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Biomedical subjects

C H Self

Publications and source records attributed to C H Self.

At least 19 recordsLinked to original sources
PubMed1998

A sandwich hybridization assay employing enzyme amplification for termination of specific ribosomal RNA from unpurified cell lysates.

We have employed the power of the cyclic NAD-based enzyme amplification system to the determination of 16S rRNA. This generally applicable system employs two oligonucleotide probes, one of which is captured on a microtiter well surface and the other labeled with alkaline phosphatase. The detection of very low levels of hybridization of the capture probe is then achieved by the means of the ultrasensitive enzyme-amplified assay system, resulting in a highly sensitive, convenient, and rapid technology which can be directly employed on unpurified samples. We have been able to demonstrate the detection of 20 amol (10(7) molecules) of pure rRNA, and specific signals from as few as 2000 bacterial cells have also been demonstrated. The total procedural time can be short-5 to 18 h-depending on the dynamic range and sensitivity required. RNA target in the range of 10(12)-10(8) molecules can be assayed within 5 h. Extending the substrate incubation time enables between 10(11) and 10(7) molecules to be determined within 18 h. The system has great potential use with respect to studying the distribution and physiological states of cellular organisms.

Enzymes↗
PubMed1998

Increased sialylation of oligosaccharides on IgG paraproteins--a potential new tumour marker in multiple myeloma.

AIMS: To investigate whether changes in carbohydrate structure of IgG are related to malignancy and stage of disease in myeloma and monoclonal gammopathy of uncertain significance (MGUS). METHODS: 61 patients were studied at diagnosis: 14 with MGUS, nine with stage I multiple myeloma, 11 with stage II, 21 with stage III, and five with solitary plasmacytoma. IgG was extracted from serum by protein G affinity chromatography. Oligosaccharides were cleaved from the protein backbone enzymatically by N-glycosidase F. Oligosaccharide analysis was performed by high pressure anion exchange chromatography with pulsed electrochemical detection (HPAE-PED). RESULTS: Up to 15 oligosaccharide peaks were identified in three major fractions: neutral, monosialylated, and disialylated. Patients with myeloma showed an increase in the proportion of sialylated oligosaccharides in comparison with patients with MGUS. The ratio of neutral to sialylated oligosaccharides (N:S) was reduced at all stages of myeloma compared with MGUS: MGUS, 11.35; myeloma stage I, 7.6 (p = 0.047); stage II, 5.20 (p = 0.035); stage III, 3.60 (p = 0.0002); plasmacytoma, 7.5 (p = 0.046). The N:S ratio was independent of paraprotein concentration (r = 0.05). CONCLUSIONS: The ratio of neutral to sialylated oligosaccharides may act as a new marker of malignancy in IgG paraproteinaemia and warrants further investigation.

Aged↗
PubMed1997

Characterization of corticotropin-releasing hormone binding sites in the human placenta.

The human placenta synthesizes and secretes large amounts of corticotropin-releasing hormone (CRH) which has been implicated in the triggering of parturition. The placental CRH was found to act in a paracrine manner to stimulate secretion of ACTH and beta-endorphin. In view of this we sought to characterize CRH binding sites in the human placenta. The specific binding of 125I-tyrosyl-ovine CRH (125I-oCRH) to placental membranes was dependent on time, temperature, pH, divalent cations and was reversible on addition of excess oCRH. Scatchard analysis revealed a high afinity binding site with a dissociation constant of approximately 0.7 nmol/L and maximum number of binding sites approximately 44 fmol/mg protein. Disuccinimidyl suberate, a chemical cross-linker, was used to covalently attach 125I-oCRH to placental membranes. The labelled placental membranes were analyzed by SDS-PAGE and autoradiography. A major radioactively labelled band with a molecular weight of 55,000 Da was identified. In this study we have identified placental binding sites for CRH with properties similar to CRH receptors described in a number of human and animal tissues and with a molecular weight similar to that of the brain CRH receptor. These binding sites may be involved in the regulation of the placental CRH/ACTH-beta-endorphin axis during pregnancy and parturition.

