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K Siddle

Publications and source records attributed to K Siddle.

At least 127 records · Page 7Linked to original sources
PubMed1983

A monoclonal antibody to human brain-type creatine kinase. Increased avidity with mercaptans.

1. A monoclonal antibody (subclass immunoglobulin G1) has been raised against human brain-type creatine kinase (CK-BB). This antibody did not cross-react with either muscle-type creatine kinase (CK-MM) or heart-type creatine kinase (CK-MB). 2. The binding constant measured with native antibody was 6 X 10(8) M-1. In the presence of 2mM-dithiothreitol this constant was some 40-50-fold greater. 3. Partial reduction and alkylation showed that the increased binding was due to a direct effect on the antibody and was associated with concomitant cleavage of the heavy-heavy interchain disulphide bonds. The binding constant measured with Fab' fragments produced from reduced and alkylated antibody was similar to that shown by the native, unreduced antibody. 4. The molecular weight of the complex found in the absence of mercaptans was consistent with one antibody and one CK-BB molecule, whereas the molecular weight estimated with reduced and alkylated antibody was consistent with a complex of two antibodies and two CK-BB molecules. 5. It is proposed that mercaptans increase the flexibility of the hinge region of the antibody molecule, allowing the formation of a higher-order complex with increased avidity for the CK-BB dimer.

Alkylation↗
PubMed1983

Immunoaffinity purification of human complement component C9 using monoclonal antibodies.

Five monoclonal antibodies to human C9 were produced. Three of the antibodies had affinities from 1.6 to 3.1 X 10(9) M-1 and 2 were of lower affinity, 0.1 to 0.3 X 10(9) M-1. Four distinct antigenic sites on C9 were detected. The lowest affinity antibody was immobilised on Sepharose 4B and used to isolate C9 from human plasma. A 1200-fold purification was obtained with yield of 30-40%. The purified antigen maintained full cytolytic activity.

Animals↗
PubMed1983

Immunoradiometric assay for human complement component C9 utilising monoclonal antibodies.

A two-site immunoradiometric assay for human C9 has been developed. The assay utilised two non-competing monoclonal antibodies to C9 in a single incubation assay protocol. The detection limit of the assay was 0.1 ng (1.4 X 10(-15) moles) in a sample volume of 100 microliters. Using this assay the C9 concentration in normal human plasma was 60.2 +/- 14.9 mg/l (mean +/- 1 standard deviation, 8.5 X 10(-10) mol/l. Significantly elevated levels were found in the plasma of patients with rheumatoid arthritis (90.4 +/- 19.9 mg/l, mean +/- 1 SD). Measurements of C9 in cerebrospinal fluid and synovial fluid were also performed. The low levels of C9 in cerebrospinal fluid (less than 1 mg/l), undetectable by previously available assay methods, were easily measurable with this highly sensitive assay.

Antibodies, Monoclonal↗
PubMed1983

Characterisation of monoclonal antibodies for human luteinising hormone, and mapping of antigenic determinants on the hormone.

Twelve mouse monoclonal antibodies for human luteinising hormone were produced. The affinities varied from 4 X 10(7) to 1 X 10(10) l/mol. The specificity of each antibody was assessed by determining the relative reactivities with luteinising hormone, thyroid stimulating hormone, follicle stimulating hormone and chorionic gonadotrophin. Six antibodies bound to the alpha-subunit as shown by similar reactivity with all hormones, and the remainder to the beta-subunit as shown by specificity for luteinising hormone. This latter group of antibodies cross-reacted only weakly with thyroid stimulating hormone (approximately 10%) and follicle stimulating hormone (approximately 3%). Three of these antibodies also showed low reactivity towards chorionic gonadotrophin (less than 10%), though the others did not (80-300%). The ability of different antibodies to bind simultaneously to luteinising hormone was examined and it was shown that several distinct antigenic determinants existed on both subunits. The characterisation of monoclonal antibody binding sites is discussed in relation to the use of antibodies in two-site immunoradiometric assays.

Animals↗
PubMed1983

Localisation of 5'-nucleotidase in a rat liver cell line using a monoclonal antibody and indirect immunofluorescent labelling.

