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R A Badley

Publications and source records attributed to R A Badley.

At least 19 recordsLinked to original sources
PubMed2001

Generation and refinement of peptide mimetic ligands for paratope-specific purification of monoclonal antibodies.

Paratope-specific purification of antibodies has distinct advantages over conventional methods of antibody purification with respect to its capacity to isolate product of high purity and immunoreactivity. The present report addresses the problems of identifying peptide ligands for the purification of antibodies reactive with nonprotein antigens. Using an anti-steroid antibody as the model, a lead sequence that bound antibody was identified from a peptide phage display library. The minimum binding unit in this sequence was deduced using a series of truncated peptides synthesized on the heads of polyethylene pins. Replacement Net analysis of the minimum binding unit identified peptides with increased affinity for the antibody. The affinity-matured peptide mimotope bound antibody in solution. By molecular modeling the peptide was superimposable onto estrone-3-glucuronide localized in the crystal structure of the antibody binding pocket. In order to resolve problems of presentation posed by the reversal of orientation of the peptide on the affinity matrix compared with the pins, the mimotope peptide was synthesized in reverse sequence using d-amino acids. The resulting affinity matrix was effective for the purification of antibody. Eluted product demonstrated molecular homogeneity and high immunoreactivity. It is concluded that the combination of biological and chemical library techniques described provides a method for the generation and affinity maturation of mimotopes for antibodies against nonprotein antigens.

Animals↗
PubMed2000

Analysis of bacterial function by multi-colour fluorescence flow cytometry and single cell sorting.

With the increased awareness of the problems associated with the growth dependent analysis of bacterial populations, direct optical detection methods such as flow cytometry have enjoyed increased popularity over the last few years. Among the analyses discussed here are: (1) Bacterial discrimination from other particles on the basis of nucleic acid staining, using sample disaggregation to provide fast reliable enumeration while minimizing data artefacts due to post sampling growth; (2) Determination of basic cell functions such as reproductive ability, metabolic activity and membrane integrity, to characterise the physiological state or degree of viability of bacteria; and (3) The use of single cell sorting onto agar plates, microscope slides or into multi-well plates to correlate viability as determined by cell growth with fluorescent labelling techniques. Simultaneous staining with different fluorochromes provides an extremely powerful way to demonstrate culture heterogeneity, and also to understand the functional differences revealed by each stain in practical applications. Analysis of bacterial fermentations showed a considerable drop (20%) in membrane potential and integrity during the latter stages of small scale (5L), well mixed fed-batch fermentations. These changes, not found in either batch or continuous culture fermentations, are probably due to the severe, steadily increasing stress associated with glucose limitation during the fed-batch process, suggesting 'on-line' flow cytometry could improve process control. Heat injured cells can already show up to 4 log of differences in recovery in different pre-enrichment media, thus contributing to the problem of viable but non-culturable cells (VBNC's). Cytometric cell sorting demonstrated decreasing recovery with increasing loss of membrane function. However, a new medium protecting the cells from intracellular and extracellular causes of oxidative stress improved recovery considerably. Actively respiring cells showed much higher recovery improvement than the other populations, demonstrating for the first time the contribution of oxidative respiration to intracellular causes of damage as a key part of the VBNC problem. Finally, absolute and relative frequencies of one species in a complex population were determined using immunofluorescent labelling in combination with the analysis of cell function. The detail and precision of multiparameter flow cytometric measurements of cell function at the single cell level now raise questions regarding the validity of classical, growth dependent viability assessment methods.

Bacteria↗
PubMed1998

Identification of epitopes within beta lactoglobulin recognised by polyclonal antibodies using phage display and PEPSCAN.

