate in 12 days at 24 degrees C) in coffee husk extract medium, a strain, L. edodes LPB 02 was selected for mushroom cultivation in SSC on coffee husk (treated and untreated), coffee spent ground, and a mixed-substrate comprising coffee husk and coffee spent ground (1:1). SSC was carried out under different conditions of moisture and spawn rate. Spawn rate of 10% and moisture level of 55-60% was found suitable for all the substrates. Treatment of the coffee husk with hot water was found useful for its utilization by the fungus. Results showed that there was an increase in the protein content and decrease in the fibre content of the substrates after SSC. Fruiting bodies were obtained from the treated coffee husk, spent ground and mixed-substrate, and the biological efficiency achieved was 85.8, 88.6 and 78.4% for these substrates, respectively. However, no fruiting body was obtained with raw coffee husk was used as the substrate. Results showed that after SSC, there was a decrease of about 27, 40 and 24% in caffeine and about 18, 49 and 12% in tannin contents in the treated coffee husk, coffee spent ground and mixed substrate, respectively. No caffeine or, tannins were found in fruiting body indicating their degradation by the fungal strain.     

Estimation of the relative affinity of B cell receptor by flow cytometry

We have developed a simple method using flow cytometry to estimate the relative affinity of B cell receptor (BCR) possessing the hapten-binding activity. Bovine serum albumin (BSA) was conjugated with a hapten, (4-hydroxy-3-nitrophenyl)acetyl (NP) and biotin (NP-BSA-bio). The interaction between NP-BSA-bio and anti-NP monoclonal antibodies (mAb) was studied as a model of the BCR reaction by surface plasmon resonance (SPR) using a biosensor chip immobilized with mAbs through anti-Fc antibody (Ab). The relative affinity of these mAbs was estimated on the basis of resonance units for the binding of NP(0.5)-BSA-bio(21) relative to that of NP(7.4)-BSA-bio(21) expressed as a ratio (NP(0.5)-BSA-bio(21)/NP(7.4)-BSA-bio(21)). In combination with streptavidin (SA)-R-phycoerythrin (PE), we measured the binding of NP-BSA-bio to BCR by flow cytometry and found that a high number of biotin molecules was necessary to improve the sensitivity of detection of the bound NP-BSA-bio without steric hindrance in the NP-BCR interaction. We demonstrated that the ratio of the mean fluorescence intensity (MFI) of NP(0.5)-BSA-bio(21)/NP(7.4)-BSA-bio(21) at a concentration of 10(-8) M could be used as a practical measure of the affinity. This method is expected to be useful for the study of affinity maturation on the cellular level.     

Molecularly imprinted based surface plasmon resonance nanosensors for microalbumin detection

Human serum albumin (HSA) is a major blood plasma protein also found in urine where its existence may be a marker of some types of liver or kidney dysfunction. Herein, we fabricated a novel surface plasmon resonance (SPR) nanosensor for selective, sensitive, and label-free microalbumin detection both in aqueous and urine sample solutions. First, HSA-imprinted nanoparticles were synthesized, which consist of ethylene glycol dimethacrylate and N-methacryloyl-L-leucine methyl ester as a cross-linker and functional monomer. The nanoparticles were characterized by zeta-size and scanning electron microscope analyses and were dropped onto the SPR chip surface to make HSA sensitive nanosensor. Characterization studies of HSA-imprinted SPR chip were carried out by atomic force microscopy, Fourier-transform infrared spectroscopy, contact angle, and ellipsometer. The limit of detection and limit of quantification values of HSA-imprinted SPR nanosensor were calculated as 0.7?pM and 1.9?pM for the concentration range of 0.15–500?nM. Selectivity studies of HSA-imprinted SPR nanosensor were achieved with hemoglobin and transferrin proteins which were chosen as competitor molecules. HSA-imprinted SPR nanosensor was displayed highly selective and sensitive to HSA.     