Autoradiography↗
PubMed1996

Enhanced specificity for small molecules in a convenient format which removes a limitation of competitive immunoassay.

Anti-complex immunoassay systems for small molecules permit the exquisite specificity achievable with monoclonal antibodies to be expressed to an extent which is not possible with competitive format immunoassay. While our previously reported anti-complex system is superior to competitive systems in terms of sensitivity, precision and specificity we have found that this specificity may be enhanced dramatically by simply interposing a wash step between the addition of sample and that of the labelled anti-complex antibody. When such a wash step was attempted with the competitive format system, after addition of sample but before addition of the labelled component, assay performance was degraded to the extent of making it unusable. We suggest, therefore, that the inherent flexibility of the anti-complex approach for small molecule assay creates an opportunity for remarkable enhancement of the functional specificity of primary antibody.

Antibody Specificity↗
PubMed1996

Advances in immunoassay technology.

Major developments continue to be reported in key areas of immunoassay technology. Following the development of excellent signal generation methods, attention has shifted to the development of immunochemical methods and instrumentation to provide convenient systems of high performance. Important advances have been made in the design of immunochemical approaches that permit the replacement of competitive format assays for small molecules, such as drugs, metabolites and pollutants, with non-competitive formats, bringing advantages previously seen only with large molecular analytes. Bispecific antibodies and recombinant proteins are also beginning to impact immunodiagnostics, with the promise of even more highly specified reagents. Improvements in automation have brought the facility of homogeneous systems to high-throughput and high-performance heterogeneous systems. Similarly, 'point of need' testing continues to progress. Through all of these advances, systems are evolving according to the needs of users in terms of operator convenience, accuracy, specificity, speed, robustness, and sensitivity.

Animals↗
PubMed1996

Light activatable antibodies: models for remotely activatable proteins.

The ability to activate biological macromolecules remotely, at specific locations and times, will allow the manipulation of a wide range of cellular activities and give rise to many practical applications. Interest has been shown in the theoretical possibility of accomplishing this by means of photochemical approaches. Photochemical changes of the guest-binding cavity of cyclodextrins has been suggested; however, these changes require organic solvent. What is needed is a widely and readily applicable method allowing activation under physiological conditions. We have developed such a method. This is based on our demonstration that relatively large amounts of the a-methyl substituted 2-nitrobenzyl alcohol, namely, 1-(2-nitrophenyl)ethanol (NPE) can be coupled to proteins using diphosgene. Previous work involved "caging" of small molecules such as ATP (ref. 5-9) and blocking amino acids in peptide synthesis with 2-nitrobenzyl compounds. For large molecules, site-specific reversible inactivation of T4-lysozyme has been reported following introduction of an aspartyl beta-nibenzyl ester into its active site by mutagenesis. In contrast, the present simple procedure allows an existing protein to be deactivated and then, when and where required, reactivated by exposure to ultraviolet-A (UV-A) light. We have employed antibodies as models for both receptors and ligands and have successfully modulated: antibody binding sites for antigen; antigen binding sites for antibody, and antibody Fc binding sites for Protein A.

Antibodies↗
PubMed1996

Ultra-specific immunoassays for small molecules: roles of wash steps and multiple binding formats.

New immunometric forms of immunoassay are much more flexible to use than competitive-format immunoassays for small molecular analytes. An example of the utility of this flexibility is the ability to wash the capture antibody after it has been exposed to analyte but before addition of the labeled reagent. This simple maneuver has a large impact on the specificity obtained from already highly specific assays. We also show that specificity can be further increased by means of our multiple binding assay approach, in which the final reading reflects analyte binding to two different primary capture monoclonal antibodies.

Antibodies, Monoclonal↗
PubMed1994

Photocleavable nitrobenzyl-protein conjugates.