A new method of labelling anti-IgG with rhodamine while protecting the IgG binding site is described. Indirect immunofluorescence of the ectoenzyme 5'-nucleotidase using a monoclonal antibody shows that it has a punctate distribution over the whole cell surface of a cultured rat liver cell line. The density of fluorescent spots representing the surface localisation of 5'-nucleotidase and of clathrin-coated pits was analysed by a computerised counting technique. The cell surface localisation of 5'-nucleotidase differs from clathrin-coated pits and microvilli. 5'-Nucleotidase within the cell is not uniquely associated with any clearly defined organelle.

5'-Nucleotidase↗
PubMed1982

Solubilization and purification of rat liver 5'-nucleotidase by use of a zwitterionic detergent and a monoclonal-antibody immunoadsorbent.

1. A variety of detergents were used to solubilize 5'-nucleotidase from rat liver plasma membranes. 2. The zwitterionic detergent Sulphobetaine 14 gave optimal solubilization by the criteria of release into a high-speed-centrifugation supernatant and the formation of the smallest and least polydisperse active enzyme observed on polyacrylamide-gel electrophoresis. 3. The Sulphobetaine 14-solubilized enzyme from rat liver was purified by using the conventional techniques of ion-exchange chromatography and gel filtration, or by an immunoaffinity step with a monoclonal antibody immunoadsorbent. 4. 5'-Nucleotidase was purified at least 12 000-fold relative to liver homogenate by the immunoaffinity purification scheme and had a specific activity in the range 285-340 mumol/min per mg of protein. The yield was in the range 9-16%. 5. The purified enzyme shows a major polypeptide band of apparent Mr 70 000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and a minor band of apparent Mr 38 000. 6. A rational approach to the general problem of the purification of minor intrinsic membrane proteins is discussed, with the use of polyacrylamide-gel electrophoresis to determine the most appropriate detergent and monoclonal antibodies in subsequent immunoaffinity purification.

5'-Nucleotidase↗
PubMed1982

Characterization of monoclonal antibodies directed against human thyroid stimulating hormone.

Monoclonal antibodies directed against human thyroid stimulating hormone (TSH) were obtained from hybrid myelomas, following fusion of mouse NSI myeloma cells with mouse spleen cells. Ten different antibodies were obtained from 4 separate fusions. Eight antibodies were of the IgG1 subclass. Affinities of antibodies for TSH were in the range 2 x 10(8)-5 x 10(10) M-1. Five of the antibodies were specific for TSH and did not react with LH, FSH or hCG. The remaining antibodies reacted with all these hormones and were assumed to recognise their common (alpha) subunit. The 5 specific antibodies fell into 3 subgroups recognising distinct antigenic determinants, whereas the 5 non-specific antibodies recognised a single determinant or closely related set of sites. It is concluded that these antibodies should be valuable reagents for use in sensitive and specific two-site immunoradiometric assays.

Animals↗
PubMed1979

Investigation of the subcellular distribution of cyclic-AMP phosphodiesterase in rat hepatocytes, using a rapid immunological procedure for the isolation of plasma membrane.

The distribution of cyclic-AMP phosphodiesterase was investigated in subcellular fractions prepared from homogenates of rat liver or isolated hepatocytes. When measured at 1 mM or 1 microM substrate concentration, approx. 35% or 50%, respectively, of enzyme activity was particulate. The soluble activity appeared to be predominantly a 'high Km' form, whereas the particulate activity had both 'high Km' and 'low Km' components. The recovery of cyclic-AMP phosphodiesterase was measured using 1 microM substrate concentraiton, in plasma membrane-containing fractions prepared either by centrifugation or by the use of specific immunoadsorbents. The recovery of phosphodiesterase was lower than that of marker enzymes for plasma membrane, and comparable with the recovery of markers for intracellular membranes. It was concluded that regulation of both 'high Km' and 'low Km' phosphodiesterase could potentially make a significant contribution to the control of cyclic AMP concentration, even at microM levels, in the liver. the 'low Km' enzyme, for which activation by hormones has been previously described, appears to be located predominantly in intracellular membranes in hepatocytes. The immunological procedure for membrane isolation allowed the rapid preparation of plasma membranes in high yield. Liver cells were incubated with rabbit anti-(rat erythrocyte) serum and homogenized. The antibody-coated membrane fragments were then extracted onto an immunoadsorbent consisting of sheep anti-(rabbit IgG) immunoglobulin covalently bound to aminocellulose. Plasma membrane was obtained in approx. 40% yield within 50 min of homogenizing cells.

3',5'-Cyclic-AMP Phosphodiesterases↗