Optical scattering artifacts observed in the development of multiplexed surface enhanced Raman spectroscopy nanotag immunoassays.

Here we describe scattering based signal suppression artifacts encountered while developing multiplex lateral flow (LF) immunoassay using surface enhanced Raman spectroscopy (SERS) "nanotags" as analyte labels. Using these SERS nanotags, we have produced a quantitative test for inflammation biomarkers that is transferable to the point of care (POC). The SERS assay shows similar performance when compared with a fluorescent nanoparticle POC test. Here, using cardiac and inflammation biomarkers, we highlight the need to carefully optimize the concentration of assay components when using SERS nanotags and a single-line multiplexing approach. We show that in certain circumstances the SERS signal may be suppressed, leading to a significant underestimation of the analyte concentrations. Using electron microscopy and optical spectroscopy, we demonstrate that the error in the measurement is associated with the light scattering properties of the nanotags. These findings will be applicable to other nanoparticle labels with high light scattering coefficients. Through careful modification of the assay to reduce the impact of light scattering, it is possible to produce quantitative assays, but potentially at the expense of multiplexing capability and assay sensitivity.

Rapid duplex immunoassay for wound biomarkers at the point-of-care.

In this study we describe a novel method of sampling and quantifying wound biomarkers for clinical settings. We believe the chosen format will allow rapid assessments of wound healing and provide biomarker evidence-based decision points for treatment of the wound at the time of presentation. The wound monitoring principle uses a proprietary sample collection tool (a thermally reversible hydrogel) to sample and isolate biomarkers within a wound environment without further sample extraction/preparation steps. We show how gel samples can be analysed in a lateral flow assay format utilising fluorescent microspheres with optically discrete emission characteristics and demonstrate quantitative detection of two analytes (duplexing) achieved in a single test line. As a model assay, the chronic wound biomarkers interleukin 6 (IL6) and tumour necrosis factor alpha (TNFα) are used. Limits of detection of 48.5 pg/mL and 55.5 pg/mL respectively in hydrogel samples and 7.15 pg/mL and 10.7 pg/mL respectively in plasma are reported. We believe this is the first literature example of quantitative detection of multiple analytes within a single test line using spectral separation to distinguish the analytes.

The diagnostic accuracy of different natriuretic peptides in the investigation of canine cardiac disease.

OBJECTIVES: We aimed to validate and determine the accuracy of a new sandwich ELISA for canine N-terminal pro-B-type natriuretic peptide (NT-proBNP) in the discrimination of canine patients with cardiac disease from those with respiratory disease and to determine the effect of confounding variables on NT-proBNP concentrations. METHODS: Validation studies for the new assay were undertaken. Concentrations of N-terminal atrial natriuretic peptide (NT-proANP) and NT-proBNP in both ethylenediaminetetraacetic acid (EDTA) plasma and serum were estimated in samples from 77 dogs at a laboratory blinded to the clinical status of the patient. The diagnostic accuracy of the each sample type and test was evaluated using receiver operating characteristic curves. The effect of age, gender and indicators of renal function was evaluated using a multivariate regression analysis. RESULTS: Concentrations of NT-proBNP in both serum and plasma accurately discriminated dogs with respiratory disease from those with cardiac disease, with an optimum cut-off concentration of 210 pmol/l. NT-proBNP concentrations were unaffected by sample type. Increasing creatinine concentration is associated with increasing concentration of NT-proBNP. Age and gender were not found to have significant effects on natriuretic peptide concentrations in this population. CLINICAL SIGNIFICANCE: Canine NT-proBNP appears to be a useful marker of the presence of cardiac disease, although concentrations must be interpreted in the light of the patient's renal function.

Clinical validation of a proANP 31-67 fragment ELISA in the diagnosis of heart failure in the dog.

