Serum antibody screening by surface plasmon resonance using a natural glycan microarray

A surface plasmon resonance (SPR) based natural glycan microarray was developed for screening of interactions between glycans and carbohydrate-binding proteins (CBPs). The microarray contained 144 glycan samples and allowed the real-time and simultaneous screening for recognition by CBPs without the need of fluorescent labeling. Glycans were released from their natural source and coupled by reductive amination with the fluorescent labels 2-aminobenzamide (2AB) or anthranilic acid (AA) followed by high-performance liquid chromatography (HPLC) fractionation making use of the fluorescent tag. The released and labeled glycans, in addition to fluorescently labeled synthetic glycans and (neo)glycoproteins, were printed on an epoxide-activated chip at fmol amounts. This resulted in covalent immobilization, with the epoxide groups forming covalent bonds to the secondary amine groups present on the fluorescent glycoconjugates. The generated SPR glycan array presented a subset of the glycan repertoire of the human parasite Schistosoma mansoni. In order to demonstrate the usefulness of the array in the simultaneous detection of glycan-specific serum antibodies, the anti-glycan antibody profiles from sera of S. mansoni-infected individuals as well as from non-endemic uninfected controls were recorded. The SPR screening was sensitive for differences between infection sera and control sera, and revealed antibody titers and antibody classes (IgG or IgM). All SPR analyses were performed with a single SPR array chip, which required regeneration and blocking of the chip before the application of a serum sample. Our results indicate that SPR-based arrays constructed from glycans of natural or synthetic origin, pure or as mixture, can be used for determining serum antibody profiles as possible markers for the infection status of an individual.     

Synthesis of peptides employing 9-fluorenylmethyl chloroformate as a coupling agent

Summary The synthesis of peptides employing 9-fluorenylmethyl chloroformate (Fmoc-Cl) as a coupling agent has been described. The method is simple, efficient and rapid. All the peptides have been obtained in good yield (70–95%). Furthermore, both the¹H NMR and the HPLC studies on Fmoc-Phg-Phe-OMe and Fmoc-D-Phg-Phe-OMe revealed that the coupling is free from racemization.     

Synthesis of peptides employing 9-fluorenylmethyl chloroformate as a coupling agent

The synthesis of peptides employing 9-fluorenylmethyl chloroformate(Fmoc-Cl) as a coupling agent has been described. The method is simple, efficient and rapid. All the peptides have been obtainedin good yield (70–95%). Furthermore, both the ¹H NMR and the HPLC studies on Fmoc-Phg-Phe-OMe and Fmoc-D-Phg-Phe-OMe revealed that the coupling is free from racemization.     

Semi-automatic liquid chromatographic analysis of olpadronate in urine and serum using derivatization with (9-fluorenylmethyl)chloroformate

The semi-automatic bioanalytical assays for olpadronate [(3-dimethylamino-1-hydroxypropylidene)bisphosphonate] involves a protein precipitation with trichloroacetic acid and a double co-precipitation with calcium phosphate for serum samples and a triple calcium co-precipitation for urine samples. These manual procedures are followed by an automated solid-phase extraction on a cation-exchange phase. The procedure is continued either directly, at high olpadronate levels in urine, or after off-line evaporation under nitrogen and reconstitution in water on the same robotic workstation. The continued automatic procedure comprehends derivatization with (9-fluorenylmethyl)chloroformate, ion-pair liquid–liquid extraction and ion-pair HPLC with fluorescence detection at 274/307 nm. The intra- and inter-day precisions for urine and serum samples are typically in the 5–8% range for different olpadronate concentrations [levels near the lower limit of quantification (LLQ) excluded]. The LLQ is 5 ng/ml olpadronate for a 2.5-ml urine sample and 10 ng/ml for a 1-ml serum sample, respectively.     

Glycan microarray profiling of parasite infection sera identifies the LDNF glycan as a potential antigen for serodiagnosis of trichinellosis

Diagnostic methods for parasite infections still highly depend on the identification of the parasites by direct methods such as microscopic examination of blood, stool and tissue biopsies. Serodiagnosis is often carried out to complement the direct methods; however, few synthetic antigens with sufficient sensitivity and specificity are available. Here we evaluated a glycan microarray approach to select for synthetic glycan antigens that could be used for serodiagnosis of parasitic infections. Using a glycan array containing over 250 different glycan antigens, we identified GalNAcβ1–4(Fucα1–3)GlcNAc-R (LDNF) as a glycan antigen that is recognized by antibodies from Trichinella-infected individuals. We synthesized a neoglycoconjugate, consisting of five LDNF molecules covalently coupled to bovine serum albumin (BSA), and used this neoglycoconjugate as an antigen to develop a highly sensitive total-Ig ELISA for serological screening of trichinellosis. The results indicate that glycan microarrays constitute a promising technology for fast and specific identification of parasite glycan antigens to improve serodiagnosis of different parasitic infections, either using an ELISA format, or parasite-specific glycan arrays.     

