Optical microchip array biosensor for multiplexed detection of bio-hazardous agents.

An optical waveguide array biosensor suitable for rapid detection of multiple bio-hazardous agents is presented. SpectroSens™ optical microchip sensors contain multiple spatially-separated waveguide channels with integral high-precision Bragg gratings sensitive to changes in refractive-index; selective surface-functionalisation of discrete sensing channels with different antibodies as bio-recognition elements enables selective multi-analyte biological detection. Interactions between target antigens in the test sample and respective surface-immobilised antibodies result in localised changes in refractive-index; the biosensor response manifests as increases in wavelength of light reflected from specific sensing channels. Multiplexed, label-free detection of 8 different biological agents, encompassing bacterial spores, vegetative cells, viruses and proteinaceous toxins has been demonstrated in real-time. Selective detection of Bacillus atrophaeus (BG) spores, Escherichia coli cells, MS2 viruses and ovalbumin (OVA) protein (simulant bio-hazardous agents) was first demonstrated as proof-of-concept; subsequently, detection of Bacillus anthracis (BA) spores (UM23CL2 strain), Franciscella tularensis (FT) cells (live vaccine strain), Vaccinia viruses (heat-killed) and ricin toxin (bio-hazardous agents) was proven. Two optical microchip sensors, each comprising 8 sensing channels were packaged into a single disposable cartridge allowing simultaneous 16-channel data acquisition. The specific antibody deposition sequence used in this study enabled detection of either 4 simulants or 4 bio-hazardous agents using a single consumable. The final device, a culmination of the multidisciplinary convergence of the fields of biology, chemistry, optoelectronics and microfluidics, is man-portable and inherently robust. The performance characteristics of the SpectroSens™ technology platform highlight its potential for exploitation as a 'detect to warn/treat' biodetector in security and defence operations.

Continuous flow microwave-assisted processing and aseptic packaging of purple-fleshed sweetpotato purees.

Pumpable purees from purple-flesh sweetpotatoes (PFSP) were subjected to microwave heating using a 60 kW, 915 MHz continuous flow system, followed by aseptic packaging in flexible containers to obtain a shelf-stable product. Initial test runs were conducted using a 5 kW 915 MHz microwave system to measure dielectric in-line properties and examine the puree temperature profiles. The results demonstrated uniformity in heating of the puree at sterilization temperatures (>121 degrees C), and the dielectric constants and loss factors were within the range of published values for orange-fleshed sweetpotato purees. The pilot-scale test runs in a 60 kW microwave unit produced shelf-stable puree packages stable at room temperature. Polyphenolic content of the PFSP purees were evaluated and the results showed that while total phenolics increased (5.9%) and total monomeric anthocyanins slightly decreased (14.5%) with microwave application, antioxidant activity determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and oxygen radical absorbance capacity (ORAC) assays did not significantly change as a result of microwave processing. Color values showed that microwave-processed samples differed from fresh puree in saturation and hue angle, but not in overall color change. PFSP purees increased in gel strength when microwave processed, packaged, and stored, but the gel could be easily disrupted into flowable purees. Overall, high-quality retention can be obtained by microwave processing and aseptic packaging of PFSP purees so that they can be used as functional food ingredients.

Feasibility of utilizing bioindicators for testing microbial inactivation in sweetpotato purees processed with a continuous-flow microwave system.

Continuous-flow microwave heating has potential in aseptic processing of various food products, including purees from sweetpotatoes and other vegetables. Establishing the feasibility of a new processing technology for achieving commercial sterility requires evaluating microbial inactivation. This study aimed to assess the feasibility of using commercially available plastic pouches of bioindicators containing spores of Geobacillius stearothermophilus ATCC 7953 and Bacillus subtilis ATCC 35021 for evaluating the degree of microbial inactivation achieved in vegetable purees processed in a continuous-flow microwave heating unit. Sweetpotato puree seeded with the bioindicators was subjected to 3 levels of processing based on the fastest particles: undertarget process (F(0) approximately 0.65), target process (F(0) approximately 2.8), and overtarget process (F(0) approximately 10.10). After initial experiments, we found it was necessary to engineer a setup with 2 removable tubes connected to the continuous-flow microwave system to facilitate the injection of indicators into the unit without interrupting the puree flow. Using this approach, 60% of the indicators injected into the system could be recovered postprocess. Spore survival after processing, as evaluated by use of growth indicator dyes and standard plating methods, verified inactivation of the spores in sweetpotato puree. The log reduction results for B. subtilis were equivalent to the predesigned degrees of sterilization (F(0)). This study presents the first report suggesting that bioindicators such as the flexible, food-grade plastic pouches can be used for microbial validation of commercial sterilization in aseptic processing of foods using a continuous-flow microwave system.