Atrial natriuretic peptide (ANP) is a polypeptide hormone found in increased concentrations in the plasma of dogs with heart failure. However, problems arise in using ANP as a diagnostic marker for heart failure because of its short half-life in plasma, proteolysis post-collection and the necessity for a radioimmunoassay. The diagnostic utility of a proANP 31-67 ELISA for the detection of heart failure in dogs was evaluated using plasma collected from 31 dogs with clinical and radiographic signs of heart failure and control samples from 40 dogs considered to be free of cardiac disease. Log proANP 31-67 levels were significantly higher in the heart failure group (P < 0.001). In this population of dogs, using a cut-off value of 1,750 fmol/ml, the sensitivity and specificity of the assay were 83.9 per cent and 97.5 per cent, respectively. Using a cut-off of 1,350 fmol/ml, the sensitivity and specificity were 93.5 per cent and 72.5 per cent, respectively. It is concluded that a proANP 31-67 fragment ELISA should prove to be a useful diagnostic aid in naturally occurring canine heart failure.

Induction of microspore-derived embryos of Brassica napus L. with polyethylene glycol (PEG) as osmoticum in a low sucrose medium.

Isolated microspores of Brassica napus were cultured on high concentrations of mannitol or polyethylene glycol (PEG 4000), with only a very limited amount of sucrose (0.08-0.1%) provided as carbohydrate source in the medium. While microspores cultured on high mannitol yielded no embryos and no embryogenic cell divisions were observed, microspores on high PEG developed into embryos within 2 weeks, and the embryo yield appeared comparable to that of the sucrose control. When placed under light, PEG embryos quickly changed color from yellow to dark green, while sucrose embryos first remained yellowish and then slowly changed color to pale green. Three-week-old PEG embryos were strikingly similar to immature zygotic embryos developed in ovulo, dissected at 14-15 days post-anthesis (DPA), while sucrose embryos differed from the latter in the size and shape, color and morphology of their cotyledons. These results demonstrate that in microspore embryogenesis of Brassica napus: (1) the level of metabolizable carbohydrate required for microspore embryo induction and formation appears to be substantially less than commonly used amounts, (2) sucrose as an osmoticum can be replaced with high-molecular-weight PEG. With further improvement the new method described here might be suitable for other Brassica species and would have a great potential application in breeding programs.

Visualization of Golgi apparatus in methacrylate embedded conifer embryo tissue using the monoclonal antibody JIM 84.

Methacrylate embedding followed by resin removal has been used for the first time to visualize a membrane-associated antigen at the tissue level. Monoclonal antibody JIM 84 was used to stain the Golgi apparatus of gymnosperm (conifer) embryos by light microscope immunocytochemistry. Specificity of labelling was confirmed by electron microscope immunocytochemistry using LR-white resin. GA staining was evident in all stages of white spruce somatic embryo development from immature to mature. Some regions of the somatic embryos (e.g. root cap/suspensor region) stained more vigorously than other regions (hypocotyl/cotyledon end). GA also stained in roots of Monterey pine and Douglas fir. Unlike the situation in most angiosperms, JIM 84 antigen appears to be absent from the conifer plasma membrane. However, it appears to be present in representatives of both major classes of higher plants.

Production of vigorous, desiccation tolerant white spruce (Picea glauca [Moench.] Voss.) synthetic seeds in a bioreactor.

This report describes a low-cost method for generating large numbers of high quality mature white spruce (Picea glauca [Moench.] Voss) somatic embryos which survived desiccation and grew to plantlets more vigorously than excised zygotic embryos cultured in vitro. Somatic embryos from suspension culture were supported within a culture chamber on a flat absorbent pad above the surface of a liquid culture medium containing 20-50 µM abscisic acid and 7.5 % polyethylene glycol. Throughout a 7 week culture period 3 L of fresh medium was pumped into one end of the chamber, while the spent medium exited by gravity from the opposite end. Over 6,300 cotyledonary stage white spruce somatic embryos were recovered after this time from a single culture chamber without manual manipulation. The somatic embryos were of excellent appearance with well developed cotyledons, and possessed high levels of storage lipids. They survived drying to about 8 % moisture content following treatment for 4 weeks at 63 % relative humidity, and following imbibition converted to normal plantlets at a frequency of 92 %, compared to 80 % for embryos grown in Petri dishes. Somatic embryos cultured within the bioreactor developed to plantlets that were 20 % longer than zygotic embryos excised from mature seed and grown in vitro, and were 38 % longer than somatic embryos cultured upon agar medium in Petri dishes.