Determination of taurine in plasma by high-performance liquid chromatography using 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride as a fluorescent labeling reagent

A sensitive high-performance liquid chromatography method for the determination of taurine in human plasma was developed. Taurine and N-methyltaurine (internal standard) were derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride to produce fluorescent sulfonamides. The labeling reaction was carried out at 70 degrees C for 20 min at pH 7.5. The fluorescent derivatives were separated on a reversed-phase column by a stepwise elution using (A) acidic phosphate buffer/acetonitrile (83/17) and (B) acetonitrile and detected by fluorescence measurement at excitation and emission wavelengths of 318 and 392 nm, respectively. The detection limit (signal-to-noise ratio=3) of taurine was 3 fmol per injection. The within-day and day-to-day relative standard deviations were 3.0-4.8 and 2.5-4.7%, respectively. The concentration (means) of taurine in normal human plasma was 48.9+/-7.5 microM.     

Development of a platform for activatable fluorescent substrates of glucose transporters (GLUTs)

We have developed a platform for activatable fluorescent substrates of glucose transporters (GLUTs). We firstly conjugated fluorescein to glucosamine via an amide or methylene linker at the C-2 position of d-glucosamine, but the resulting compounds, FLG1 and FLG2, showed no uptake into MIN6 cells. So, we changed the fluorophore moiety to a fluorescein analogue, 2-Me TokyoGreen, which is less negatively charged. TokyoGreen-conjugated glucosamines TGG1 and TGG2 were successfully taken up into cells via GLUT. We further derivatized TGG1 and TGG2, and among the synthesized compounds, 2-Me-4-OMe TGG showed weak fluorescence under the acidic conditions of the extracellular environment inside tumors and in gastric cancers, and strong fluorescence at the intracellular physiological pH, under the control of a photoinduced electron transfer (PeT) process. This fluorogenic platform should be useful for developing a range of activatable fluorescent substrates targeting GLUTs, as well as derivatives that would be fluorescently activated by various intracellular enzymes, such as esterases, β-galactosidase and bioreductases.     

9-fluorenylmethyl chloroformate as a fluorescence-labeling reagent for derivatization of carboxylic acid moiety of sodium valproate using liquid chromatography/tandem mass spectrometry for binding characterization: a human pharmacokinetic study

In High Performance Liquid Chromatographic (HPLC) determination of chemicals with acidic functions, different labeling agents are used to improve sensitivity of the assay. 9-Fluorenylmethyl chloroformate (FMOC-Cl), on the other hand, is a suitable labeling agent, which reacts with both primary and secondary amines and less readily with hydroxyl groups in alkaline conditions. However, the reagent has not been applied in labeling of chemicals with acidic function yet. In this study which is the first report on application of FMOC-Cl in derivatization and analysis of a drug with acidic function, valproic acid (VPA), one of a series of fatty carboxylic acids with anticonvulsant activity, was derivatized using the reagent and quantified in serum samples by HPLC with fluorescence detection. In addition, to document the reaction between the labeling agent and carboxylic acid moiety of the drug, we developed a liquid chromatography-tandem MS/MS (LC-MS/MS) method. Following liquid-liquid extraction, derivatization of the drug and an internal standard was achieved in alkaline medium. The elute was monitored by a fluorescence detector with respective excitation and emission wavelengths of 265 and 315 nm. The present method is more sensitive comparing with other published HPLC procedures for analysis of VPA. The assay is sensitive enough to measure drug levels obtained in human single dose studies with a limit of quantification of 0.01 μg/mL. Also the method is linear over the concentrations range of 0.01-32 μg/mL of VPA in human serum using 100 μL serum sample and 5 μL injection. The coefficient variation values of both inter and intra day analysis were less than 12% and the percentage error was less than 4%. The method performance was studied and the validated procedure applied in a randomized cross-over bioequivalence study of two different VPA preparations in 24 healthy volunteers.     

