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K Siddle

Publications and source records attributed to K Siddle.

At least 109 records · Page 6Linked to original sources
PubMed1987

Replacement of lysine residue 1030 in the putative ATP-binding region of the insulin receptor abolishes insulin- and antibody-stimulated glucose uptake and receptor kinase activity.

To test whether the tyrosine kinase activity of the insulin receptor is crucial for insulin action, we have constructed mutations of the human insulin receptor at Lys-1030, which is in the presumed ATP-binding region. By using oligonucleotide-directed mutagenesis, this lysine residue was replaced with either methionine, arginine, or alanine. Chinese hamster ovary cells were transfected by mutant cDNAs and the expressed insulin receptors were characterized. We show here that none of these mutants exhibited insulin-activated autophosphorylation and kinase activity in vitro. They also do not mediate insulin- and antibody-stimulated uptake of 2-deoxyglucose. The tyrosine kinase activity is thus required for a key physiological response of insulin.

Adenosine Triphosphate↗
PubMed1987

Characterization and use in immunoradiometric assay of monoclonal antibodies directed against human proinsulin.

Proinsulin in human serum is heterogenous. Existing assay methods do not distinguish between the various forms intermediate on the pathway of processing from proinsulin to insulin. We report the production of mouse monoclonal antibodies against human proinsulin with biosynthetic human proinsulin (produced by recombinant DNA technology) as immunogen. Four monoclonal antibody-producing cell lines were obtained from two separate fusions. Two of the antibodies had affinity constants for intact proinsulin of 3.1 X 10(10) and 5.6 X 10(10) L/mol and bound all forms of proinsulin and insulin. Cross-reactivity with intact proinsulin was greater than with 65-66 and 32-33 split proinsulins and des 64-65 and des 31-32 proinsulins. The third antibody had an affinity constant for intact proinsulin of 6.3 X 10(10) L/mol and reacted only with intact proinsulin, 65-66 split proinsulin, and des 64-65 proinsulin; there was no reaction with 32-33 split proinsulin, des 31-32 proinsulin, or C-peptide. The fourth antibody reacted only with intact proinsulin and had an affinity constant of 4.1 X 10(8) L/mol. We report the use of two antibodies in a sensitive two-site immunoradiometric assay for intact proinsulin. Insulin, C-peptide, and 32-33 split proinsulin did not react in the assay up to a concentration of 100 pM. The 65-66 split proinsulin and des 64-65 proinsulin reacted with a potency of approximately 55% relative to intact proinsulin. Serum proinsulin concentrations measured with this assay were compared with those determined by an alternative method that detects only split proinsulins.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1986

Monoclonal antibodies reacting with multiple epitopes on the human insulin receptor.

Monoclonal antibodies for the human insulin receptor were produced following immunization of mice with IM-9 lymphocytes and/or purified placental receptor. Four separate fusions yielded 28 antibodies, all of which reacted with receptor from human placenta, liver and IM-9 cells. Some antibodies cross-reacted to varying degrees with receptor from rabbit, cow, pig and sheep, but none reacted with rat receptor. At least 10 distinct epitopes were recognized as indicated by species specificity and binding competition experiments. All of these epitopes appeared to be on extracellular domains of the receptor as shown by binding of antibodies to intact cells. In some cases the epitopes were further localized to alpha or beta subunits by immunoblotting. Several antibodies inhibited binding of 125I-insulin to the receptor, some had no effect on binding, and others enhanced the binding of 125I-insulin. It is concluded that these antibodies will be valuable probes of receptor structure and function.

Animals↗
PubMed1986

Rat and mouse monoclonal antibodies to human myelin basic protein.

