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A Bradbury

Publications and source records attributed to A Bradbury.

At least 19 recordsLinked to original sources
PubMed2001

Antibodies in haystacks: how selection strategy influences the outcome of selection from molecular diversity libraries.

Antibodies against most antigens can be isolated from high quality phage antibody libraries. However, not all antibodies binding a particular antigen are necessarily found when standard selections are performed. Here we investigate the effect of two different selection strategies on the isolation of antibodies against a number of different antigens, and find that these different strategies tend to select different antibodies, with little overlap between them. This indicates that the full diversity of these libraries is not tapped by a single selection strategy and that each selection strategy imposes different selective criteria in addition to that of antigen binding. To fully exploit such libraries, therefore, many different selection strategies should probably be employed for each antigen. The use of alternative strategies should be considered when selection apparently fails, or when the number of different antibodies recognizing an antigen needs to be maximised. Furthermore, the microtitre selection strategy developed is likely to prove useful in the application of phage antibody libraries to the human genome project, allowing the high throughput selection of antibodies against multiple antigens simultaneously.

Antibodies↗
PubMed2001

Molecular dissection of the tissue transglutaminase autoantibody response in celiac disease.

Celiac disease (CD) is an intestinal malabsorption characterized by intolerance to cereal proteins accompanied by immunological responses to dietary gliadins and tissue transglutaminase, an autoantigen located in the endomysium. Tissue transglutaminase belongs to the family of enzymes that catalyze protein cross-linking reactions and is constitutively expressed in many tissues as well as being activated during apoptosis. The role of gliadins in eliciting the immune response in CD and how transglutaminase is linked to the primary reaction are still unclear. In this work, we report the production and analysis of six phage Ab libraries from the peripheral and intestinal lymphocytes of three CD patients. We were able to isolate Abs to transglutaminase from all intestinal lymphocytes libraries but not from those obtained from peripheral lymphocytes. This is in contrast to Abs against gliadin, which could be obtained from all libraries, indicating that the humoral response against transglutaminase occurs at the local level, whereas that against gliadin occurs both peripherally and centrally. Abs from all three patients recognized the same transglutaminase epitopes with a bias toward the use of the V(H)5 Ab variable region family. The possible role of these anti-transglutaminase Abs in the onset of CD and associated autoimmune pathologies is discussed.

Adult↗
PubMed2000

Early expression of GABA(A) receptor delta subunit in the neonatal rat hippocampus.

The cDNA library screening strategy was used to identify the genes encoding for GABA(A) receptor subunits in the rat hippocampus during development. With this technique, genes encoding eleven GABA(A) receptor subunits were identified. The alpha5 subunit was by far the most highly expressed, followed by the gamma2, alpha2 and alpha4 subunits respectively. The expression of the beta2, alpha1, gamma1, beta1 and beta3 subunits was moderate, although that of the alpha3 and delta subunits was weak. In situ hybridization experiments, using digoxigenin-labeled cRNA probes, confirmed that the delta subunit was expressed in the neonatal as well as in the adult hippocampus, and is likely to form functional receptors in association with other subunits of the GABA(A) receptor. When the more sensitive RT-PCR approach was used, the gamma3 subunit was also detected, suggesting that this subunit is present in the hippocampus during development but at low levels of expression. The insertion of the delta subunit into functional GABA(A) receptors may enhance the efficacy of GABA in the immediate postnatal period when this amino acid is still exerting a depolarizing and excitatory action.

Animals↗
PubMed2000

Mass spectral analysis of a protein complex using single-chain antibodies selected on a peptide target: applications to functional genomics.

Genome projects are identifying an ever-increasing number of genes, accelerating the need for reagents to study the expression of these genes and elucidate the function and cellular location of the gene products. Our goal was to develop a strategy to allow human single-chain variable fragment (scFv) antibodies to be used for these endeavors. A library containing 7x10(9) individual variants was displayed by bacteriophage and selected against a biotinylated peptide corresponding to the C-terminal 15 amino acid residues of Ku86, one component of a heterodimer involved in double-stranded DNA break repair. Four unique scFv antibodies were recovered that not only recognized the selected peptide, but also the intact protein. Three of the scFv antibodies were expressed in soluble form and recognized Ku86 by Western analysis. The affinity of one of the scFv antibodies for Ku86 was 16 nM as measured by BIAcore analysis. scFv immunoprecipitation of Ku86 also isolated the other component of the heterodimer, Ku70, as determined by Western analysis and mass spectrometry. These results demonstrate the utility of scFv antibodies as invaluable reagents for functional genomics.

Antibodies↗
PubMed2000

'Specific' oligonucleotides often recognize more than one gene: the limits of in situ hybridization applied to GABA receptors.

As exquisite probes for gene sequences, oligonucleotides are one of the most powerful tools of recombinant molecular biology. In studying the GABA receptor subunits in the neonatal hippocampus we have used oligonucleotide probes in in situ hybridization and cloning techniques. The oligonucleotides used and assumed to be specific for the target gene, actually recognized more than one gene, leading to surprising and contradictory results. In particular, we found that a GABA(A)-rho specific oligonucleotide recognized an abundant, previously unknown, transcription factor in both in situ and library screening, while oligos 'specific' for GABA(A) subunits were able to recognize 30 additional unrelated genes in library screening. This suggests that positive results obtained with oligonucleotides should be interpreted with caution unless confirmed by identical results with oligonucleotides from different parts of the same gene, or cDNA library screening excludes the presence of other hybridizing species.