Two different epitope mapping techniques were used to identify linear epitopes recognised by polyclonal IgG antibodies from rabbits immunised with bovine beta lactoglobulin (BLG), which is generally regarded as a major allergen in milk. The first, PEPSCAN, was used to investigate the binding of several rabbit polyclonal antisera to sequential overlapping peptides (12-mers) across the sequence of BLG. Each peptide was synthesized on a different polypropylene PIN, and a standard ELISA procedure was used to locate which of these peptides bound the antibodies under investigation. Comparisons of PEPSCANs for antisera from six different rabbits showed that each rabbit recognized a similar set of epitopes within BLG. PEPSCAN analysis also showed that polyclonal antibodies from the mouse recognize a set of epitopes similar to those recognized by the rabbit. The second epitope mapping technique is known as phage display and utilizes libraries of randomized short peptides fused to the coat proteins of filamentous phage as a source of epitopes for analysis. A gene VIII phage display library was used in this study with constrained nonapeptides, which were screened for epitopes recognized by affinity purified rabbit anti-BLG IgG. Immobilised rabbit anti-BLG IgG was screened in two separate experiments, each consisting of three rounds of panning. For each separate experiment, a sensitive phage ELISA was used to screen several hundred single phage clones for binding to anti-BLG IgG immobilised on microtiter plates. As a result, a number of positive phage were identified from the two separate screens of the library (19 different peptides were isolated, which resembled four different regions of BLG). The identified sequences were found to constitute a subset of the linear epitopes recognized by the PEPSCAN technique. The coordinates of the crystal structure of BLG were used to display mapped epitopes on its structure. This study has permitted detailed mapping of the major linear antigenic regions within BLG recognised by IgG antibodies from immunised rabbits and mice.

Amino Acid Sequence↗
PubMed1998

High-density immobilization of an antibody fragment to a carboxymethylated dextran-linked biosensor surface.

There are numerous chemical methods published that enable protein coupling to carboxymethyl (CM) dextran. Here we have taken traditional amine coupling using N-hydroxysuccinimide (NHS) and N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and coupled an antibody fragment (scFv) to CM dextran at a very high density. Using an upgraded BIAlite from Biacore AB, more than 7000 RU of scFv was coupled to a CM dextran biosensor chip. In addition, scanning electron microscopy was performed on CM dextran biosensor chips following amine coupling of 30 nm gold anti-IgG particles. This showed that amine coupling was uniform across the biosensor chip surface. Calculations show that 7620 RU of an scFv coupled to such a surface results in a mean distance between binding sites of 8.8 nm. This equates to a packing volume of approximately 20% of the available space occupied by the antibody fragment. Comparisons made with densities of covalently coupled IgG show that a greater number of antibody fragment molecules can be coupled per unit area. This is most likely due to the smaller size of an antibody fragment (scFv), which has a volume of less than 20% of an IgG molecule. The significance of these findings is discussed.

Animals↗
PubMed1998

Assessment of bacterial viability status by flow cytometry and single cell sorting.

Rapid bacterial detection and viability measurements have been greatly enhanced by recent advances in the use of fluorescent stains in cytometry. It has previously been shown that four physiological states can be distinguished: reproductively viable, metabolically active, intact and permeabilized. Previous sorting experiments have shown that not all intact cells readily grow, but some intact cells can grow even when they fail to show metabolic activity, as determined by esterase turnover. To circumvent the limitations imposed by active dye extrusion or cell dormancy on viability measurements used to date (e.g., enzyme activity or cell polarization), a fast triple fluorochrome staining procedure has been developed that takes account of these problems. This allows further cellular characterization of intact cells by: active exclusion of ethidium bromide (EB) (metabolically active cells), uptake of EB but exclusion of bis-oxonol (BOX) (de-energized but with a polarized cell membrane) and uptake of both dyes (depolarized). Permeabilized cells were identified by propidium iodide (PI) uptake. The method was validated using an electronically programmable single cell sorter (EPICS Elite) and aged Salmonella typhimurium cells. Reproductive viability was determined by sorting single cells to their staining pattern directly onto agar plates. Most polarized cells could be recovered as well as a significant fraction of the depolarized cells, demonstrating that depolarization is a sensitive measure of cell damage but a poor indicator of cell death.

Bacteriological Techniques↗
PubMed1995

Pre-treatment serum levels of tumour markers in metastatic breast cancer: a prospective assessment of their role in predicting response to therapy and survival.