Comparison of sensory, microbiological, and biochemical parameters of microwave versus indirect UHT fluid skim milk during storage.

Shelf-stable milk could benefit from sensory quality improvement. Current methods of heating cause flavor and nutrient degradation through exposure to overheated thermal exchange surfaces. Rapid heating with microwaves followed by sudden cooling could reduce or eliminate this problem. The objectives for this study were focused on designing and implementing continuous microwave thermal processing of skim fluid milks (white and chocolate) to compare sensory, microbiological, and biochemical parameters with conventionally prepared, indirect UHT milks. All test products were aseptically packaged and stored at ambient temperature for 12 mo. Every 3 mo, samples were taken for microbiological testing, reactive sulfhydryl determinations, active enzyme analysis, instrumental viscosity readings, color measurements, and descriptive sensory evaluation. Microbiological plate counts were negative on all milks at each time point. Enzymatic assays showed that plasmin was inactivated by both heat treatments. 5,5'-dithio-bis(2-nitrobenzoic acid) analysis, a measure of reactive sulfhydryl (-SH-) groups, showed that the initial thiol content was not significantly different between the microwave-processed and UHT-treated milks. However, both heating methods resulted in an increased thiol level compared with conventionally pasteurized milk samples due to the higher temperatures attained. Sulfhydryl oxidase, a milk enzyme that catalyzes disulfide bond formation using a variety of protein substrates, retained activity following microwave processing, and decreased during storage. Viscosity values were essentially equivalent in microwave- and UHT-heated white skim milks. Sensory analyses established that UHT-treated milks were visibly darker, and exhibited higher caramelized and stale/fatty flavors with increased astringency compared with the microwave samples. Sweet aromatic flavor and sweet taste decreased during storage in both UHT and microwave milk products, whereas stale/fatty flavors increased over time. Sensory effects were more apparent in white milks than in chocolate varieties. These studies suggest that microwave technology may provide a useful alternative processing method for delivery of aseptic milk products that retain a long shelf life.

Recombinant NY-ESO-1 cancer antigen: production and purification under cGMP conditions.

The cancer-testis antigen, NY-ESO-1, has been engineered into a bacterial expression plasmid which incorporates a His6-tag. The plasmid was transfected into E. coli strain BL21 and Master and Working cell banks generated from this expression system. Three 15-litre fermentations were performed under cGMP (code of Good Manufacturing Practice) conditions and the crude NY-ESO-1 tagged protein isolated as solubilised inclusion bodies. A three-step cGMP chromatography process (immobilised metal affinity, anion exchange, and hydrophobic interaction) was utilised to purify the protein. The purified NY-ESO-1 is being used in early stage human cancer vaccine trials in Australia and the U.S.A.

Engineering and characterisation of chimeric monoclonal antibody 806 (ch806) for targeted immunotherapy of tumours expressing de2-7 EGFR or amplified EGFR.

We report the generation of a chimeric monoclonal antibody (ch806) with specificity for an epitope on the epidermal growth factor receptor (EGFR) that is different from that targeted by all other anti-EGFR therapies. Ch806 antibody is reactive to both de2-7 and overexpressed wild-type (wt) EGFR but not native EGFR expressed in normal tissues at physiological levels. Ch806 was stably expressed in CHO (DHFR -/-) cells and purified for subsequent characterisation and validated for use in preliminary immunotherapy investigations. Ch806 retained the antigen binding specificity and affinity of the murine parental antibody. Furthermore, ch806 displayed enhanced antibody-dependent cellular cytotoxicity against target cells expressing the 806 antigen in the presence of human effector cells. Ch806 was successfully radiolabelled with both iodine-125 and indium-111 without loss of antigen binding affinity or specificity. The radioimmunoconjugates were stable in the presence of human serum at 37 degrees C for up to 9 days and displayed a terminal half-life (T(1/2beta)) of approximately 78 h in nude mice. Biodistribution studies undertaken in BALB/c nude mice bearing de2-7 EGFR-expressing or amplified EGFR-expressing xenografts revealed that (125)I-labelled ch806 failed to display any significant tumour retention. However, specific and prolonged tumour localisation of (111)In-labelled ch806 was demonstrated with uptake of 31%ID g(-1) and a tumour to blood ratio of 5 : 1 observed at 7 days postinjection. In vivo therapy studies with ch806 demonstrated significant antitumour effects on established de2-7 EGFR xenografts in BALB/c nude mice compared to control, and both murine 806 and the anti-EGFR 528 antibodies. These results support a potential therapeutic role of ch806 in the treatment of suitable EGFR-expressing tumours, and warrants further investigation of the potential of ch806 as a therapeutic agent.