Identification of the core residues of the epitope of a monoclonal antibody raised against glycoprotein D of herpes simplex virus type 1 by screening of a random peptide library

Random peptide libraries (RPL) displayed on the surface of a filamentous bacteriophage can be used to identify peptide ligands that interact with target molecules. We have used a 15-amino acid residue RPL displayed on bacteriophage M13 to identify the core residues within the epitope of a monoclonal antibody (mAb) A16 which interacts with a continuous epitope restricted to amino acid residues 9 to 19 in the N-terminal region of glycoprotein D of herpes simplex virus type 1 (gD-1). The single peptide sequence obtained after three rounds of selection contained identical residues at three positions compared to the authentic gD-1 sequence. Synthetic peptides were prepared based on the sequence of the original epitope and the phage-derived epitope. The binding constants (K_a) with mAb A16 were determined using surface plasmon resonance (SPR) biosensor technology. The RPL-derived peptide and peptide 9–19 of gD-1 had approximately the same affinity for mAb A16. This suggests that those residues within the epitope that are essential for binding were identified. The synthesis of shorter versions of the RPL-derived peptide restricted the binding region to seven amino acid residues. These results show that minimal information retrieved from the screening of an RPL combined with peptide synthesis can characterize the epitope of an mAb with high resolution. Immunization of mice with the phage-derived peptide protected against a challenge with a lethal dose of herpes simplex virus type 1 equally well as the gD-1 derived peptide.     

Antitumor activity and macrophage nitric oxide producing action of medicinal herb, Crassocephalum crepidioides

Background

Drugs dedicated to alleviate neurodegenerative diseases like Parkinson??s and Alzheimer??s have always been associated with debilitating side effects. Medicinal mushrooms which harness neuropharmacological compounds offer a potential possibility for protection against such diseases. Pleurotus giganteus (formerly known as Panus giganteus) has been consumed by the indigenous people in Peninsular Malaysia for many years. Domestication of this wild mushroom is gaining popularity but to our knowledge, medicinal properties reported for this culinary mushroom are minimal.

Methods

The fruiting bodies P. giganteus were analysed for its nutritional values. Cytotoxicity of the mushroom??s aqueous and ethanolic extracts towards PC12, a rat pheochromocytoma cell line was assessed by using 3-[4,5-dimethythiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Neurite outgrowth stimulation assay was carried out with nerve growth factor (NGF) as control. To elucidate signaling mechanisms involved by mushroom extract-induced neurite outgrowth, treatment of specific inhibitor for MEK/ERK and PI3K signalling pathway was carried out.

Results

The fruiting bodies of P. giganteus were found to have high carbohydrate, dietary fibre, potassium, phenolic compounds and triterpenoids. Both aqueous and ethanolic extracts induced neurite outgrowth of PC12 cells in a dose- and time-dependant manner with no detectable cytotoxic effect. At day 3, 25???g/ml of aqueous extract and 15???g/ml of ethanolic extract showed the highest percentage of neurite-bearing cells, i.e. 31.7?±?1.1% and 33.3?±?0.9%; respectively. Inhibition treatment results suggested that MEK/ERK and PI3K/Akt are responsible for neurite outgrowth of PC12 cells stimulated by P. giganteus extract. The high potassium content (1345.7?mg/100?g) may be responsible for promoting neurite extension, too.

Conclusions

P. giganteus contains bioactive compounds that mimic NGF and are responsible for neurite stimulation. Hence, this mushroom may be developed as a nutraceutical for the mitigation of neurodegenerative diseases.     

表面等离子体共振传感技术及其在临床检验中的应用

表面等离子体共振(SPR)传感技术具有实时、快速、无需标记、无背景干扰和样品无损等优点,被广泛应用于生物技术、医学、环境科学及药物检测等领域.着重介绍了SPR传感相关技术的研究进展,特别总结了SPR芯片的表面修饰技术和SPR传感器联用技术,其中联用技术重点从分子印迹联用技术、免疫分析联用技术及核酸联用技术3个方面及在医疗等临床检验领域中的应用进行了综述.     

Enhanced sensitivity of surface plasmon resonance (SPR) immunoassays using a peroxidase-catalyzed precipitation reaction and its application to a protein microarray

We describe a method to improve the sensitivity of surface plasmon resonance (SPR) immunoassays using a horseradish peroxidase (HRP)-catalyzed precipitation reaction. The precipitation reaction catalyzed by HRP bound to the SPR biosensor surface via a sandwich immunoassay induced a shift in the SPR angle. Human interferon (IFN)-gamma at concentrations ranging from 0.01 to 100 ng/ml was detectable by this method. We also show that this biocatalytic signal amplification method can be applied to SPR imaging (SPRI), in an immunoassay of multiple proteins on a protein microarray format.     