Advances in the use of biotinylated diaminopyridine (BAP) as a versatile fluorescent tag for oligosaccharides

We recently described a novel fluorescent compound, 2-amino,6-amidobiotinyl-pyridine(BAP), that allows the tagging of oligosaccharides, their fractionationby reversed-phase HPLC with picomole scale detection, and theformation of functional neoglycoprotein equivalents with (strept)avidin for the detection of receptors and the generation ofmonospedflc antibodies (Rothenberg etaL, Proc. Natl Acad. SetUSA, 90,11939-11943,1993). Here, we describe the enhancementof this approach by the following, (i) A simple one-step purificationof BAP from its synthetic precursors and other reactants. (ii)Development of HPLC sizing column methods to quickly purifyBAP-coupled oligosaccharides away from free BAP and other reactants.(iii) Development of anion-exchange and amine-adsorption HPLCprocedures for the fractionation of BAP-oligo-saccharide adductsby charge and size, respectively, (iv) Investigation of theaffinity of BAP-oligosaccharides for (strept)avidin, confirmingthe formation of stable complexes, (v) The use of BAP for sensitivemonosaccharide compositional analysis of glycoproteins. (vi)Formation of stable BAP adducts without reduction and its implicationsfor the mechanism of adduct formation. These advances make availablea multitude of techniques for the fractionation of BAP-coupledoligosaccharides based on several different physical parameters.Distinct species of BAP-coupled oligosacharides can be isolatedand subjected to detailed structural analysis. Such definedmolecules form stable complexes with streptavidin that are effectivelyneo-glycoproteins, which can be used in a variety of biologicalapplications. Notably, all of these approaches require relativelyinexpensive materials and conventional equipment available Inmost laboratories. analysis biotin fluorescence neoglycoconjugates     

Site-specific biosynthetic incorporation of a fluorescent tag into proteins via cysteine-tRNA(Cys)

Site-specific incorporation of biophysical probes into proteins during translation can permit structure/function studies on selected proteins in heterogeneous environments. We report here a procedure for incorporating a fluorescent tag into proteins via Escherichia coli Cys-tRNA(Cys) during in vitro protein synthesis. Naturally occurring Cys-tRNA(Cys) is an attractive vehicle for fluorophore incorporation since it can be readily prepared in quantity and reacted with commercially available fluorophores. Moreover, proteins can often be constructed with a single Cys so that fluorophore incorporation results in a tag at a unique site.     

Tracing Vibrio parahaemolyticus in oysters (Tiostrea chilensis) using a Green Fluorescent Protein tag

Oysters feed by removing particles from the water. This food is composed of complex mixtures of living microorganisms, detritus, and inorganic particles that widely range in size. It has been speculated that some marine heterotrophic microorganisms, such as Vibrio parahaemolyticus, could enter in this digestive process and persist in the oyster tissue. Since some strains of V. parahaemolyticus are pathogenic for humans, these bacteria are considered to be a constant menace for health and aquaculture. In order to improve the safety of marine products it is imperative to obtain more knowledge about Tiostrea chilensis and its interactions with V. parahaemolyticus. In this study V. parahaemolyticus ATCC 17802 was tagged using plasmid pKV111, which carries the gfp gene that codifies a Green Fluorescent Protein (GFP), thereby allowing these strains (VpGFP) to be detected under epifluorescence microscopy. Results obtained showed that T. chilensis can filter VpGFP directly from sea water and suggested that most of them were digested by oysters. However, in the postharvest stage, a small fraction can remain in oyster tissues after depuration and VpGFP can rapidly grow if the bivalves are stored at room temperature.     

Homogeneous noncompetitive assay of protein via Förster-resonance-energy-transfer with tryptophan residue(s) as intrinsic donor(s) and fluorescent ligand as acceptor

Homogeneous noncompetitive assay of a protein in biological samples based on Förster-resonance-energy-transfer (FRET) was proposed by using its tryptophan residues as intrinsic donors and its specific fluorescent ligand as the FRET acceptor that was defined as an analytical FRET probe. Conjugate of a suitable fluorophore, which should have an excitation peak around 340 nm but an excitation valley around 280 nm, with a moiety binding to a protein of interest gave an analytical FRET probe to the protein. To test this method, N-biotinyl-N′-(1-naphthyl)-ethylenediamine (BNEDA) was used as an analytical FRET probe for homogeneous noncompetitive assay of streptavidin (SAV). The occurrence of FRET between the bound BNEDA and tryptophan residues was supported by the modeled geometry of the complex. By excitation at 280 nm, free BNEDA produced negligible fluorescence at 430 nm, but the bound BNEDA produced much higher stable fluorescence at 430 nm after 2 min of binding reaction. The competitive binding between BNEDA and biotin gave the dissociation constant of (16 ± 3) fM for BNEDA (n = 3). By excitation at 280 nm, fluorescence at 430 nm of reaction mixtures containing 32.0 nM BNEDA responded linearly to SAV subunit concentrations ranging from 0.40 to 30.0 nM with the desirable resistance to common interferences in biological samples. Therefore, by using tryptophan residue(s) in a protein of interest as intrinsic donor(s) and its fluorescent ligand as the corresponding FRET acceptor, this homogeneous noncompetitive assay of the protein in biological samples was effective and advantageous.     

