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Biomedical subjects

J H Skerritt

Publications and source records attributed to J H Skerritt.

At least 19 recordsLinked to original sources
PubMed2000

Development of immunoassays for tyramine and tryptamine toxins of Phalaris aquatica L.

The leaves of the perennial pasture grass Phalaris aquatica L. (phalaris) contain two groups of known toxins, indole alkaloids, primarily dimethyltryptamines and N-methyltyramines, which cause illnesses in grazing animals, especially sheep. Using amino-reactive and phenolic hydroxyl-reactive homobifunctional reagents, simple methods were devised for coupling toxins representative of those in phalaris to carrier proteins and enzymes for ELISA development. ELISAs were produced for both groups of toxins. Dimethyltryptamines were most sensitively detected [lower limit of detection (LLD) of 1 microg/L for bufotenine] using rabbit anti-bufotenine antibodies, coupled to ovalbumin using divinyl sulfone, with detection using a peroxidase conjugate prepared using the same hapten coupled with 1, 4-butanediol diglycidyl ether. The assay cross-reacted with other toxins of the same class (N,N-dimethyltryptamine and N, N-dimethyl-5-methoxytryptamine) but not with the structurally related amino acids histidine and tryptophan. The most sensitive N-methyltyramine assay (LLD of 1 microg/mL for N-methyltyramine) utilized antisera to tyramine with N-methyltyramine coupled to peroxidase. Significant cross-reaction was seen with the low-grade toxin hordenine, but detection of tyramine was poorer, whereas the amino acid tyrosine was not detected. These assays could be applied to the analysis of simple extracts of Phalaris leaves with minimal interference. A good correspondence was observed between toxin levels by ELISA and estimates from a more tedious thin-layer chromatography method. The method has now been incorporated in a Phalaris breeding program.

Alkaloids↗
PubMed2000

Rapid antibody-based field test to distinguish between Helicoverpa armigera (Lepidoptera: Noctuidae) and Helicoverpa punctigera (Lepidoptera: Noctuidae).

Helicoverpa armigera (Hübner) and Helicoverpa punctigera (Wallengren) are the two most important insect pests of cotton production in Australia and require application of insecticides to control them. H. armigera has developed resistance to several insecticides but H. punctigera has not. Cost-effective management of insecticide resistance requires that growers be able to determine the proportion of H. armigera eggs or young larvae present on their crop before applying insecticides. This is impossible visually. We generated two monoclonal antibodies that reacted with the insect protein "lipophorin" and were capable of discriminating individuals of the two species at all life-stages. The antibodies were incorporated into a rapid test kit that was tested under field conditions over two growing seasons. Results obtained with the kit agreed closely with those obtained by rearing larvae through to second instar.

Animals↗
PubMed1999

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.

Animals↗
PubMed1999

Immunoassay for wheat processing quality: utilization of a sandwich assay incorporating an immobilized single-chain fragment.

A single-chain fragment (scFv) was engineered from a monoclonal antibody to high molecular weight glutenin subunits (HMW-GS), wheat flour polypeptides that play a major role in determining the mixing- and extension strength-related properties of dough and its subsequent baking performance. The scFv was expressed in a thioredoxin mutant Escherichia coli strain that allows disulfide bond formation in the cytoplasm and incorporated into a diagnostic test for wheat quality. Although the scFv lacks the more highly conserved antibody constant regions usually involved with immobilization, it was able to be directly immobilized to a polystyrene microwell solid phase without chemical or covalent modification of the protein or solid phase and utilized as a capture antibody in a double-antibody (two-site) immunoassay. In the sandwich assay, increasing HMW-GS concentrations produced increasing assay color, and highly significant correlations were obtained between optical densities obtained in the ELISA using the scFv and the content of large glutenin polymers in flours as well as measures of dough strength as measured by resistance to dough extension in rheological testing. The assay using the scFv was able to be carried out at lower flour sample extract dilutions than that required for a similar assay utilizing a monoclonal capture antibody. This research shows that engineered antibody fragments can be utilized to provide superior assay performance in two-site ELISAs over monoclonal antibodies and is the first application of an engineered antibody to the analysis of food processing quality.

Antibodies, Monoclonal↗
PubMed1994

Cloning of a wheat 15-kDa grain softness protein (GSP). GSP is a mixture of puroindoline-like polypeptides.

