Development of a Field Enzyme-Linked Immunosorbent Assay (ELISA) for Detection of α-Amylase in Preharvest-Sprouted Wheat

A sandwich enzyme-linked immunosorbent assay (ELISA) has been developed for detection of α-amylase in preharvest sprouted wheat and adapted to rapid field-use formats requiring 15–20 min to perform. Polyclonal and monoclonal antibodies were prepared to detect a mixture of high and low pI isozymes of α-amylase and high pI isozymes only. All antibodies detected α-amylase on immunoblots of either a crude wheat extract or of purified enzyme, but only the polyclonal antibodies functioned in a sandwich ELISA. Depending on the antibody combination, the tube ELISA detected either the high and low pI isozymes of α-amylase or the high pI isozymes only with a detection limit of ≈0.5–1.0 ng/mL of amylase. Wheats with falling numbers (FN) of <350 sec could be discriminated from sound wheats, with decreasing FN producing increasing assay color. Using 130 wheat grain samples, ELISA absorbances for detection of both high and low pI isozymes and of high pI isozymes only were highly positively correlated with amylase enzyme activity and negatively correlated with FN. The correlations were similar for detection of both isozyme families and for detection of high pI isozymes only. Analyses of three sets of wheat samples from different environments demonstrated that the relationship between ELISA absorbance and FN had little dependence on wheat cultivar. The precision of sample analysis using the field ELISA was similar to the precision of FN test apparatus.     

Canine and feline intracranial meningiomas: an updated review

Meningiomas are the most common primary brain tumours in dogs and cats. There are several morphological phenotypes of this extra-axial neoplasm and they show predilections for certain anatomical locations. There have been a number of attempts to apply the current World Health Organization (WHO) classification for human meningiomas to dogs and cats and to obtain a universal classification scheme for domestic animals. Recently, certain enzymes involved in tumour growth have been recognised as biological markers and have been related to degrees of malignancy. The secondary effects of meningiomas have also been investigated, and vascular endothelial growth factor and peritumoural oedema have been reported to reduce survival rate. Breed and age predisposition are recognised in both dogs and cats and the presenting clinical signs are quite consistent. Magnetic resonance imaging and computed tomography are the techniques of choice for the presumptive diagnosis of meningiomas in domestic animals but advanced imaging techniques are constantly being developed and applied. Treatment methods for meningiomas involve a combination of de-bulking surgery, chemotherapy and radiotherapy, and detailed accounts of several treatment protocols have been reported.     

Development of a compound-specific ELISA for flufenoxuron and an improved class-specific assay for benzoylphenylurea insect growth regulators.

This study describes immunochemical approaches for the compound-specific detection of flufenoxuron and class-specific detection of benzoylphenylurea (BPU) insecticides. With the aim of developing a highly specific immunoassay for flufenoxuron, a hapten was synthesized by introducing a spacer arm at the 2,6-difluoro substituent aromatic ring of a flufenoxuron derivative. An IC(50) value of 2.4 ppb was obtained for flufenoxuron, with detection of the other four BPUs being more than 4000-fold less sensitive. For the development of class-specific ELISA for five BPUs, a new approach was used for the hapten preparation in which a butanoic acid linkage was introduced into the 3,5-dichloro-substituted aniline ring of chlorfluazuron analogue. Although the resultant ELISA still exhibited slightly differing cross-reactions for these five BPUs, this method had broader specificity than the previously reported polyclonal antibody-based ELISA. Spike and recovery studies for five BPUs in soil and water indicated that both the compound- and class-specific ELISAs were able to quantitatively detect BPU residues in soil and water. This study also provided additional insights into the influence of the immunizing hapten structure on the specificities of the antibodies obtained.     

Modification of accessory activity of sheep monocytes in vitro by a coenurus antigen from Taenia multiceps.

A factor in Taenia multiceps coenurus fluid (TMCF) has previously been shown to modify the accessory activity of murine macrophages in vivo and in vitro. The factor (TMCF-F24) has been purified by ion exchange in a fast protein liquid chromatography (FPLC) system. This study was conducted to determine whether TMCF-F24 is an antigen in naturally occurring cerebral coenuriasis, and whether it can also modify normal sheep blood monocytes. Specific IgG antibodies to TMCF-F24 were detected, using ELISA, in serum and cerebrospinal fluid of sheep with clinical coenuriasis. Alterations in monocyte accessory activity were detected by an assay which measured the rate of increase in mitogen-induced lymphocyte transformation caused by addition of increasing numbers of the monocytes. Normal monocytes caused a positive increase in lymphocyte transformation. Monocytes incubated with TMCF-F24 caused progressive inhibition of transformation. This factor may therefore modify monocyte-T cell interaction in natural infection.     