Generation of antibodies recognizing an aberrant glycoform of human tissue inhibitor of metalloproteinase-1 (TIMP-1) using decoy immunization and phage display

Aberrant glycosylation of human tissue inhibitor of metalloproteinase-1 (TIMP-1) by N-acetylglucosaminyltransferase-V (GnT-V) was previously reported to be related to cancer progression. Here, we report on the antibodies recognizing the structural features initiated by an addition of N-linked β(1,6)-N-acetylglucosamine (GlcNAc) branch by GnT-V on TIMP-1. Two glycoforms of TIMP-1, TIMP1-L produced in Lec4 cells without GnT-V activity and TIMP1-B in GnT-V overexpressing transfectant cells, were purified from culture supernatant and used for generation of antibodies. TIMP1-L was injected in the left hind footpad of mice as decoy and TIMP1-B in the right hind footpad as immunogen. Phage-displayed scFv library was constructed from the B cells retrieved from the right popliteal lymph nodes and subjected to panning and screening. Phage ELISA of individual clones revealed the scFv clones with preferential binding activity to TIMP1-B, and they were converted into an scFv-Fc format for further characterization of binding specificity. Glycan binding assay of an antibody, 1-9F, revealed its differential specificity toward an extension of glycan structure initiated with β(1,6)-GlcNAc linkage and terminally decorated with a sialic acid. This study demonstrates feasibility of a new strategy combining decoy immunization with phage display for the efficient generation of antibodies tracking down structural features of different glycoforms.     

Evaluation of a fluorescent amplified fragment length polymorphism (FAFLP) methodology for the genotypic discrimination of Aeromonas taxa

A fluorescent amplified fragment length polymorphism (FAFLP) fingerprinting assay is evaluated for its ability to differentiate DNA hybridization groups in the genus Aeromonas. After empirical determination of optimal assay conditions using a limited set of strains, 98 well-characterized type and reference strains encompassing all known Aeromonas taxa were subjected to FAFLP fingerprinting using the standardized protocol. The present study clearly indicates that the use of fluorescent dye-labeled primers does not significantly affect the high capacity of this technique to differentiate among genotypically closely related Aeromonas taxa. Compared to the original AFLP protocol involving the application of radio-isotopes, the new FAFLP technology offers a better performance when considering speed of analysis and user safety. On the other hand, FAFLP fingerprints exhibited a significant reduction in the relative number of bands compared to the corresponding autoradiographic patterns. In our hands, the omission of the preselective amplification step and the use of a size standard mix enhanced the cost effectiveness and the reproducibility of the technique. Cluster analysis of FAFLP band patterns generated from Aeromonas type and reference strains demonstrated once more the high correlation of AFLP-generated data with DNA-DNA homology data.     

Detection of Legionella species in potting mixes using fluorescent in situ hybridisation (FISH)

This study used Fluorescent in situ Hybridisation (FISH) with rRNA targeted oligonucleotide probes combined with scanning confocal laser microscopy to successfully detect Legionella spp. in commercially available potting mix. A range of techniques were explored to optimise the FISH method by reducing background fluorescence and preventing non-specific binding of probes. These techniques included the use of a blocking agent, UV light treatment, image subtraction of a nonsense probe and spectral unmixing of specific probes fluorescence and autofluorescence dependent on the specific emission spectra of probe fluorophores.Spectral unmixing was the best microscopy technique for reducing background fluorescence and non-specific binding of probes was not observed. The rapid turnaround time and increased sensitivity of the FISH provides as an alternative to traditional culture methods, which are tedious and often give varied results. FISH is also advantageous compared to PCR methods as it provides information on the structure of the microbial community the bacteria is situated in. This study demonstrates that FISH could provide an alternative method for Legionella detection and enumeration in environmental samples.     

