A membrane-based ELISA assay and electrochemical immunosensor for microcystin-LR in water samples.

We describe within this paper the development of an affinity sensor for the detection of the cyanobacterial toxin microcystin-LR. The first stage of the work included acquiring and testing of the antibodies to this target. Following the investigation, a heterogeneous direct competitive enzyme-linked immunosorbent assay (ELISA) format for microcystin-LR detection was developed, achieving a detection limit, LLD(80) = 0.022 µg L(-1). The system was then transferred to an affinity membrane sorbent-based ELISA. This was an amenable format for immunoassay incorporation into a disposable amperometric immunosensor device. This membrane-based ELISA achieved a detection limit, LLD(80) = 0.06 µg L(-1). A three-electrode immunosensor system was fabricated using thick-film screen-printing technology. Amperometric horseradish peroxidase transduction of hydrogen peroxide catalysis, at low reducing potentials, versus Ag/AgCl reference and carbon counter electrodes, was facilitated by hydroquinone-mediated electron transfer. A detection limit of 0.5 µg L(-1) for microcystin-LR was achieved. Similar levels of detection could be obtained using direct electrochemical sensing of the dye produced using the membrane-based ELISA. These techniques proved to be simple, cost-effective, and suitable for the detection of microcystin-LR in buffer and spiked tap and river water samples.

Specific protease activity indicates the degree of Pseudomonas aeruginosa infection in chronic infected wounds.

Chronic non-healing wounds are a major health problem with resident bacteria strongly implicated in their impaired healing. A rapid-screen to provide detailed knowledge of wound bacterial populations would therefore be of value and help prevent unnecessary and indiscriminate use of antibiotics-a process associated with promoting antibiotic resistance. We analysed chronic wound fluid samples, which had been assessed for microbial content, using 20 different fluorescent labelled peptide substrates to determine whether protease activity correlated with the bacterial load. Eight of the peptide substrates showed significant release of fluorescence after reaction with some of the wound samples. Comparison of wound fluid protease activities with the microbiological data indicated that there was no correlation between bacterial counts and enzyme activity for most of the substrates tested. However, two of the peptide substrates produced a signal corresponding with the microbial data revealing a strong positive correlation with Pseudomonas aeruginosa numbers. This demonstrated that short fluorescent labelled peptides can be used to detect protease activity in chronic wound fluid samples. The finding that two peptides were specific indicators for the presence of P. aeruginosa may be the basis for a diagnostic test to determine wound colonisation by this organism.

Effects of chelating agent and environmental stresses on microbial biofilms: relevance to clinical microbiology.

AIMS: To determine the effect of pH, temperature, desiccation, ethylenediaminetetraacetic acid (EDTA) and desferrioxamine B (DFO) on Panton-Valentine leukocidin-positive community acquired methicillin-susceptible Staphylococcus aureus (PVL +ve CA-MSSA) biofilm formation. METHODS AND RESULTS: Biofilms from PVL +ve CA-MSSA (clinical isolate) were subjected to pH, temperature, desiccation, EDTA and DFO. PVL +ve CA-MSSA were more resistant to pH and heat than their planktonic equivalents. Desiccation studies demonstrated that PVL +ve CA-MSSA biofilms were more refractory to the treatment than planktonic cells. Significant inhibition of PVL +ve CA-MSSA biofilm formation was observed in the presence of 1 mmol l(-1) EDTA. Low concentrations (2·5 µmol l(-1) ) of DFO enhanced the growth of PVL +ve CA-MSSA biofilms. At higher concentrations (1 mmol l(-1) ), DFO did inhibit the growth but not as much as EDTA. A combination of EDTA and DFO inhibited PVL +ve CA-MSSA biofilm formation at lower concentrations than either alone. CONCLUSIONS: This study demonstrates that PVL +ve CA-MSSA biofilms are resistant to environmental stress but their growth can inhibited effectively by a mixture of EDTA and DFO. SIGNIFICANCE AND IMPACT OF THE STUDY: The inhibition of biofilm formation by PVL +ve CA-MSSA using chelating agents has not been previously reported and provides a practical approach to achieve the disruption of these potentially important biofilms formed by an emerging pathogen.