Interleukin-12- and interferon-gamma-mediated natural killer cell activation by Agaricus blazei Murill

Dried fruiting bodies of Agaricus blazei Murill (A. blazei) and its extracts have generally used as complementary and alternative medicines (CAMs). Here, we report that the oral administration of A. blazei augmented cytotoxicity of natural killer (NK) cells in wild-type (WT) C57BL/6, C3H/HeJ, and BALB/c mice. Augmented cytotoxicity was demonstrated by purified NK cells from treated wild-type (WT) and RAG-2-deficient mice, but not from interferon-gamma (IFN-gamma) deficient mice. NK cell activation and IFN-gamma production was also observed in vitro when dendritic cell (DC)-rich splenocytes of WT mice were coincubation with an extract of A. blazei. Both parameters were largely inhibited by neutralizing anti-interleukin-12 (IL-12) monoclonal antibody (mAb) and completely inhibited when anti-IL-12 mAb and anti-IL-18 mAb were used in combination. An aqueous extract of the hemicellulase-digested compound of A. blazei particle; (ABPC) induced IFN-gamma production more effectively, and this was completely inhibited by anti-IL-12 mAb alone. NK cell cytotoxicty was augmented with the same extracts, again in an IL-12 and IFN-gamma-dependent manner. These results clearly demonstrated that A. blazei and ABPC augmented NK cell activation through IL-12-mediated IFN-gamma production.     

Flow-stress-induced discrimination of a K-ras point mutation by sandwiched polymer microsphere-enhanced surface plasmon resonance

The highly sensitive detection of a K-ras point mutation with the aid of DNA-carrying microspheres as a flow-stress receptor is proposed at the surface of a surface plasmon resonance (SPR) biosensor. Single-stranded DNAs were immobilized onto epoxy-group-derivatized gold surfaces and the hybridization of DNA targets was monitored. The subsequent interaction with DNA-carrying micospheres enhanced the SPR response. The increase of flow rate during the event of dissociation changed the amount of detachment of the DNA-carrying microspheres for the mismatched pair. In addition, the viscosity was changed by addition of glycerol to the buffer. The increase of shear stress from the flow resulted in detachment of DNA-carrying microspheres hybridized with the mismatched sequence and increased the ability to discriminate a point mutation. This is a new method which not only increases the lower detection limit of evanescent wave-based biosensors, but also the ability to discriminate a point mutation which is a critical factor for ultrasensitive DNA detection in flow devices.     

Characterization of a novel single-stranded RNA mycovirus in Pleurotus ostreatus

A mycovirus, named oyster mushroom spherical virus (OMSV), was isolated from cultivated oyster mushrooms with a severe epidemic of oyster mushroom Die-back disease. OMSV was a 27-nm spherical virus encapsidating a single-stranded RNA (ssRNA) of 5.784 kb with a coat protein of approximately 28.5 kDa. The nucleotide sequence of the virus revealed that its genomic RNA was positive strand, containing 5784 bases with seven open reading frames (ORF). ORF1 had the motifs of RNA-dependent RNA polymerases (RdRp) and helicase. ORF2 encoded a coat protein. ORF3 to 7 could encode putative polypeptides of approximately 12, 12.5, 21, 14.5, and 23 kDa, respectively, but none of them showed significant similarity to any other known polypeptides. The 5' end of the viral RNA was uncapped and the 3' end was polyadenylated with 74 bases. Genomic structure and organization and the derived amino acid sequence of RdRp and helicase domain were similar to those of tymoviruses, a plant virus group.     

Development and validation of a biosensor-based immunoassay for progesterone in bovine milk

We have developed a rapid automated immunoassay, using the BIACORE surface plasmon resonance (SPR) biosensor, to measure progesterone in bovine milk. The assay was designed as an inhibition assay with progesterone covalently immobilised to the carboxymethyl dextran matrix of a CM5 sensor chip. A fixed amount of monoclonal anti-progesterone antibody 39C5H7 was mixed 9:1 with the sample and the amount of free antibody was then determined using biomolecular interaction analysis (BIA) by injection of the mixture over the immobilised progesterone sensor surface. The assay was designed to cover the concentration range 0.5 to 50 ng/ml. The limit of detection (LOD) was 3.56 ng/ml. Reproducibility of the assay was very good with both intra-assay and inter-assay coefficients of variation <5%. As results become available within minutes of injection and the procedure involves fully automated instrumentation, we believe that this BIA assay for progesterone in milk could be used in-line in the milking parlour and, thus, provide an important tool for reproductive management of dairy cattle to detect heat and predict pregnancy.     