Identification of Mycoplasmas using a fluorophore-free microarray and infrared chemical imaging (IRCI)

A novel application of mid-infrared chemical imaging (IRCI) for the fluorophore-free detection and identification of mycoplasma species is reported for the first time. The PCR-amplified biotinylated targets hybridized to microarray probes were treated with streptavidin-gold nanoparticles followed by silver enhancement. This modification has the potential to expand the implementation of DNA microarray techniques in laboratories involved in the detection of cell substrates, other biological products, and clinical materials for the presence of mycoplasmas.     

Biodiesel production from rubber seed oil using poly (sodium acrylate) supporting NaOH as a water-resistant catalyst

Poly (sodium acrylate) supporting NaOH (NaOH/NaPAA) was prepared by in situ polymerization of aqueous solution of acrylic acid with an over-neutralization by adding excess of NaOH. NaOH/NaPAA presented a promising selectivity for water absorbency and good water retention with negligible swelling capacity in the organic solvents of methanol, glycerol, rubber seed oil methyl esters, and rubber seed oil. NaOH/NaPAA catalysts showed a basic strength of 15.0 < H_ < 18.4 and their basicity increased with the increase of the NaOH loading amount. NaOH/NaPAA catalysts exhibited almost the same catalytic activity in the transesterification of rubber seed oil with methanol under the optimized reaction conditions compared to conventional homogeneous NaOH catalyst. Furthermore, the functional absorbent/catalyst system presented a good water resistance in the transesterification which retained high catalytic activity when a water concentration in the reaction system was less than 2 wt.%.     

Benzoxazinone — kanamycin derivative: a new fluorescent probe for flow cytometry analysis of bacteria (Agrobacterium tumefaciens)

Abstract A new polycation fluorescent dye (BVC-kinamycin-conjugate) has been synthesized and used to detect alive bacteria by flow cytometry. This fluorescent chromophore has the noteworthy property of being excited at the same wavelength as fluoresceinylated conjugates (488 nm) and to show a much longer emission wavelength (616 nm) than fluoresceinylated derivates (520 nm); furthermore its fluorescence intensity is not quenched at low pH in contrast with fluorescein. In such conditions, bacteria can easily be detected with cytofluorimeter equipped with a single excitation wavelength beam. The binding of fluoresceinylated lectins and antibodies onto a strain of Agrobacterium tumefaciens has been studied by this method.     

Visualization of sialyl Lewis(X) glycosphingolipid microdomains in model membranes as selectin recognition motifs using a fluorescence label

Selectin-induced leukocyte rolling along the endothelial surface is an essential step in the cellular immune response. For efficient recognition, the relevant carbohydrate epitope sialyl Lewis(X) (sLe(X); alpha-Neup5Ac-(2-->3)-beta-Galp-(1-->4)-[alpha-Fucp-(1-->3)]GlcpNAc) has to be arranged in clusters. We describe the synthesis of the sLe(X)-glycosphingolipid (sLe(X)-GSL) with a NBD fluorescence label in the tail region, which allows the direct visualization of sLe(X)-GSL microdomains to very low concentrations (0.01mol%) in various planar phosphocholine matrices by fluorescence microscopy. Cell rolling experiments of E-selectin expressing cells along these membranes confirmed that the fluorescence analog behaves similar to the naturally occuring sLe(X)-GSL. This is direct evidence for recent hypotheses on multivalent sLe(X) binding as molecular basis for selectin-mediated cell rolling.     

Formation of the nanostructure on a silica surface as mercury(II) ions adsorption sites

The present work investigates the adsorptive interactions of Hg(II) ions with hydroxylated silica, aminopropylsilica and silica chemically modified by β-cyclodextrin in aqueous medium. Batch adsorption studies were carried out with various agitation time and mercury(II) concentration. The maximum adsorption was observed within 15-30 min of agitation. The kinetics of the interactions, tested with model of Lagergren for pseudo-first and pseudo-second order equations, showed better agreement with first order kinetics (k₁ = 3.4 ± 0.2 to 5.9 ± 0.3 min⁻¹). The adsorption data gave good fits with Langmuir isotherms. The results have shown that β-cyclodextrin-containing adsorbent has the largest adsorption specificity to Hg(II) : K_L = 14 400 ± 700 L/mg. “β-Cyclodextrin-” inclusion complexes with ratio 1:1 and super molecules with composition С₄₂H₇₀O₃₅ · 3Hg(NO₃)₂ are formed on the surface of β-cyclodextrin-containing silica.