Asexual sporulation signalling regulates autolysis of Aspergillus nidulans via modulating the chitinase ChiB production.

AIMS: Elucidation of the regulation of ChiB production in Aspergillus nidulans. METHODS AND RESULTS: Mutational inactivation of the A. nidulans chiB gene resulted in a nonautolytic phenotype. To better understand the mechanisms controlling both developmental progression and fungal autolysis, we examined a range of autolysis-associated parameters in A. nidulans developmental and/or autolytic mutants. Investigation of disorganization of mycelial pellets, loss of biomass, extra-/intracellular chitinase activities, ChiB production and chiB mRNA levels in various cultures revealed that, in submerged cultures, initialization of autolysis and stationary phase-induced ChiB production are intimately coupled, and that both processes are controlled by the FluG-BrlA asexual sporulation regulatory pathway. ChiB production does not affect the progression of apoptotic cell death in the aging A. nidulans cultures. CONCLUSIONS: The endochitinase ChiB plays an important role in autolysis of A. nidulans, and its production is initiated by FluG-BrlA signalling. Despite the fact that apoptosis is an inseparable part of fungal autolysis, its regulation is independent to FluG-initiated sporulation signalling. SIGNIFICANCE AND IMPACT OF THE STUDY: Deletion of chiB and fluG homologues in industrial filamentous fungal strains may stabilize the hyphal structures in the autolytic phase of growth and limit the release of autolytic hydrolases into the culture medium.

Analysis of herbicides: demonstration of the utility of enzyme immunoassay verification by HPLC.

Evidence has accumulated that herbicides in the environment present a significant health hazard to the population. Therefore, the levels of heavily used substances such as atrazine and simazine and their metabolites need to be regularly assessed. The objective was to develop a rapid and simple tube ELISA procedure suitable for use in field studies and non-specialized laboratories. The antisera used were polyclonal antibodies raised in sheep against atrazine or simazine amido caproic acid conjugated to bovine serum albumin. The antibodies were first used to construct a two-step competitive ELISA procedure in 96-well microtitre plates. The 96-well format was then adapted to a coated-tube enzyme immunoassay, by immobilization of hapten-gelatine conjugates on polystyrene tubes. This enabled the colour to be read using a basic spectrophotometer. Soil samples were collected from agricultural and non-agricultural sites in Poland. Atrazine and simazine were extracted by liquid extraction from soil and assayed by tube ELISA. In addition, the samples were extracted by solid-phase extraction before analysis by HPLC. The immunoassays and chemical analysis were carried out by different individuals who were unaware of each other's results, which were then compared at the end of the study. Correlation of the two methods was excellent, with R=98.7 and 81.3 for atrazine and simazine, respectively. The immunoassay yielded the same order of results without having to perform solid-phase extraction before analysis. The study has demonstrated that the simple antigen-coated tube assay provides a cost-effective and valuable screening test. Comparison with the more elaborate, heavily labour-intensive HPLC analysis demonstrated that the results obtained by the simpler enzyme-immunoassay tests were within the same order.

Monitoring an antibody affinity chromatography with a label-free optical biosensor technique.

In the production of polyclonal antibody, a purification step is necessary which is often done by affinity chromatography. We present a biosensor system based on reflectometric interference spectroscopy (RIfS) to monitor the quantity and quality in terms of affinity and kinetic constants of the antibody during this procedure. Biosensors are rapid compared with ELISA, which is done in practice and can work fully automated. They provide additional information about the active antibody to protein concentration ratio and the affinity of the antibody. We show how to determine these values very accurate. In addition, we describe a new rapid method to monitor the affinity chromatography in process. This gives the possibility to select antibody fractions with best properties in respect to the application.

Polycystin-1: immunoaffinity isolation and characterisation by mass spectrometry.