We have developed methods to allow the reversible binding of up to 15 nitrobenzyl residues per bovine serum albumin molecule and show 95% of these residues can be removed by exposure to UV light for 10 min. The general non-specific coating method is presented by means of a model system but is applicable to a wide range of proteins with important biological functions. Potentially, any protein could be coated with sufficient photo-removable groups to inhibit its biological function. Its activity may then be restored at will by exposure to UV light removing the coupled 2-nitrobenzyl groups.

Nitrobenzenes↗
PubMed1994

Monosaccharide composition of haptoglobin in liver diseases and alcohol abuse: large changes in glycosylation associated with alcoholic liver disease.

Liver damage through prolonged intake of high amounts of alcohol is a serious problem that affects many members of the population. The aim of this study was to investigate changes in the glycosylation of haptoglobin (Hp) resulting from alcoholic liver diseases. The monosaccharide composition was measured in Hp isolated from 48 healthy individuals, 15 alcohol abusers (AA); 25 patients with alcoholic liver disease, including those with alcoholic cirrhosis (ALD/AC), and 17 other patients with either chronic active hepatitis (AH) or primary biliary cirrhosis (BC). Fucose was elevated per mol of Hp in 70%, 44%, and 33% of the individuals in the ALD/AC, BC and AA groups, respectively. Fucose was not elevated in the AH group. N-acetylglucosamine was also elevated in the ALD/AC group. Expressing results per 3 mol of mannose suggested the presence of higher branching with increased fucose content in Hp from all the abnormal groups except the AH group. More structural information is required to develop the diagnostic potential of carbohydrate measurements of Hp in alcoholic liver diseases.

Acetylglucosamine↗
PubMed1994

Glycation of monoclonal antibodies impairs their ability to bind antigen.

As elevated levels of glycated IgG have been detected in the plasma of patients with diabetes mellitus, a disease associated with increased susceptibility to infection, we have investigated whether glycation of MoAbs affects the kinetics and/or affinity of antigen binding. Three mouse MoAbs were incubated with 0.5 M glucose at pH 7.4 for 14-21 days at 37 degrees C. Control MoAbs were incubated using identical conditions but with no added glucose. Using a surface plasmon resonance technique we found that glycation significantly increased the rate of dissociation (kdiss) of the antigen-antibody complex for all three MoAbs (P < 0.05, n = 4), but had no significant effect on the rate of association (kass). For one of the MoAbs, against human IgG (Fab), we also measured kdiss by an alternative method utilizing radiolabelled antigen, which confirmed that glycation of the antibody significantly increases kdiss (P < 0.001, n = 8). We also found using an ELISA-based method that glycation of the same MoAb significantly increased the equilibrium dissociation constant (Kd) (P < 0.05, n = 6). A significant increase in kd was observed after glycation using glucose concentrations consistent with those found in poorly controlled diabetics (P < 0.02, n = 5). We conclude that in vitro glycation can significantly lower the affinity of an antibody for its antigen, and significantly increases the rate of dissociation of the antigen-antibody complex.

Animals↗
PubMed1994

High-performance assays of small molecules: enhanced sensitivity, rapidity, and convenience demonstrated with a noncompetitive immunometric anti-immune complex assay system for digoxin.

We report the first clinical application of a noncompetitive immunometric assay system that provides advantages for the rapid and robust assay of small molecules similar to those realized for larger molecules with two-site immunometric assays. This anti-immune complex assay is based on the interaction of a receptor such as a primary antibody with its ligand, such that new binding sites, recognizable by a secondary antibody, are formed. In this report the system is applied to the measurement of digoxin in serum. Utilizing an anti-complex antibody that recognizes a digoxin-bound primary antibody with affinity > 2000-fold over its binding to the primary antibody alone, we show that this anti-complex assay system provides a high-performance assay for serum samples, being conveniently simple (immobilized primary antibody binds digoxin and then labeled secondary antibody so that when excess unbound label is washed away the immunometric readout reflects the digoxin concentration), rapid (incubation time 1-10 min), sensitive (detection limit 30 ng/L), precise (3-4% within-run CV, 1-8% total CV), and free from interference from digoxin-like immunoreactive factors.