Application of an immunosensor for the detection of the β-lactam antibiotic, cephalexin

Public concern surrounding antibiotic contamination in food and food products has made it imperative to develop analytical methods for their detection. Polyclonal antibodies were used in the development of a surface plasmon resonance (SPR)-based inhibition immunoassay for cephalexin. A conjugate consisting of cephalexin-bovine serum albumin (BSA) was immobilized on the dextran gel surface of the sensor chip. Binding/regeneration studies of antibody to immobilized cephalexin were studied and dissociation of the antibody from the immobilized cephalexin was easily achieved with 10 mmol l^-1 NaOH. Forty surface regeneration cycles were carried out and found to be reproducible with only a 7.4% decrease in binding over this number of regenerations. Model inhibition immunoassays for cephalexin were developed in PBS and spiked milk samples with detection ranges of 4.88 to 2,500 ng ml^-1 and 244 to 3,906 pg ml^-1, respectively.     

Detection of egg yolk antibodies reflecting Salmonella enteritidis infections using a surface plasmon resonance biosensor

A surface plasmon resonance (SPR) biosensor assay was developed on the basis of a lipopolysaccharide antigen of Salmonella enterica serovar enteritidis (S. enterica serovar enteritidis) to detect egg yolk antibodies against S. enterica serovar enteritidis. This biosensor assay was compared to two commercial ELISA kits based on LPS antigen and flagellar antigen. A number of 163 egg yolk and combined egg white and yolk samples from chickens experimentally infected with S. enterica serovar enteritidis and 90 egg yolk and combined egg white and yolk samples from uninfected chickens were analyzed. Receiver operating characteristic analysis of the data calculated a diagnostic sensitivity of 82% and a diagnostic specificity of 100%. The within-day coefficient of variation of a positive internal-control egg yolk was 1%. The SPR biosensor assay was able to detect antibodies in a significantly higher percentage of known positive samples than the commercial ELISA's. The anticipated use of the SPR biosensor assay is to determine the S. enterica serovar enteritidis serostatus of non-vaccinated layer hens.     

Immunobiosensor analysis- of clenbuterol in bovine hair

Due to the potential speed and flexibility of surface plasmon resonance (SPR)-biosensor analysis, the technique is currently being exploited for detection of residues in food. In the present study an antibody-based inhibition assay for analysis of clenbuterol in cattle hair was developed using a Biacore 2000 instrument. Analysis of clenbuterol with and without enhancement with a secondary antibody was compared. A fast extraction method for hair, which enabled direct analysis in the biosensor with no clean up other than microscale ultrafiltration was developed. In contrast to most of the published methods in the area, no organic solvent or solid phase extraction step was needed. The biosensor method was validated by analysis of hair samples from animals treated with clenbuterol and the results were compared to results from GC-MS analysis. The correlation was good (r²=92.7%). The detection limit of the new method was 10 ng g^-1 hair.     

Epitopic characterization of native bovine beta-lactoglobulin

Two monoclonal antibodies (mAbs) (mAb 97 and mAb 117) selected from a panel of 52 mAbs directed against beta-lactoglobulin (BLG) have previously been used to develop a two-site enzyme immunometric assay (EIA) specific for the native form of the protein [J. Immunol. Methods 220 (1998) 25]. In the present work, the conformational epitopes recognized by these two mAbs and by the 50 others have been studied. Firstly, an epitope map was drawn using a surface plasmon resonance (SPR) biosensor: the epitopes were organized in a circle of 11 overlapping and 1 nonoverlapping antigenic regions. Secondly, 55 site-directed BLGA mutants were prepared and tested by ELISA and competitive immunoassay to localize these 12 antigenic regions on the protein molecule. Among them, 20 mutants showed a 10- to 7500-fold decrease in relative affinity for the mAbs of one or several neighbouring regions: their circular dichroism (CD) spectra were identical to the spectrum of wild-type (WT) BLGA. At least one mutant was found for each of the 11 overlapping antigenic regions which circled the molecule and for the nonoverlapping one which was localized near the entrance of the calyx. The two mAbs initially chosen were each directed towards very conformation-dependent epitopes and were thus suitable for monitoring native BLG in food products and manufacturing processes. Other mAb pairs could be used to follow the fate of specific regions of the molecule during denaturation or proteolytic digestion.