Quality-related Epitopes of High Mr Subunits of Wheat Glutenin

Three hundred and eighty four immobilised overlapping nonapeptides, corresponding to the full amino acid sequences of three high M_r subunits of glutenin from bread wheat (Triticum aestivum) grain, were used to determine the linear epitopes recognised by four monoclonal antibodies. These antibodies were selected on the basis of significant and positive correlations between their binding to wheat flour extracts in a two-site ('sandwich') enzyme immunoassay and rheological measures of dough strength, an important aspect of bread wheat quality. The antibodies did not bind to a single, specific sequence but bound a series of related peptides in each high M_r glutenin subunit examined. The sequences recognised were not identical for the four antibodies, but in each case were in the central repeating domain of the high M_r glutenin subunits, and usually comprised regions that overlapped the degenerate repeat nonamer and hexamer sequences. High M_r glutenin subunits that have been associated with greater dough strength, such as the D-genome allelic products 1Dx5 and 1Dy10, displayed an increased number of the epitope sequences. The location of the epitopes in sequences of overlapping β-turns in the repetitive region supports the hypothesis that dough elasticity arises partly from β-turn-forming secondary structure in the repeat regions of the M_r glutenin subunits. Additional β-turn within high M_r subunits may extend their structure to allow increased interaction between the glutenin subunits and with the other proteins of the gluten complex, thus improving dough strength.     

Spinal nephroblastoma in a crossbreed dog

A three-year-old, male crossbreed dog presented with progressive hindlimb paresis. Magnetic resonance imaging revealed an intramedullary spinal cord lesion of 1.5 cm diameter at the levels of the first and second lumbar vertebrae. Following surgical excision of the mass, there was resolution of the neurological signs. Twelve months later, hindlimb paresis was again evident. A second surgical procedure restored ambulatory status for a further five months before signs recurred and the dog was euthanased. A diagnosis of spinal nephroblastoma was made on the basis of signalment, lesion location and histopathological analysis of biopsy specimens.     

Direct Evidence that the Number and Location of Cysteine Residues affect Glutenin Polymer Structure

In this study, the possible roles of three well-characterised model prolamins in the structure of the glutenin macropolymer were examined. Model prolamins were labelled with fluorescein isothiocyanate (FITC), and incorporated into the glutenin macropolymer of a base flour using a partial reduction-oxidation scheme. The effect of incorporation of the model prolamins on dough behaviour was determined by assessing differences in polymer size distribution, mixing properties, and distribution of the model prolamins in a dough after incorporation. Using this approach, the prolamins capable of forming inter-chain disulphide bonds were shown to be incorporated into the glutenin macroÍpolymer, while prolamins that were not capable of forming inter-chain disulphide bonds were retained as monomers. The distribution of fluorescently-labelled prolamins after their incorporation into the glutenin macropolymer of the dough was examined by confocal light scanning microscopy, in order to determine the possible roles of ω-gliadins and glutenin-like subunits with varied cysteine residue compositions in the structural organisation. The role of the model prolamins was a function of the disulphide-bonding capabilities of the polypeptides. Model ω-gliadins were retained as monomers and functioned as space fillers; model glutenin-like subunits containing a single cysteine residue incorporated into the glutenin macropolymer but functioned as chain terminators; and model glutenin-like subunits containing two cysteine residues incorporated into the glutenin macropolymer and acted as chain extenders.     

Mapping the distribution of DDT residues as DDE in the soils of the irrigated regions of northern New South Wales,Australia using ELISA and GIS

An enzyme-linked immunosorbent assay (ELISA) specific for DDE [1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene] has been used to map DDT [1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane)] residues in the top 10 cm of soil in three river valleys of northern New South Wales, Australia. Despite being almost 20 years since DDT was last applied for cotton growing in these areas, the relationship between sites of greatest application and current residue levels was strong. DDE concentrations in the range 0-2 ppm were found, although most the 389 soil samples examined contained less than 0.2 ppm of DDE. Although some relationship between mode of land use and current residue levels was apparent, this varied from valley to valley and may have reflected different farming practices and times of application. The study demonstrates that the combination of ELISA and geographical information system (GIS) analysis provides an effective means of displaying levels of soil contamination by a pesticide and the possible need for remediation.