Antibodies, Anti-Idiotypic↗
PubMed1993

Glycation increases the vascular clearance rate of IgG in mice.

As elevated levels of glycated IgG have been detected in the plasma of diabetics we have investigated whether glycation of IgG affects its vascular clearance rate, using a mouse model system. Polyclonal mouse IgG was aseptically incubated for 14-19 days with 0.5 M glucose in 0.1 M phosphate buffer (pH 7.4) at 37 degrees C. As control, IgG was incubated under identical conditions but with no added glucose. After incubation, both forms were labelled with 125I and injected intravenously into BALB/c mice. The rate of vascular clearance of the glycated IgG was found to be significantly higher than the control IgG in the periods 5-24 h (P < 0.001, n = 6) and 24-48 h (P < 0.01, n = 6) after injection. After 2-3 days the mice were killed and the major organs were harvested. With glycated IgG there was a significant increase in the 125I accumulated in the kidney (P < 0.02). In later experiments, dual labelling with 131I and 125I allowed mixtures of glycated and unglycated IgG to be injected into the same mouse so that the vascular clearance of both forms of IgG could be followed simultaneously. These experiments confirmed that glycation of the IgG significantly increases its vascular clearance rate.

Animals↗
PubMed1993

Effects of pyrogenic immunomodulators on the release of corticotrophin-releasing factor-41 and prostaglandin E2 from the intact rat hypothalamus in vitro.

1. The actions of the following pyrogens: lipopolysaccharide (LPS), polyinosinic:polycytidylic acid (Poly-I:C), human interleukin (IL)-1 alpha and IL-1 beta, human IL-6 and rat interferon (INF) on corticotrophin-releasing factor-41 (CRF-41) and prostaglandin E2 (PGE2) release from the intact rat hypothalamus in vitro have been studied. 2. Rat hypothalami were incubated in vitro in an artificial cerebrospinal fluid. Immunoreactive (ir)-CFR-41 and PGE2 released into the medium were measured by two-site enzyme amplified immunometric assay (EAIA) and radioimmunoassay (RIA) respectively. 3. Human IL-6 (1 to 10,000 IU ml-1) caused a dose-dependent release of irCRF-41, rising to a maximal 3-4 fold increase over basal at the highest dose tested. Human IL-1 alpha (1 to 1000 IU ml-1), human IL-1 beta (1 to 1000 IU ml-1), poly-I:C (10 pg ml-1 to 100 micrograms ml-1) and rat INF (1 to 10,000 IRu ml-1) all failed to alter irCRF-41 release. 4. LPS (1 mg ml-1) caused a 35% decrease in irCRF-41 release; however, over the dose-range of 0.1 microgram ml-1 to 100 micrograms ml-1, LPS failed to alter irCRF-41 release. The decreased irCRF-41 release in response to LPS (1 mg ml-1) was accompanied by a decrease in the subsequent 56 mM KCl stimulation of irCRF-41. 5. Human IL-1 alpha and IL-1 beta (1000 IU ml-1) were able to stimulate the release of irPGE2 from intact hypothalami, causing a 2 fold increase over basal release. Poly-I:C (100 microg ml-1), LPS (0.1 microg ml-1 to 1 mg ml-1), rat INF (10,000 IRu ml-1) and human IL-6 (1 to 10,000 iu ml-1) all failed to alter irPGE2release.6. In conclusion, these results suggest that the in vitro release of CRF-41 and PGE2, in response to pyrogens, are mediated via different cytokines. In view of this it is possible that different cytokines may mediate the temperature, prostaglandin and hypothalamo-pituitary-adrenocortical axis activation seen during pyrogenic stimulation in vivo.

Adjuvants, Immunologic↗