Topographic mapping of the brain activity of perceived motion.

The study compared electrical brain activity of 6 subjects in 5 different conditions. Absolute theta and alpha power in the frontal, parietal, and occipital areas were analyzed, with significant differences found only in the frontal area. Results suggested that the perception of movement requires higher order cognitive processing outside the occipital area. Implications for education and cognitive research are discussed.

Self-actualization of youth in a summer camp.

The self-actualization scores of 57 youths who attended a summer day camp for gifted students were assessed using the Reflections Of Self by Youth (ROSY). Significant sex differences were confirmed. Contrary to Lewis's significant difference (1996) in mean self-actualization between Grades 7 and 8, self-actualization scores in this study were uncorrelated with grade.

Bond strength of rebonded orthodontic brackets.

A study was undertaken to determine the bond strength of brackets rebonded with a no-mix resin system or a paste-paste resin system. The efficacy of plastic conditioner and Enhance adhesion booster (Reliance Orthodontic Products, Inc., Itasca, Ill.) as an aid in rebonding was also evaluated. Sixty extracted human premolars were divided into two groups based on the two adhesive systems used. Both groups of 30 were subdivided and (1) initial bond, (2) rebond, and (3) rebond using plastic conditioner and adhesion booster. Samples were stressed to bond failure using an Instron machine. Bond separation occurred in the majority of samples at the enamel/resin interface. Mean bond strengths ranged from 78.8 kg cm-2 for rebonding with a no-mix adhesive and no other conditioners, to 182.7 kg cm-2 for initial bonding using a paste-paste adhesive. Rebonding using a paste-paste adhesive with no other conditioners produced a bond strength statistically indistinguishable from initial bonding with either system. Plastic conditioner and adhesion booster failed to improve rebond strength. The data suggest that, given certain circumstances, rebonding is a viable option when a bracket has been debonded.

Confirmed allergic reactions to amide local anesthetics.

Allergic reactions to ester local anesthetics are not uncommon. It is generally accepted that similar reactions to the newer amides do not occur. This assumption should be reconsidered. A confirmed case of true amide allergy is reported.

Conventional immunoassays underestimate anti-GST antibody titre.

This paper highlights problems associated with the quantitation of serum antibody levels to recombinant glutathione S-transferase (GST). Measurement of anti-GST antibodies in conventional immunoassays, where GST is bound directly to the ELISA plate, was found to substantially underestimate the amount of GST-specific antibody levels in test sera. This insensitivity in immunoassay of anti-GST antibodies can be overcome by using any one of several recombinant GST fusion proteins as the coating antigen in ELISA rather than simply GST. Comparison of anti-GST antibody titres assessed by the two procedures indicated that use of unfused GST underestimated the anti-GST antibody titre by more than ten-fold.

Monoclonal anti-H1 histone autoantibodies from unimmunized Balb/c mice. Specificity and VH and VL domain sequences.