Polycystin-1 is a putative 460 kDa membrane protein with a unique structure and is possibly representative of a new family of proteins. Its structure suggests an involvement in cell signalling and cell-matrix interactions. The amino acid sequence of polycystin-1 has to date been predicted from its gene sequence. This, to our knowledge, is the first report of the isolation and analysis of polycystin-1 at the protein level using mass spectrometry to confirm its predicted structure. The availability of purified polycystin-1 will allow a new approach to unravelling the complexity of the cell-cell and cell-matrix interactions of this large molecule in normal cells and its perturbation in disease.

A model to assess the infection potential of jet injectors used in mass immunisation.

Jet injectors are needleless injectors that penetrate skin with high-pressure fluid. They have potential advantages over needles and syringes in mass immunisation programs, but concerns over their capacity to transfer blood-borne viruses have been a barrier to acceptance. Hepatitis B infection can transmit in 10 pl of blood; detection of such low volumes presents severe difficulties to such assessments. A model to assess jet injector safety was developed using injection of an inert buffer into calves and assaying the next injector discharge, representing the next dose of vaccine, for blood using a highly sensitive ELISA. Four injectors were tested: two with reusable heads and direct skin contact, one with single-use injector heads and one where the injector head discharged at a distance from the skin. All injectors tested transmitted significant (over 10 pl) volumes of blood; the volumes and frequency of contamination varied with injector. The source of the contamination was consistent with contamination by efflux of injected fluid and blood from the pressurised pocket in tissue that is formed during injection. This insight should inform the design of safe jet injectors.

Label-free biochemical detection coupled on-line to liquid chromatography

The on-line coupling of a label-free optical biosensor to a HPLC system is described by combining the separation power of HPLC with the specificity of the biosensor system. A highly cross-reactive antibody against the pesticide isoproturon was used as model for affinity proteins. The binding strength of the antibody to the utilized pesticides was characterized with the biosensor, first. In the on-line coupling setup, the eluate of the HPLC was mixed continuously with the antibodies. The presence of antigens was detected by a reduction of the antibody binding to the transducer. This reduced binding was quantified by a differentiation of the sensor signal by applying a Savitzky-Golay algorithm. Limits of detection were found to be in the femtomole range without preconcentration, which is comparable to a study using fluorescence-based biochemical detection.

Safety testing of needle free, jet injection devices to detect contamination with blood and other tissue fluids.

Needle free jet injection guns have been used extensively in both veterinary and human health to deliver both vaccine and drugs, but in recent years, concerns have mounted for their potential to transmit blood borne disease agents among consecutive vaccinates. A Ped-O-Jet type jet injection device was used to deliver serial subcutaneous injections of 0.5 mL saline (as a surrogate for vaccine) into calves and pigs, with intervening ejectates collected in vials to represent what the next vaccinate would have received. An enzyme linked immunosorbant assay was developed to detect species specific albumin as a marker for blood, using calibration standards from known dilutions of bovine or porcine blood. Assay sensitivity of 20 pL/mL corresponded to the estimated minimal chimpanzee infectious dose of 10 pL for hepatitis B virus. The methodology and available results for evaluating the safety of jet injector devices are reported.

Integrated optical surface plasmon resonance immunoprobe for simazine detection.

This paper presents the detailed design and characterisation of a regenerable integrated optical surface plasmon resonance immunoprobe as a detector for the triazine herbicide simazine. A sensor design theoretically optimised for use in the aqueous environment is presented and its fabrication described. Experimental results on the sensitivity to changes in bulk refractive index of the analyte and on non-specific binding of ovalbumin are presented. Binding inhibition immunoassays were conducted for simazine and the lower limit of detection determined to be 0.16 microgram/l using anti-simazine IgG antibodies and 0.11 microgram/l using anti-simazine Fab fragments. A sample test cycle of 20 min was established.

Cross-reactivity between the rheumatoid arthritis-associated motif EQKRAA and structurally related sequences found in Proteus mirabilis.