Age Factors↗
PubMed2000

Exploiting recombination in single bacteria to make large phage antibody libraries.

The creation of large phage antibody libraries has become an important goal in selecting antibodies against any antigen. Here we describe a method for making libraries so large that the complete diversity cannot be accessed using traditional phage technology. This involves the creation of a primary phage scFv library in a phagemid vector containing two nonhomologous lox sites. Contrary to the current dogma, we found that infecting Cre recombinase-expressing bacteria by such a primary library at a high multiplicity of infection results in the entry of many different phagemid into the cell. Exchange of Vh and Vl genes between such phagemids creates many new V h/Vl combinations, all of which are functional. On the basis of the observed recombination, the library is calculated to have a diversity of 3x1011. A library created using this method was validated by the selection of high affinity antibodies against a large number of different protein antigens.

Antibodies↗
PubMed2000

The neuronal microtubule-associated protein tau is a substrate for caspase-3 and an effector of apoptosis.

We have identified a class of tau fragments inducing apoptosis in different cellular contexts, including a human teratocarcinoma-derived cell line (NT2 cells) representing committed human neuronal precursors. We have found a transition point inside the tau molecule beyond which the fragments lose their ability to induce apoptosis. This transition point is located around one of the putative caspase-3 cleavage sites. This is the only site that can be effectively used by caspase-3 in vitro, releasing the C-terminal 19 amino acids of tau. These results establish tau as a substrate for an apoptotic protease that turns tau itself into an effector of apoptosis. Accordingly, tau may be involved in a self-propagating process like what has been predicted for the pathogenesis of different neurodegenerative disorders.

3T3 Cells↗
PubMed2000

Health outcomes.

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Humans↗
PubMed2000

Reply

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Journal Article↗
PubMed2000

The relationship between lower limb symptoms and superficial and deep venous reflux on duplex ultrasonography: The Edinburgh Vein Study.

BACKGROUND: Previous work from this group has demonstrated the relationships between lower limb symptoms and the presence and severity of trunk varicose veins as seen on clinical examination to be generally weak, symptom specific, and gender dependent. OBJECTIVE: This study was undertaken to investigate the relationships in the general population between lower limb symptoms and the presence of superficial or deep venous reflux. METHODS: A cross-sectional study was made of an age-stratified random sample of 1566 subjects (699 men and 867 women) aged 18 to 64 selected from 12 general practices in Edinburgh, Scotland. Subjects completed a self-administered questionnaire regarding symptoms (heaviness/tension, a feeling of swelling, aching, restless legs, cramps, itching, tingling) and underwent duplex ultrasound examination of the superficial and deep venous systems of both legs. Reflux of 0.5 seconds or greater was considered pathologic. Deep venous reflux was defined as reflux in at least the popliteal vein. RESULTS: There was a significant positive relationship between isolated superficial reflux and the presence of heaviness/tension (P <.025, both legs) and itching (P =.002, left leg) in women. Isolated superficial reflux in men was not significantly positively associated with any symptom. Isolated deep venous reflux was not significantly related to any symptom in either leg in either sex. Combined reflux was related to a feeling of swelling (P =.018, right leg; P =.0022, left leg), cramps (P =.0049, left leg) and itching (P =.0043, left leg) in men, and aching (P =.03, right leg) and cramps (P =.026, left leg) in women. CONCLUSION: In the general population, only certain lower limb symptoms were related to the presence of reflux on duplex ultrasound scanning. The strongest relationships were observed in the left legs of men with combined superficial and deep reflux.

Adolescent↗
PubMed2000

Human recombinant tissue transglutaminase ELISA: an innovative diagnostic assay for celiac disease.

OBJECTIVE: Tissue transglutaminase is the autoantigen recognized by the sera of celiac patients. An enzyme-linked immunosorbent assay (ELISA) based on guinea-pig tissue transglutaminase was recently used to measure serum tissue transglutaminase antibodies for the diagnosis of celiac disease. We determine the sensitivity and specificity of an ELISA test based on the use of human recombinant transglutaminase, compared with the guinea pig transglutaminase ELISA and IgA antiendomysium antibodies. METHODS: Serum samples were tested from 65 patients with intestinal biopsy proven celiac disease, from 10 patients with Crohn's disease, and from 150 healthy blood donors. RESULTS: Human transglutaminase ELISA identified 64 of 65 celiac patients, whereas the guinea pig transglutaminase ELISA and IgA antiendomysium antibodies identified 58 of 65 and 60 of 65 subjects, respectively. The three tests showed comparable specificity. CONCLUSIONS: These results proved that the human tissue transglutaminase-based ELISA represents a cost-effective strategy for identifying both symptomatic and atypical forms of celiac disease and could mean that intestinal biopsy need no longer be the gold standard for diagnosing this clinical condition. Furthermore, early identification and treatment of patients with celiac disease in an outpatient setting could have significant implications for reducing long-term morbidity and can produce major savings in future health care costs.

Adolescent↗