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P Tighe

Publications and source records attributed to P Tighe.

11 recordsLinked to original sources
PubMed2002

An immuno-precipitation assay for determining specific interactions between antibodies and phage selected from random peptide expression libraries.

Libraries of random peptides displayed by bacteriophage can be screened to select phage expressing peptides that specifically bind antibodies, so that the peptide sequence motifs expressed by the phage can help to define the epitopes of the antibodies. It is often desirable to screen antibody-selected phage for binding of the selecting antibody in an immunoassay in order to verify the specificity of the interaction. Enzyme-linked immunosorbent assays (ELISAs) are commonly used for this purpose. However, for many antibodies, the best techniques for measuring specific, high affinity interactions are immuno-precipitation assays. Immuno-precipitation was therefore investigated as a means of measuring interactions between antibodies and phage clones selected from random peptide display libraries. Three mouse monoclonal antibodies specific for glutamic acid decarboxylase were used to select peptides as 9-mers on T7 phage, linear 12-mers on pIII of M13 phage, or constrained 15-mers on pVIII of M13 phage. Following the cloning and sequencing of selected phage, mixtures of antibody and phage were incubated in solution and the immune complexes were precipitated with Protein G bound to Sepharose beads. In order to detect and quantitate the phage that had formed immune complexes and been precipitated, advantage was taken of the biological properties of the phage by inducing infection of Escherichia coli by the precipitated phage. The aim was to quantitate the phage precipitated by determining the number of plaques produced, which would therefore be proportional to the degree of interaction between the phage and the antibody in solution. The results presented here indicate that this method of measuring monoclonal antibody interactions with phage selected for expression of peptides recognised by the monoclonal antibody is highly specific and sensitive.

Amino Acid Sequence↗
PubMed1999

Laboratory-based quality assurance programme for near-patient urine dipstick testing, 1990-1997: development, management and results.

The need for quality assurance (QA) programmes covering near-patient testing technologies has been acknowledged since the mid-1980s. However, most effort has been directed towards capillary blood glucose monitoring techniques. The most common biochemical near-patient testing device is the dry reagent strip or 'dipstick' for urinalysis. This report describes a quality assurance programme for urinalysis, used in the wards and clinics of a district general hospital, and in some of the general practitioner surgeries within its catchment area. The project describes the preparation of an aqueous 'urine' sample, the design of a report form, the dispatch of the sample and report forms to the ward/clinic/health centre, the receipt and scoring of the returned results, and the assessment of the results, both in terms of management information and sources of error. Samples were spiked to give a target value midway between two colour blocks for each analyte. Results were scored as +/- 1 if adjacent colour block to the target, +/- 2 for results two colour blocks (error) and +/- 3 (gross error) for results three or more colour blocks from the target value. For urine glucose, results showed an overall 14.7% error and 2.6% gross error rate. Similar scoring gave the following error and gross error rates for the other analytes: bilirubin (1.0%, 3.3%); ketone (4.3%, 0.3%); specific gravity (13.4%, 3.1%); pH (11.2%, 6.5%); blood (7.7%, 2.9%); protein (9.7%, 2.3%); and nitrite (gross errors 4.9%). Investigation of the results of the programme showed four types of error in dipstick testing-timing, misalignment, misunderstanding and transcription. Analysis of the results showed a reduction in error rates when an electronic reader was used (errors 2.0%, gross errors 0.75%), compared to reading against the colour blocks on the side of the bottle (7.7%, 1.6%) or using the colour blocks on a flat card reader (7.4%, 1.7%). The report demonstrates the importance of a QA scheme in providing a reliable screening service. It gives confidence to the users of dipsticks, information on training needs to clinical managers, and an understanding of both types of error and how they can be reduced.

England↗
PubMed1995

Selection of peptide ligands for the antimucin core antibody C595 using phage display technology: definition of candidate epitopes for a cancer vaccine.