Cross-reactivity or molecular mimicry may be one of the underlying mechanisms involved in the etiopathogenesis of rheumatoid arthritis (RA). Antiserum against the RA susceptibility sequence EQKRAA was shown to bind to a similar peptide ESRRAL present in the hemolysin of the gram-negative bacterium Proteus mirabilis, and an anti-ESRRAL serum reacted with EQKRAA. There was no reactivity with either anti-EQKRAA or anti-ESRRAL to a peptide containing the EDERAA sequence which is present in HLA-DRB1*0402, an allele not associated with RA. Furthermore, the EQKRAA and ESRRAL antisera bound to a mouse fibroblast transfectant cell line (Dap.3) expressing HLA-DRB1*0401 but not to DRB1*0402. However, peptide sequences structurally related to the RA susceptibility motif LEIEKDFTTYGEE (P. mirabilis urease), VEIRAEGNRFTY (collagen type II) and DELSPETSPYVKE (collagen type XI) did not bind significantly to cell lines expressing HLA-DRB1*0401 or HLA-DRB1*0402 compared to the control peptide YASGASGASGAS. It is suggested here that molecular mimicry between HLA alleles associated with RA and P. mirabilis may be relevant in the etiopathogenesis of the disease.

Evaluation of ELISA microtiter plate-based assays for the direct determination of isoproturon in water samples and soil extracts.

Trials were carried out on the commercially available Millipore isoproturon ELISA microtiter plate tests and on laboratory assays developed by Hirst as part of a Joint European Union research project (BIOPTICAS). The lowest detectable dose (LDD) was determined as three times the standard deviation of the blanks. Depending on the calibration curves obtained on different days with different plates, the LDD varied from 0.020 to 0.064 microgram/L for the Millipore test and from 0.080 to 0.329 microgram/L for the Hirst test. The mean coefficients of variation within a single plate for triplicate determinations of standard solutions in the 0.05 to 0.5 microgram/L range were 5.5 and 3.6% for Millipore and Hirst respectively. Cross-reactivity was studied for mono- and di-demethylated isoproturon, chlortoluron, diuron and linuron. The highest cross-reactivity with both tests was that of mono-demethylated isoproturon (22% for Millipore, 4% for Hirst). This molecule was the only one to show significant cross-reactivity in the Hirst test, whereas in the Millipore test, the di-demethylated isoproturon also cross-reacted (4%). Natural water samples, 19 ground-, 53 lysimetric plate and 47 suction cup water samples, and 32 soil samples were also analysed with the ELISA tests. HPLC with a diode array detector was used as a validated control technique for the natural samples. For each water type, ELISA concentrations of both tests were significantly correlated with the HPLC values (r > or = 0.937; p < 0.001). For the soil extracts, the correlations were also significant (p < 0.001), but the scatter in the data was greater. Overall, the Millipore correlation coefficients were higher than those of Hirst.

Electrochemiluminescence flow injection immunoassay for atrazine.

Antibodies to atrazine were labelled with glucose oxidase and used in colorimetric enzyme linked immunosorbent assays. Transparent aminosilanized indium tin oxide coated glass electrodes were derivatized with aminodextran covalently modified with atrazine caproic acid. The labelled antibodies were used to investigate the derivatized electrodes colorimetrically and the electrodes were use in an electrochemiluminescence flow injection analyser. Electrochemiluminescence immunoassay for atrazine in the range 0-10 ppb showed that it was possible to detect less than 0.1 ppb, the precautionary limit for pesticides in drinking water recommended by the European Commission.

Biochemical aspects of biosensors.

A review of the major types of biological molecules and systems (including antibodies, enzymes and whole cells) on which biosensors can be based is presented. Specific emphasis is placed on a critical assessment of the relative strengths and weaknesses of the respective technologies and on analysis of the importance of practical considerations such as sample interference, signal-to-noise ratio and biomolecule stability. The importance of efficient coupling of the biological and transducer components of a biosensor is highlighted. Future trends and directions in biosensor research and commercial aspects of the technology are also discussed. The article concludes with a summary of current biosensor research activities at the GEC-Marconi Hirst Research Centre.

AMC-anti-FITC conjugates: novel reagents for amplified immunochemical techniques. Immunofluorescent staining of human fibroblasts.