The value of pre-treatment serum tumour marker levels in 85 consecutive patients of newly diagnosed metastatic breast cancer was prospectively assessed for predicting response to therapy and survival. The markers studied were carcinoembryonic antigen (CEA), orosomucoid (ORO), erythrocyte sedimentation rate (ESR), C reactive protein (CRP), ferritin (FERR), human milk fat globule membrane 1 and 2 (HMFG1 and 2), CA 15-3 and NCRC-11. There was no correlation between serum marker levels and response to therapy. Only serum concentrations of CRP (P = 0.02), FERR (P = 0.001), HMFG1 (P = 0.004) and HMFG2 (P = 0.04) were predictive for survival. The prognostic significance of HMFG1 was restricted to a minority of patients (7%) with extreme values of these serum markers.

Aged↗
PubMed1993

The value of the human milk fat globule membrane antigen HMFG2 in epithelial ovarian cancer monitoring: comparison with CA125.

We assayed serum HMFG2 in serial samples from 215 primary epithelial ovarian cancer patients using an 'in-house' single determinant ELISA, 45% of patients with stage I, 54% with stage II, 61% with stage III and 75% with stage IV disease had elevated serum HMFG2. Post-operative levels were significantly related with residual tumour volume (P < 0.005), and fell in the majority of responders, although the association with response to first-line chemotherapy was not significant. HMFG2 had a sensitivity of 50% specificity of 83%, accuracy of 61%, PVP of 86% and PVN of 45% for disease at second-look laparotomy. Serial levels gave a lead time to clinical relapse in 47% of patients who responded to therapy, including one patient with negative CA125 levels. HMFG, paralleled CA125 in many respects, although it was elevated in fewer patients. In a stepwise discriminant analysis, HMFG2 added to the discrimination of CA125 (r = 0.183, P < 0.005), although additional accurate information was only given in patients with advanced poorly differentiated serous cystadenocarcinoma. Given that HMFG2 is expressed in few patients who are CA125 negative it is unlikely that it will have a significant clinical impact upon patient management.

Antibodies, Monoclonal↗
PubMed1993

Continuous chemotherapy in responsive metastatic breast cancer: a role for tumour markers?

A biochemical response index comprising ESR, CEA and CA 15.3 was evaluated in 67 patients with systemic breast cancer treated by chemotherapy; 55 were assessable by UICC criteria and the response index (96% of all UICC assessable patients). Marker changes at 2 and 4 months showed a highly significant correlation with the UICC assessed response at 3 and 6 months (P < 0.001); sensitivity 100%, specificity 87%; positive predictive value 85%; negative predictive value 100%. This index was then used to select out truly responsive patients and to prospectively direct their chemotherapy. Twenty-six responding (biochemical/clinical) patients were randomised to discontinue cytotoxics after 6 months and move to maintenance hormones (n = 13) or continue chemotherapy whilst the biochemical markers kept falling or remained within the normal range. Biochemical progression prompted a change of chemotherapy. Continuous chemotherapy in biochemically defined responders was associated with a significant lengthening of remission duration and an improved quality of life and survival. We are now using the index to routinely direct chemotherapy and select out true responders for maintenance chemotherapy.

Adult↗
PubMed1993

Construction of a reshaped HMFG1 antibody and comparison of its fine specificity with that of the parent mouse antibody.

A human antibody with milk mucin specificity was obtained by transferring the complementarity determining regions (CDR) of the mouse antibody HMFG1 onto carefully selected human framework regions. The resulting reshaped human antibody, HuHMFG1, showed no difference in relative affinity for its antigen compared with the parent mouse HMFG1. The minimum epitope recognized by both the mouse and reshaped antibodies was demonstrated by epitope mapping to be identical, and consists of the tetramer PDTR. In a replacement net analysis, in which each of the amino acids was replaced in turn with the 19 other residues, it was determined that mouse HMFG1 and HuHMFG1 reacted with this series of synthetic peptides in an equivalent manner, indicating retention of identical fine specificity in the HuHMFG1 antibody. In contrast to other published reports, this was achieved without involvement of any framework residues in the binding site transfer. These data demonstrate that if well-matching human framework regions are employed grafting the CDR only can be sufficient to confer desired specificities to human antibodies and can, indeed, provide human analogues of mouse antibodies with virtually indistinguishable affinities and fine specificities relative to the mouse parent antibodies.

Amino Acid Sequence↗
PubMed1991

Tumour localisation with a radioactively labelled reshaped human monoclonal antibody.