Scanning electron microscopy of hydrated and desiccated mature somatic embryos and zygotic embryos of white spruce (Picea glauca [Moench] Voss.).

Four scanning electron microscope techniques for preparing somatic and zygotic embryos of white spruce (Picea glauca [Moench] Voss.) were compared. Direct sputter coating without critical point drying worked well for desiccated embryos while conventional methods using chemical fixation were appropriate for hydrated somatic embryos. Low temperature scanning electron microscopy and plastic replicas provided excellent specimens of all embryos studied. Plastic replicas were used to document cotyledon formation and growth during maturation of somatic embryos. Apart from some differences in embryo size, orientation of cotyledons and surface wrinkling, the general morphology of mature somatic embryos of white spruce was very similar to zygotic embyros at a similar stage of development.

Manipulation of conditions for the culture of somatic embryos of white spruce for improved triacylglycerol biosynthesis and desiccation tolerance.

In order to enhance post-germinative vigour, somatic embryos of Picea glauca (Moench) Voss. were matured under in-vitro conditions that stimulated triacylglycerol (TAG) biosynthesis. In P. glauca seeds over 90% of the TAG was stored within the megagametophyte, and isolated zygotic embryos contained twice the amount of TAG of somatic embryos cultured for four weeks on basal medium containing 16 µM abscisic acid (ABA). Polyethylene glycol-4000 (PEG) as a non-permeating osmoticum with ABA promoted TAG biosynthesis by somatic embryos and sustained maturation throughout an eight-week culture period. Treatments that promoted TAG biosynthesis also prevented precocious germination and promoted desiccation tolerance. Thus, the optimal culture conditions for maturation, desiccation survival, and plantlet regeneration were 16-24 µM ABA and 7.5% PEG for eight weeks, followed by desiccation. Under these conditions the levels of TAG per somatic embryo were raised ninefold to about five times the zygotic-embryo level, and the TAG fatty-acid composition became similar to that of zygotic embryos. A study of sectioned material, using light and transmission electron microscopy, showed that the structure and distribution of lipid bodies within these somatic embryos and the degree of embryo development were similar to mature zygotic embryos. Up to 81% of the desiccated somatic embryos regenerated to plantlets during which time the TAG was utilised in a manner similar to zygotic seedlings.

The isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca (moench) voss.

Conditions were standardized for the isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca. A combination of 0.5% Cellulase R-10, 0.25% Macerozyme, 0.25% Driselase, 0.25% Rhozyme HP-150 with 0.5M mannitol and 5 mM CaCl2.2H2O produced an average of 4.5 × 10(6) protoplasts per gram fresh weight of cells. Of the several protoplast culture media tested, von Arnold and Eriksson and Kao and Michayluk (KM8P) media best supported mitotic divisions of protoplasts. A density of 10(5) protoplasts per ml and the addition of 5 mM glutamine to the culture medium was necessary to induce sustained divisions and microcallus formation. Microcalli grew into subculturable callus using a nurse culture technique.

Regeneration of somatic embryos from protoplasts isolated from an embryogenic suspension culture of white spruce (Picea glauca).