Fluorescein antibodies were labelled with 7-aminocoumarin (AMC) derivatives, the 3-acetic acid and the 3-propionic acid N-hydroxysuccinimide esters. The labelled antibodies were used in conjunction with fluorescein isothiocyanate (FITC) and carboxyfluorescein-conjugated primary and secondary antibodies to develop novel immunofluorescent staining procedures. These methods combine the advantages of the fluorescence properties of AMC and the ready availability of FITC-labelled antisera to provide an amplified fluorescence signal as well as overcoming the photobleaching problems in FITC staining. The method is easy to perform and is expected to make an important contribution to the improvement of the quality of staining achieved with immunofluorescence. Details of the procedure used to stain human fibroblasts with antifibronectin antibodies are reported in order to illustrate the method.

Reduction of the rate of fluorescence decay of FITC- and carboxyfluorescein-stained cells by anti-FITC antibodies.

Anti-fluorescein antibodies were found to prevent the fading of emitted fluorescence from fibroblasts stained with fluorescein-labelled fibronectin antibodies. The prevention of fading is the result of specific binding of the fluorochromes present on the stained cells by the anti-fluorescein antibodies. The sheep anti-FITC antibody used in this study was equally effective in preventing the fading of both FITC- and carboxyfluorescein-labelled fibronectin antibodies. The method is simple, effective, does not interfere with the primary immune reaction, and in addition to preventing the fading of fluorescence it reduced the background fluorescence of the specimens. The procedure is expected to make an important contribution to improving the quality of fluorescence immunohistochemical techniques used in diagnosis.

A single-step method for the purification of anti-FITC antibodies by use of a coumarin immunosorbent.

Monoclonal anti-fluorescein isothiocyanate (FITC) antibody cross-reacts with 7-hydroxy coumarin derivatives conjugated to BSA. This property permitted the affinity purification of monoclonal anti-FITC antibodies from ascitic fluid using an-immunosorbent consisting of a 7-hydroxy coumarin derivative linked to Sepharose 4B. Ascitic fluid was applied to the immunosorbent column and, after washing, the bound antibody was eluted under extremely mild conditions using 3 M MgCl2. Antibody eluted in this manner was greater than 96% pure as assessed by SDS-PAGE. A polyclonal sheep anti-FITC antibody was also purified from serum on the same immunosorbent to greater than 94% purity. This simple and rapid method for the purification of anti-FITC antibodies will find applications in both immunodiagnostic procedures and in studies of hapten-antibody interactions. The affinity constant of the purified monoclonal anti-FITC antibody conjugated to horseradish peroxidase was assessed by ELISA and was found to be 1.5 x 19(9) M-1.

Three-color fluorescence in situ hybridization for the simultaneous detection of multiple nucleic acid sequences.

A method is described for visualizing three nucleic acid sequences simultaneously by in situ hybridization using a new blue immunofluorescent label, amino methyl coumarin acetic acid (AMCA), in combination with green and red fluorescing FITC and TRITC. Three chromosome-specific repetitive probes labeled with either amino acetyl fluorene (AAF), mercury, or biotin were hybridized simultaneously to metaphase chromosomes prepared from human blood lymphocytes or to interphase tumor nuclei. Conditions for the combined use of three immunocytochemical affinity systems as well as the optimal spectral separation of the three fluorescing labels have been determined. Three-color in situ hybridization was applied to the study of numerical chromosome abnormalities as occur in human solid tumors. Further applications of this method in prenatal diagnosis for the detection of aneuploidy of the most frequently involved autosomes, as well as for the quantification of gene copy number and mRNA expression, are discussed.

A sensitive method of detecting proteins on dot and Western blots using a monoclonal antibody to FITC.

Monoclonal antibodies to FITC were produced and shown to be specific for the fluorochrome. Molecular weight marker proteins labelled with FITC could be detected after SDS-PAGE and transfer onto nitrocellulose using anti-FITC followed by an anti-mouse IgG-alkaline phosphatase conjugate. The molecular weight of an antigen common to Legionella pneumophila and recognised by a monoclonal antibody could be determined accurately on a Western blot when FITC labelled markers were used as internal standards. The FITC-anti-FITC system was shown to be extremely sensitive, detecting 23.7 amol of BSA-FITC conjugate (equivalent to 1.42 x 10(7) molecules of FITC) in a dot blot assay.