A genetically reshaped human IgG1 monoclonal antibody (Hu2PLAP) with anti-tumour specificity, was radiolabelled with Indium-111 by chelation with a new macrocyclic compound (DOTA) which allows the production of stable radioimmunoconjugates for in vivo application. This was used to image seven patients with malignant disease, of whom two had been previously exposed to mouse monoclonal antibodies and had developed human anti-mouse antibodies (HAMA). Successful tumour localisation was seen in the four patients with active disease and antigen positive tumours. No patient showed any antibody responses against Hu2PLAP, but three out of six patients tested showed an immune response against the macrocycle DOTA. Reshaped human monoclonal antibodies with anti-tumour specificity may facilitate repeated administrations of radioactive antibodies, thus allowing new possibilities, both in the diagnosis and treatment of cancer.

Adult↗
PubMed1990

Assessment of four monoclonal antibodies as serum markers in breast cancer.

Four monoclonal-antibody-defined serum markers (CA15-3, HMFG1, HMFG2 and NCRC-11) were examined in five groups of subjects: controls, benign breast disease and stage I/II, stage III and metastatic breast cancer. None of the markers were significantly elevated in primary breast cancer (i.e. stage I/II or stage III) compared with controls or patients with benign breast disease. These markers therefore have no role in screening or in the diagnosis of primary breast cancer. CA15-3, HMFG2 and NCRC-11 were significantly increased in the patients with metastatic breast cancer (P less than 0.001), indicating a potential use in the diagnosis of symptomatic metastases. In patients with metastases, sequential changes in CA15-3 correlated significantly with clinical response to therapy. Thus CA15-3 is a powerful marker of response and in combination with other markers, may provide an objective measurement of response to therapy in patients with advanced breast cancer.

Adult↗
PubMed1989

HMFG1 antigen: a new marker for carcinomatous meningitis.

Carcinomatous meningitis is a devastating metastatic complication of systemic carcinoma, which may occur insidiously, accompanied by a confusing spectrum of clinical symptoms and signs. In the absence of reliable diagnostic tumour markers, the diagnosis is established by the demonstration of malignant cells within the cerebrospinal fluid (CSF). Cytological techniques requiring skillful interpretation are occasionally negative in the presence of established disease, and when positive may indicate leptomeningeal malignancy of such advanced nature that effective palliation is difficult. Biochemical tumour marker technology offers the potential of reliable diagnosis in early disease states, prior to the appearance of exfoliated malignant cells. In a series of 100 patients, we assayed for an epithelial associated glycoprotein (HMFGI antigen) in CSF obtained at lumbar puncture. In 18 of 20 patients with carcinomatous meningitis, this high-molecular-weight glycoprotein was detectable in the CSF. The antigen was also present in 2 patients with neoplastic meningitis complicating lymphoma and medulloblastoma, but was not detected in the CSF of the remaining 78 patients.

Antigens, Neoplasm↗
PubMed1987

Optical biosensors for immunoassays: the fluorescence capillary-fill device.

This paper reports, for the first time, details of a novel type of optical biosensor for immunoassays, the fluorescence capillary-fill device (FCFD). This is based on a straightforward adaptation of the technology used to mass manufacture liquid-crystal display (LCD) cells to give cheap disposable immunosensors. These merely require contact by the sample to give a result in about a minute, and use certain principles of optical fibres and waveguides to avoid the need for operator attention, for physical separation methods or for washing steps. After a very brief introductory review and classification of optical biosensors, the main features of the FCFD and its associated instrumentation are described. The optical characteristics of the FCFD are then described, followed by accounts of the immunoassay method, the measurement system used in the experiments, the fabrication of FCFD sensors and a detailed description of the design of a competitive immunoassay for human immunoglobulin G (hIgG). The experimental details and the results of a first attempt at such an assay are then presented and discussed. It is concluded that the demonstration of this assay is a significant achievement, because the format of the FCFD, its manufacturing process and its instrumentation are completely novel. Certain problem areas have been identified and quantified; intended further work on these is outlined.

Animals↗
PubMed1985

Actomyosin organization in stationary and migrating sheets of cultured human endothelial cells.