The wheat starch 15-kDa protein (called grain softness protein or GSP) consists of a major polypeptide and several minor polypeptides. An antiserum raised against GSP was used to screen a wheat cDNA library. A cDNA family encoding approximately 15-kDa proteins that included a heptapeptide sequence previously isolated from protease digests of GSP was identified. A partial cDNA was used in a prokaryotic expression system to produce a fusion protein which reacted strongly against the original anti-GSP serum. A new antiserum raised against the fusion protein produced a weak reaction against a 15-kDa polypeptide extracted from wheat seeds. The results suggest that the proteins encoded by the cDNA family form a minor component of the mixture of 15-kDa polypeptides defined as GSP. RNA complementary to the cDNAs could be extracted from both soft and hard wheat grains from about half-way through grain filling. The encoded proteins are novel members of the 2S superfamily of seed proteins, a diverse family of proteins which maintain a characteristic framework of cysteine residues. The deduced proteins show the highest similarity to the oat 16-kDa avenin and to wheat puroindoline (a lipid-binding 15-kDa protein from wheat). Review of previously published data shows that puroindoline is also closely related to the major polypeptide of GSP, suggesting that the lipid-binding properties of GSP polypeptides may influence grain softness.

Amino Acid Sequence↗
PubMed1994

Studies of in vitro gamma-interferon production in coeliac disease as a response to gliadin peptides.

The effects of certain fractions of a peptic-tryptic-pancreatinic (PTP) digest of wheat gliadin and of synthetic peptides on the production of gamma interferon (gamma-IFN) in cultures of whole blood from adult patients with coeliac disease (CD) have been studied using a sandwich enzyme immunoassay. The most active peptides were fraction 9, its two principal sub-fractions (sub-fractions 1 and 2) and a synthetic peptide of sequence RPQQPYPQPQPQ (peptide V) corresponding to the principal peptide obtained from reversed-phase HPLC of fraction 9. Results with blood from the control group of subjects also indicated some response to these antigens, in most cases at similar levels to those observed with the coeliacs. Fraction 1 of the PTP digest and the other nine synthetic peptides tested were inactive with both coeliacs and controls. These results are in agreement with the results of in vivo and in vitro toxicity tests. They provide evidence of a link between toxicity and cell-mediated immune response in CD, and suggest that peptide V represents one of the active parts of the gliadin molecule.

Adult↗
PubMed1993

Isolation and characterization of wheat triticin cDNA revealing a unique lysine-rich repetitive domain.

Polyclonal antibodies were raised against a purified 22 kDa triticin polypeptide (delta) and were used to screen a wheat seed cDNA library in the Escherichia coli expression vector lambda gt11. The isolated cDNA clones were grouped into three families based on their cross-hybridization reactions in DNA dot-blot studies. Southern blots of genomic DNAs extracted from ditelocentric and nullisomic-tetrasomic lines of Chinese Spring wheat, probes with the excised cDNA inserts, indicated that one of the three families (9 clones) had triticin clones. This was finally confirmed by comparing the predicted amino acid sequences of two of these clones (lambda Tri-12, lambda Tri-25) with the published tryptic peptide sequences of triticin. The Southern blots also showed that there is at least one triticin gene located on the short arm of each of the homoeologous group 1 chromosomes (1A, 1B, 1D), although till now no triticin protein product has been identified for the chromosome 1B. The nucleotide sequence of the largest triticin cDNA clone lambda Tri-25 (1567 bp) is presented here, and its predicted amino acid sequence shows strong homology with the legumin-like proteins of oats (12S globulin), rice (glutelin) and legume seeds. A unique feature of the triticin sequence is that it contains a lysine-rich repetitive domain, inserted in the hypervariable region of the typical legumin-like genes. Northern blotting of total RNA extracted from different stages of the developing wheat seed revealed that the triticin gene expression is switched on 5-10 days after anthesis (DAA). There was a steady increase in the level of triticin mRNA until 20 DAA, after which it started decreasing. The maximum mRNA accumulation occurred between 17 and 20 DAA. These observations conform closely with the published data on triticin protein accumulation during grain development.

Amino Acid Sequence↗
PubMed1992

Co-existence of coeliac disease and insulin-dependent diabetes mellitus in children: screening sera using an ELISA test for gliadin antibody.

The prevalence of coeliac disease in children with insulin-dependent diabetes mellitus was investigated using a screening test of serum for antigliadin antibody by ELISA. One hundred and eighty (180) unselected diabetic children were screened for IgA and IgG class antigliadin antibodies (AGA); children with either grossly elevated or slightly elevated AGA had small bowel biopsies. The four children with the highest IgA AGA had total villous atrophy. These four children were considered to have unsuspected coeliac disease. The prevalence of coeliac disease in this group of children was one in 45. Anti-gliadin IgA and IgG tests are suitable for screening children at high risk of having coeliac disease.

Adolescent↗
PubMed1992

Differentiation of coeliac disease and other malabsorption diseases using specific serum antigliadin IgG subclass profiles and IgA1 levels.