Aims-To further define the specificity of the antimucin core antibody C595 by fitting it with a family of hexapeptide ligands by immunoselection of filamentous bacteriophage from a gene III display library of approximately 6.4 x 10(7) random hexapeptides.Methods-Three rounds of immuno-selection were used to enrich for C595 binding phage. DNA sequencing revealed the hexapeptides expressed. Bacteriophage and corresponding synthetic hexapeptides were used in ELISA assay to determine binding affinities.Results-Twenty nine clones from this selected population were analysed. Seven contained the natural epitope RPAP, encoded by two different DNA sequences; 17/29 contained the motif RLPP. In all, 28/29 clones contained the motif RXXP and one clone (RVRPAP) contained the motif RXXP in two peptidic registers; 24/28 clones (6/8 DNA sequences) contained a hydrophobic residue (V or I) at position 1 relative to the RXXP motif. In addition the proximity of RXXP to glycine (position 5) suggests that this contributes in the natural epitope to antibody/antigen binding, which was not detected by chemical synthetic methods. One clone, KSKAGV, bears no obvious relationship to the natural epitope and therefore qualifies as a weakly binding mimotope.Conclusions-This approach has rapidly defined the specificity of this antibody in unprecedented detail, and provides a more comprehensive molecular basis for exploring the immune recognition of the MUC1 mucin by the C595 antibody. Importantly, the novel but related epitopes seen provide peptide specificities and a strategy which may prove useful in generating cancer vaccine candidates.

Journal Article↗
PubMed1995

Immunocytochemical study of the ontogeny of the marsupial Didelphis albiventris immune system.

Marsupials have considerable merits as models for studying the developmental dynamics of the mammalian immune system, but until recently there has been a conspicuous lack of specific immune probes to facilitate such studies. To begin a precise study of the ontogeny of the marsupial Didelphis albiventris we have used cross-reactive polyclonal antibodies raised against evolutionarily highly conserved peptides which form part of the antigen specific receptor complexes of human differentiated lymphocytes. Moreover, because of antigen receptor conservation, the antibodies also recognise specifically the immunocompetent T and B lymphocytes of other species including those in the organs of the opossum. Use of the antipeptide antibodies together with other cross-reacting antibodies has allowed us to study the cellular immunology of T and B cells and antigen presenting cells (APC) during the development of thymus, skin, lymph nodes and spleen in the Brazilian white-belly opossum. The molecular nature and identity of the T cell antigens detected in opossum tissues were confirmed by immunoblotting. These findings indicate that it is now possible to exploit these antibody probes for comparative mammalian studies, and indeed to investigate interesting features of the opossum, such as reaction of the immature immune system of the pouch young to antigenic stimulation.

Animals↗
PubMed1993

Reinfection with hookworm after chemotherapy in Papua New Guinea.

Reinfection with hookworm (Necator americanus) following chemotherapy was studied over 2 years in a rural village in Madang Province, Papua New Guinea. The prevalence of hookworm infection had returned to pre-treatment levels after 2 years, and the geometric mean hookworm burden had returned to 58% of the pre-treatment value. The rate of acquisition of adult worms was independent of host age, and was estimated as a geometric mean of 2.9-3.3 worms/host/year (arithmetic mean 7.9-8.9 worms/host/year). There was significant predisposition to hookworm infection; the strength of this predisposition did not vary significantly between age or sex classes.

Age Factors↗
PubMedDate not supplied

Immunocytochemical study of Peyer's patches follicular-associated epithelium in the marsupial, Didelphis albiventris.

The lack of probes defining leukocyte subpopulations has restricted ontogenetic studies of the opossum gut. We report for the first time the organization of the gut cellular immune components using species cross-reactive antibodies. Mouse monoclonal antibodies against human HLA-DR were used together with immunocytochemistry to demonstrate MHC class II-like antigens in the opossum Peyer's patches (PP). Positive staining was obtained in the M cell and enterocytes comprising the follicular-associated epithelium (FAE). Rabbit polyclonal antibody against human CD3 stained opossum thymocytes and T-cell dependent areas of spleen, lymph node, and PP interfollicular zones, but failed to stain intraepithelial lymphocytes in the FAE. In contrast rabbit polyclonal antibody against human IgA stained B-cell immunocytes and plasma cells present in the M-cell lateral invaginations. It is surmised that B-cell activation could occur in the opossum M-cell niches by thymus independent antigens, bypassing T-helper-cell function.

Animals↗