We used immunofluorescence microscopy to study the organization of actin, myosin and vinculin in confluent endothelial cells and in cells migrating into an experimental wound and interference reflection microscopy to assess the cell-substratum adhesion pattern in these cells. In confluent stationary endothelial cell monolayers actin showed a distinct cell-to-cell organization. Myosin, on the other hand, was diffusely distributed and was clearly absent from cell peripheries. Vinculin was confined as linear arrays to cell-cell contact areas. Interference reflection microscopy revealed areas of close and distant adhesion but no focal adhesion sites in these cultures. Twelve hours after experimental wounding a distinct zone of advancing cells was seen at the wound edge. These cells showed a spreadout morphology and, in contrast to stationary cells, had a stress fibre-type organization of both actin and myosin. Vinculin was in the migrating cells seen as plaques at the ventral cell surface. In interference reflection microscopy numerous focal adhesions were seen. The results indicate that the actomyosin system forms the structural basis for monolayer organization of endothelial cells and responds by reorganization upon cell migration.

Actomyosin↗
PubMed1984

Distinct cytoskeletal domains revealed in sperm cells.

Antibodies against different cytoskeletal proteins were used to study the cytoskeletal organization of human spermatozoa. A positive staining with actin antibodies was seen in both the acrosomal cap region and the principal piece region of the tail. However, no staining was obtained with nitrobenzoxadiazol-phallacidin, suggesting that most of the actin was in the nonpolymerized form. Most of the myosin immunoreactivity was confirmed to a narrow band in the neck region of spermatozoa. Tubulin was located to the entire tail, whereas vimentin was only seen in a discrete band-like structure encircling the sperm head, apparently coinciding with the equatorial segment region. Surface staining of the spermatozoa with fluorochrome-coupled Helix pomatia agglutinin revealed a similar band-like structure that co-distributed with the vimentin-specific staining. Instead, other lectin conjugates used labeled either the acrosomal cap region (peanut and soybean agglutinins), both the acrosomal cap and the postacrosomal region of the head (concanavalin A), or the whole sperm cell surface membrane (wheat germ and lens culinaris agglutinins and ricinus communis agglutinin l). In lectin blotting experiments, the Helix pomatia agglutinin-binding was assigned to a 80,000-mol-wt polypeptide which, together with vimentin, also resisted treatment with Triton X-100. Only the acrosomal cap and the principal piece of the tail were decorated with rabbit and hydridoma antibodies against an immunoanalogue of erythrocyte alpha-spectrin (p230). p230 appeared to be the major calmodulin-binding polypeptide in spermatozoa, as shown by a direct overlay assay of electrophoretic blots of spermatozoa with 125I-calmodulin. The results indicate that spermatozoa have a highly specialized cytoskeletal organization and that the distribution of actin, spectrin, and vimentin can be correlated with distinct surface specializations of the sperm cells. This suggest that cytoskeleton may regulate the maintenance of these surface assemblies and, hence, affect the spermatozoan function.

Acrosome↗
PubMed1984

Syncytioskeletons in choriocarcinoma in culture.

Indirect immunofluorescence microscopy using anti-actin serum has been used to investigate the distribution of actin-containing polymers in BeWo cells. This cell line, derived from a human choriocarcinoma, contains tissue that, like its tissue of origin, is partly syncytial. The syncytial nature has been inferred from study of Nomarski optical sections and from transmission electron microscopy. The multinucleated plaques of tissue possess a syncytioskeleton with a number of actin-containing features characteristic of cultured cells. These include stress fibres, cortical layers and ruffled membranes. Other actin-containing structures are more typical of the related non-pathological syncytiotrophoblast. These include a dense population of microvilli. The overall organization of the actin syncytioskeletons bears no obvious relationship to the number or position of nuclei in the syncytium. Indirect immunofluorescence microscopy has also been employed to localize the protein tubulin in BeWo cells. The microtubules do not appear to be spatially organized by a particular nucleus. Rather, there are numerous microtubule-organizing centres (MTOCs) that exist in the cytoplasm and do not have the expected numerical and positional relationship to nuclei. From these data it appears that polymeric cytoskeletal elements in these syncytia are organized in a manner not immediately subordinate to syncytial nuclei.

Actins↗