Examination of the nuclear reactivities of monoclonal IgM kappa autoantibodies, secreted by GFM-5 1B12 and NU-6 1F12 hybridomas derived from germ-free and nude mice, respectively, demonstrated homogeneous nuclear immunofluorescence staining patterns consistent with the recognition of histones. Under these conditions, GFM-5 1B12 and NU-6 1F12 mAbs produced species non-specific binding to components within the nuclei of mouse, human and Drosophila melanogaster cells. Immunoblotting confirmed the binding of these two autoantibodies to autologous H1 histones as well as bovine and insect H1 histones. Identification of the epitopes bound by GFM-5 1B12 and NU-6 1F12 mAbs within the D. melanogaster H1 histones was undertaken using 248 overlapping octapeptides encompassing the entire sequence of D. melanogaster H1 histones. GFM-5 1B12 mAbs bound several octapeptides derived from the amino- and carboxyl-terminal regions of D. melanogaster H1 histones with accessible KT, AT or VT amino acids. NU-6 1F12 mAbs, which stained nuclei within sections of D. melanogaster lavae, failed to bind to any of the 248 linear octapeptides, implying recognition of a conformational H1 histone epitope by this autoantibody. ELISA analysis of the polyspecific binding properties of GFM-5 1B12 and NU-6 1F12 mAbs demonstrated that both antibodies exhibited unique polyspecificity profiles. GFM-5 1B12 mAbs recognized bovine carbonic anhydrase and mouse IgG1, while NU-6 1F12 bound bovine cardiolipin, rat cytochrome c, Escherichia coli beta-galactosidase, toxoid from Clostridium tetani, mouse IgG1 and the haptens, DNP and FITC from the 24 antigen test panel. Comparison of the VH and VL domain sequences of GFM-5 1B12 and NU-6 1F12 mAbs demonstrated that the variations in autoreactivity and polyspecificity profiles resulted from amino acid variations in the CDRs of the VH and VL domains of these autoantibodies. Significantly, major differences in the VH domain sequences of the NU-5 1F12 and GFM-5 1B12 mAbs suggest that the VH domains may preferentially contribute to the unique specificities of the two anti-H1 histone autoantibodies.

Chancroid in Dallas: new lessons from an old disease.

In June 1986, an unusual number of cases of darkfield negative, nonvesicular, painful genital ulcers were noted in men presenting to the Sexually Transmitted Diseases Clinic of the Dallas County Health Department. Serologic findings were routinely nonreactive in these patients. This clinical presentation was consistent with a diagnosis of chancroid, and empiric therapy with erythromycin proved quite efficacious. A retrospective review of charts revealed several similar presentations in May. After 3 weeks of experimentation with culture media, positive cultures for Haemophilus ducreyi were obtained and confirmed by the Centers for Disease Control, definitively establishing the presence of chancroid in Dallas. By year's end, 383 cases of chancroid had been diagnosed.

Concomitant infection with Neisseria gonorrhoeae and Chlamydia trachomatis in pregnancy.

Gonorrhea is an important marker for endocervical chlamydial infections in nonpregnant women. Concomitant infection rates as high as 50% have been reported. There are few data on concomitant infection rates in pregnant patients. The purpose of this study was to examine the prevalence of endocervical chlamydial infections in pregnant women with gonorrhea. Patients with cervical cultures positive for Neisseria gonorrhoeae at their initial prenatal visit had endocervical specimens for Chlamydia trachomatis culture obtained before anti-gonorrheal therapy. Control patients were selected at random from the same prenatal population. The prevalence of C trachomatis in patients with gonorrhea was significantly greater than that in the control population (46 versus 5%; P less than .001). Patients with gonorrhea were younger, less often married, and more often black than the control population, but these demographic differences did not account for the large difference in the chlamydial prevalence. Erythromycin 500 mg four times daily provided an excellent cure rate without intolerable side effects. Pregnant patients being evaluated or treated for gonorrhea should also be considered at high risk for concomitant cervical chlamydial infection.

High-performance liquid chromatography of amino acids, peptides and proteins. XCIII. Comparison of methods for the purification of mouse monoclonal immunoglobulin M autoantibodies.