Regeneration of white spruce (Picea glauca) somatic embryos from protoplasts derived from an embryogenic suspension culture was accomplished using a culture medium containing 2 mgl(-1) 2,4-D and 1 mgl(-1) 6-BAP. Divisions within 2 days led to plating efficiencies in the order of 24% after 9 days. A reduction in the osmoticum, necessary for sustained growth, was carried out gradually over 30 days. Embedding in agarose and culture in 5 cm petri dishes prior to transfer of agarose blocks to a bead type culture, led to the formation of somatic embryos as early as 23 days after isolation and yielded plating efficiencies in the order of 5-10% after 35 days culture.

Hormone levels in the foetal and neonatal prostate.

The prostates of 41 foetuses and neonates have been assayed for testosterone, 5 alpha-dihydrotestosterone (DHT), oestradiol and oestriol and the presence of these hormones has been demonstrated in all cases. Oestriol was found to be present in the highest concentration. A highly significant correlation was found between testosterone and DHT as well as between DHT and oestradiol. The correlation between oestriol and oestradiol was also significant. It has been pointed out that the presence of these hormones is essential for the growth and development of the prostate gland.

An evaluation of Ficoll density gradient centrifugation as a method for eliminating microbial contamination and purifying plant protoplasts.

Ficoll density gradient centrifugation was used to generate sterile protoplasts from non-sterile Pteridium cultures. The method was found to drastically reduce levels of fungal and bacterial contamination introduced prior to centrifugation whilst concentrating and purifying protoplasts.

Oestrogen-related changes in sex hormone binding globulin levels during normal and gonadotrophin-stimulated menstrual cycles.

Previous studies have identified no consistent change in sex hormone binding globulin (SHBG) levels during normal menstrual cycles. This is despite marked cyclical changes in plasma oestradiol concentration, and the observation that SHBG level increases in pregnancy, and after administration of exogenous gonadotrophin or synthetic oestrogens. The level of SHBG was measured in 19 normal females at 2 d intervals from day -8 to +10 where the preovulatory peak of oestradiol was designated as occurring on day 0. SHBG levels increased by a mean 15% +/- 4 (SEM) between the follicular and luteal phases (P less than 0.001) and this was due entirely to an increment between day 0 and +2. The change in SHBG levels was correlated with the change in oestradiol levels between days -8 and 0 (P less than 0.001). Fifty-six infertile patients were also studied. Twenty-seven received Pergonal alone whilst the other 29 received Pergonal after a preceding 5 d on Clomid. In both groups peak preovulatory oestradiol levels were greater than 3 times higher than in normal cycles. SHBG levels showed no change in the follicular phase but rose markedly during the luteal phase. These increases were significantly correlated with peak preovulatory oestradiol concentration but showed no relationship with mid-luteal progesterone concentration. We conclude that supranormal levels of oestradiol cause marked increases in SHBG binding capacity and increases in SHBG level of a lower order occur shortly after the preovulatory peak of oestradiol in the normal menstrual cycle.

Plasmolysis of Pteridium protoplasts: A study using light and scanning-electron microscopy.

A study was undertaken using gametophytes of the fern Pteridium aquilinum to examine the effects of plasmolysis on the topography of protoplasts. Methods are described whereby the surfaces of non-isolated protoplasts can be observed in the plasmolysed condition using scanning electron microscopy. Plasmolysed gametophytes were also examined in the light microscope using differential interference contrast and ultra-violet fluorescence microscopy after staining with fluorescein diacetate. With scanning electron microscopy, plasmolysed protoplast surfaces appeared smooth with no evidence of wrinkling or infolding of excess membrane. The formation of irregular-shaped protoplasts, protoplasmic threads, subprotoplasts, and protoplasmic networks covering internal wall surfaces all provided evidence for strong wall adhesion of the protoplasm. The availability of membrane for uptake into folds or vesicles is therefore thought to be minimal. Transmission electron microscopy showed some protoplasmic threads to be plasmodesmata, the remainder being cell-wall contact points. Remnants of these threads were occasionally observed on isolated protoplasts in both the light and electron microscopes.