The differential diagnostic potential of serum gliadin-specific IgG subclass antibodies was assessed by comparing the antigliadin IgG1, 2, 3, 4 profile at different stages of coeliac disease with that of gastro-intestinal infection and also conditions associated with increased intestinal permeability. The IgG subclass profile of untreated coeliac disease was found to be the same as in healthy controls (IgG1 approximately IgG2 > IgG3 > IgG4), with only the magnitude of the individual subclass responses being increased in coeliac patients. Coeliac adults and children on gluten-free diets had different antigliadin IgG subclass profiles with IgG2 being elevated. Increased intestinal permeability or recent gastro-intestinal infection did not alter the antigliadin subclass profile from that observed in healthy individuals. Assessment of the diagnostic potential of antigliadin IgA1 and IgG1-4 measurements in screening for coeliac disease demonstrated that measurement of subclasses of gliadin-specific IgA and IgG was less sensitive and specific compared with the combined use of total antigliadin IgA and IgG. Therefore it is suggested that IgG subclasses should not be used for routine screening for coeliac disease.

Adolescent↗
PubMed1991

Cellular and humoral responses in coeliac disease. 1. Wheat protein fractions.

The humoral and cellular immune response of coeliac individuals to various wheat protein fractions was studied using serum antibody ELISA assays and the indirect leucocyte migration inhibition factor (LMIF) assays. Greater migration inhibition factor activity was seen in coeliacs on a gluten-free-diet having low serum antibody titres, and using purified T-cells instead of total peripheral blood mononucleocytes. Gliadin was the most active fraction in both assays. Raised antibodies to low-molecular weight and high-molecular weight glutenin polypeptides was observed, though these proteins had little migration inhibition factor activity. No cellular or humoral response was seen to albumins or globulins. Proteins associated with the granules of well-washed wheat starch are distinct from gluten proteins and had little T-cell activity, correlating with clinical observations that properly prepared wheat starch is devoid of coeliac toxicity. The greater specificity of the humoral response for individual wheat protein fractions in this study, compared with the earlier reports, likely results from cross-contamination in the earlier work of each fraction with gliadin.

Adult↗
PubMed1991

Cellular and humoral responses in coeliac disease. 2. Protein extracts from different cereals.

The humoral and cellular immune responses to grain protein extracts from coeliac-toxic and non-toxic cereals were compared by use of a number of ELISA and immunoblotting methods and the indirect leucocyte migration inhibition factor (LMIF) assay. Both adult and child coeliacs had elevated levels of serum antibody to proteins from the coeliac-toxic cereals, namely bread wheat, durum wheat, rye and barley and low levels of proteins from other cereals. Using protein blotting techniques, antibody binding was greatest to gliadins/low mol mass glutenin subunits and homologous prolamins from rye and barley, consistent with the ELISA findings. Competition ELISA and preabsorption tests indicated that antibody reaction to maize storage proteins did not simply result from cross-reaction of antigliadin antibodies. In LMIF assays, only the wheat extracts had activity in coeliac patients. This is most likely partly due to loss of some of T-cell epitopes from the extraction technique required for these proteins, as well as the relatively small effects seen for even very active fractions in the LMIF assay.

Adult↗
PubMed1991

Self-management of dietary compliance in coeliac disease by means of ELISA "home test" to detect gluten.

To improve compliance with a gluten-free diet in coeliac disease a simple prototype test kit was developed to detect gluten in foods for use at home. The test is based on monoclonal antibodies to heat-stable gluten proteins which crossreact appropriately with barley and rye proteins. It is suitable for use with a wide range of raw or cooked foods. The food is extracted with dilute hydrochloric acid and 1 drop of the extract transferred to an antibody-coated tube; enzyme-labelled gluten detection antibody is added and after 3 min the tube is washed and colour developer is added. The reaction is stopped after 2 min, stabilising the blue colour. The home kit was compared with a quantitative laboratory kit, and the qualitative agreement was very good. The kit could distinguish foods with trace gluten contents (acceptable for a "gluten-free" diet) from those with a slightly higher but unacceptable gluten content. In a trial of the prototype kit by 47 coeliac disease patients of diverse ages and educational backgrounds, 93% of tests correctly identified foods as acceptable or unacceptable.

Adolescent↗
PubMed1991

A reliable screening test for coeliac disease: enzyme-linked immunosorbent assay to detect anti-gliadin antibodies in serum.