A comparison of methods for the purification of naturally occurring mouse monoclonal autoantibodies, of the immunoglobulin M (IgM) isotype, has been performed to determine the optimal strategies for the isolation of IgM from ascites fluid and in vitro tissue culture hybridoma supernatants. In order to quantify each purification procedure, the concentration of IgM in eluted fractions was determined by using a double-sandwich mu-chain-specific anti-IgM enzyme-linked immunosorbent assay, and the purity of the IgM was determined by a bicinchoninic acid-based protein assay and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The most efficient single-step purification was based on size-exclusion chromatography on high-resolution Superose 6 HR 10/30 fast protein liquid chromatography (FPLC) columns. This procedure resulted in recoveries of monoclonal IgMs of ca. 71-86% with purities between 68 and 86%. Single-step chromatography of monoclonal IgM, on Superose 6 FPLC columns resulted in a 21-fold purification of IgM, prepared by the in vitro culture of hybridoma cells in dialysis membrane. Size-exclusion chromatography, performed with Sephacryl S-300 columns, resulted in reduced resolution of monoclonal IgM, with yields of ca. 57-80% and purity of ca. 42-58% compared with the high-resolution Superose 6 FPLC columns. "Non-ideal" size-exclusion chromatography on Superose 6 FPLC columns resulted in selective retention of monoclonal IgMs and elution of IgM with high-ionic-strength buffers in the trailing peak. Recovery of IgM with this strategy was high (ca. 82-92%) but the purity was not comparable to the single-step fractionation of IgM on Superose 6 FPLC columns. Single-step anion- and cation-exchange and mixed-mode hydroxyapatite chromatography resulted in only partial purification of monoclonal IgM with the applied procedures. With these latter separation techniques, monoclonal IgM was eluted with a variety of other ascites fluid or supernatant proteins, including those with apparent molecular weights identical to those of mouse IgG and albumin. Sequential purification of monoclonal IgMs by Mono Q anion exchange, followed by Superose 6 FPLC columns, resulted in a 2- to 3-fold purification of IgM but did not separate IgM from high-molecular-weight contaminants with apparent molecular weights similar to those of alpha 2-macroglobulin and IgG. Enrichment of monoclonal IgM from ascites fluid by ammonium sulphate precipitation revealed increasing IgM recovery with increasing ammonium sulphate final concentrations up to 60%.(ABSTRACT TRUNCATED AT 400 WORDS)

Fluorescent monoclonal antibody compared with carbohydrate utilization for rapid identification of Neisseria gonorrhoeae.

A commercially available fluorescein-conjugated monoclonal antibody (MAb) (Syva Co., Palo Alto, Calif.; Genetic Systems, Seattle, Wash.) against Neisseria gonorrhoeae was compared with a standard cystine Trypticase agar (CTA) sugar utilization method and with three rapid carbohydrate utilization tests, including the Minitek (BBL Microbiology Systems, Cockeysville, Md.), Neisseria-Stat (Richardson Scientific, Dallas, Tex.), and Neisseria-Kwik (Micro-Biologics, St. Cloud, Minn.) systems for the identification of Neisseria species. The MAb correctly identified all 86 clinical isolates of N. gonorrhoeae. Of these 86 isolates, 28 were found later (48 h after the initial inoculation) to be contaminated with non-Neisseria bacteria. In the other four test systems studied, the identification rates for pure and contaminated N. gonorrhoeae cultures were, respectively, as follows: CTA sugars, 88 and 32%; Minitek, 67 and 50%; Neisseria-Stat, 97 and 96%; and Neisseria-Kwik, 80 and 74%. The MAb did not identify any of the 50 nongonoccocal Neisseria isolates tested. The most expensive test system was the MAb, followed by the Neisseria-Kwik, Minitek, Neisseria-Stat, and CTA sugars systems. The MAb appears to be a rapid and accurate method to identify in vitro isolates of N. gonorrhoeae.

H-2Kk and vesicular stomatitis virus G proteins are not extensively associated in reconstituted membranes recognized by T cells.

It is shown that liposomes containing (i) a fluorescein-labeled murine histocompatibility antigen (FITC-H-2Kk) and the G protein of vesicular stomatitis virus or (ii) H-2Kk and fluorescein-labeled viral protein (FITC-G) can elicit H-2-restricted syngeneic antiviral cytotoxic T cells as assayed by 51Cr release from appropriate virus-infected target cells. Fluorescence recovery after photobleaching was used to measure the diffusion coefficients of these reconstituted proteins in four different samples: (i) FITC-H-2Kk; (ii) FITC-H-2Kk and G; (iii) FITC-G; and (iv) FITC-G and H-2Kk. The same rate of lateral diffusion (D = 1 x 10(-8) cm2/sec at 37 degrees C in 25% cholesterol/75% dimyristoylphosphatidylcholine) was obtained in every case. Both proteins, fluorescent as well as nonfluorescent, could be patched by using specific antibodies. When G was patched with antibody, FITC-H-2Kk did not copatch. When H-2Kk was patched with antibody FITC-G did not copatch. These diffusion and patching measurements rule out the possibility that these proteins have either extensive oligomeric associations or strong specific pairwise associations.