A simple, rapid, highly reproducible enzyme-linked immunosorbent assay detecting anti-gliadin antibodies in serum to screen for coeliac disease (CD) is described. By combining the results of anti-gliadin IgA and IgG determinations the overall sensitivity of the assay was found to be 100% and the specificity 96% for children and 99% for adults. Significantly elevated antigliadin IgA and IgG antibodies were detected in all 20 children and all 25 adults with untreated CD. False positive results were found in 1/79 histologically normal control and 5/86 disease control children, while for adults false positive rates were 0/74 and 1/34 for the healthy and disease control groups, respectively. Anti-gliadin IgA and IgG was measured in serum samples from 52 coeliac patients (11 children and 41 adults) treated with a gluten-free diet (GFD). Each of the children and 28 of the adults who followed a strict GFD had significantly lower IgA and IgG levels than untreated CD patients. The serum anti-gliadin IgA and IgG levels of the 13 adults not complying with a GFD were similar to those found for untreated CD patients. This assay is recommended as a screening test for CD as well as a tool for follow-up of treated patients.

Adolescent↗
PubMed1991

Identification of reactive synthetic gliadin peptides specific for coeliac disease.

Gluten intolerance (coeliac disease) is characterised by the development of a small intestinal lesion following exposure to the gliadin fraction after consumption of wheat and related cereals. Cellular immune mechanisms are thought to be responsible for gliadin toxicity, but the toxic sequence/s within gliadin have not been clearly established. A panel of synthetic gliadin peptides was tested using peripheral blood mononuclear cells from coeliac patients and two assays for cell-mediated immunity. Using the indirect leucocyte migration inhibition factor and the macrophage procoagulant activity assays, gliadin peptides which were located in the aminoterminal or the proline-rich domain of the alpha/beta gliadin molecule were coeliac-active. Peptides predicted by T cell algorithms or on the basis of homology to adenovirus Ad12 Elb protein and which were located in the proline-poor gliadin domains were inactive. Protein sequence studies which indicate significant homology in the proline-poor gliadin domains with a number of non-coeliac-toxic seed proteins also supported the hypothesis that the proline-rich domains may be more important in the pathogenesis of coeliac disease.

Algorithms↗
PubMed1990

Antibody response reveal differences in oral tolerance to wheat and maize grain protein fractions.

The influence of diet on humoral immune responses to gluten- and maize-derived proteins was examined using ELISA and protein blotting techniques. Mice raised on the maize-based (gluten-free) diet responded well to parenteral immunization with each of six gluten-derived protein preparations (whole gliadin, two omega-gliadin fractions, wheat salt-soluble proteins, a peptic-tryptic digest and a subtilisin digest of gluten), as serum antibody levels increased at least 300-fold in each case. In contrast, mice raised on the wheat-based diet responded poorly to immunization with either whole gliadin or omega-gliadin and were virtually non-responsive to enzymic digest of gluten. Diet had little effect on the magnitude of the antibody response to wheat salt-soluble proteins, with both groups showing a 300-fold increase in titre. Similarly, tolerance to alpha-zeins, the alcohol-soluble proteins of maize, did not occur on either diet. However, some oral tolerance was observed to maize glutelin. The specificity of the various antibody responses was then analysed by immunoblotting. Following immunization with gluten proteins or digests, antibodies from the maize-fed mice bound more or less equally to each of the main gliadin bands and to the glutenins while the mice on the wheat-based diet had antibody specific for omega-gliadin proteins. Serum antibodies from the maize-fed mice, immunized with either alpha-zein or maize glutelin, showed even labelling of the major maize endosperm proteins while antibodies from mice on the wheat diet showed strong labelling of the Mr 27,000 and 58,000 bands. These results show that diet influenced the specificity, as well as the magnitude of serum antibody responses to cereal proteins. In addition, oral tolerance appeared to affect the humoral response to some cereal proteins more than others. Both of these findings have important implications for our understanding of coeliac disease.

Animals↗
PubMed1990

Macrophage procoagulant activity as an assay of cellular hypersensitivity to gluten peptides in coeliac disease.

Gluten and casein digests were tested for their ability to induce a cellular immune response on peripheral blood mononuclear cells (PBM) obtained from coeliac patients as well as healthy volunteers and disease control patients using the macrophage procoagulant assay. PBM from coeliac patients who had been on a gluten-free diet for less than 2 years responded strongly to gluten peptides, while coeliac patients at diagnosis or who had maintained a strict gluten-free diet for longer than 5 years showed weaker responses. PBM from healthy volunteers did not respond to gluten or casein peptides, whereas those from patients with Crohn's disease displayed weak reactivity to gluten and casein peptides. Our study using the macrophage procoagulant assay confirms previous findings that lymphoid cells from patients with coeliac disease exhibit a specific cellular immune response to gluten. This assay represents an alternative measure for cell-mediated immunity and is technically much simpler than the previously described leucocyte migration inhibition assay. Macrophage procoagulant activity is measured using a simple plasma recalcification time assay or spectrophotometrically using commercially available chromogenic substrates.

Adult↗