BALB/c mice and Lewis rats were immunized with human myelin basic protein and its N- and C-terminal fragments. Mouse X mouse fusions produced seven monoclonal antibodies, all of the IgG class and directed against the N-terminal fragment. Five of the antibodies seemed to be against the same epitope, between amino acid residues 92 and 118. One antibody bound between residues 45 and 91, and the remaining antibody reacted with both peptides 1-44 and 45-91. Three monoclonal antibodies, all of the IgM class, were obtained by rat X rat hybridization. Two monoclonal antibodies, raised against whole myelin basic protein and the C-terminal fragment, respectively, each bound to peptide 118-178. The remaining antibody, raised against the N-terminal fragment, bound to peptide 45-91. These monoclonal antibodies are of interest for use in clinical radioimmunoassays and for immunohistochemical investigation of the structural relationships of the myelin sheath.

Animals↗
PubMed1985

An enzyme-amplified monoclonal immunoenzymometric assay for prostatic acid phosphatase.

An immunoassay for prostatic acid phosphatase is described in which a high degree of specificity for the prostatic isoenzyme, obtained by the use of monoclonal antibodies, is combined with great sensitivity, made possible by enzyme-amplified measurement of the combination of the isoenzyme with its antibody. The increase in sensitivity thus achieved is of the order of 170 times that of conventional methods of measurement. The advantages of the enzyme-amplified method have been shown to be particularly useful in detecting and monitoring small abnormalities of prostatic acid phosphatase levels in patients with prostatic cancer.

Acid Phosphatase↗
PubMed1984

A rapid, sensitive two-site immunometric assay for TSH using monoclonal antibodies: investigation of factors affecting optimisation.

Twenty-three monoclonal antibodies specific for human thyrotropin (TSH) have been prepared and characterised. Four different epitopes have been identified, 3 of which are sufficiently distinct to allow simultaneous binding of the corresponding antibodies. Only 1 epitope is expressed on free TSH beta-subunit. The best antibodies have affinities for TSH approaching 10(11) M-1 and cross-reactions with lutropin and chorionic gonadotropin of 0.2% or less. A 2-site immunoradiometric assay for TSH was established using 2 monoclonal antibodies, one of which was absorbed to plastic tubes and the other labelled with 125I. Several parameters affecting assay performance were investigated, including conditions for adsorption of antibodies to various plastics, selection of antibody combinations, concentration of labelled antibody and incubation protocol. The optimised assay covered a working range of 1-60 mU/1 (0.17-10 ng/ml) TSH in a 4 h, single incubation protocol, with no significant interference from other glycoprotein hormones at their maximum physiological or pathological concentrations.

Adsorption↗
PubMed1984

Red cell-labelled monoclonal antibodies for assay of human chorionic gonadotropin and luteinising hormone by reverse passive haemagglutination.

The assay of human chorionic gonadotropin and luteinising hormone by reverse passive haemagglutination reaction, using monoclonal antibodies coupled to red cells, is described. Quantitation is achieved by end-point determination for serial dilutions of standard or sample, the haemagglutination reaction being observed after settling under gravity for 90 min. Red cell-labelled antibodies were stabilised with glutaraldehyde without loss of sensitivity and allowing long term storage. Various antibody combinations were assessed, and the best combination under optimum conditions gave a positive haemagglutination reaction down to 0.2 ng/ml with HCG.

Antibodies, Monoclonal↗
PubMed1984

The existence and properties of two dimers of rat liver ecto-5'-nucleotidase.

Immunoaffinity-purified rat liver 5'-nucleotidase contained two subunits of Mr 70 000 (alpha) and 38 000 (beta). Charge-shift electrophoresis and chemical cross-linking revealed that approx. 80% of the solubilized enzyme activity occurred as an alpha alpha-dimer of Mr 140 000. The remaining 20% was an alpha beta-dimer of Mr 108 000. The beta-subunit did not possess enzymic activity. Peptide mapping and immunoblotting with antibodies against the alpha- and beta-subunits showed that the beta-subunit was homologous with a part of the alpha-subunit. Three monoclonal antibodies against rat liver 5'-nucleotidase were characterized as binding to the extracellular domain of the enzyme. All three monoclonal antibodies and concanavalin A bound to the alpha-subunit, but no binding could be detected to the beta-subunit. It was therefore concluded that the beta-subunit was a fragment of an alpha-subunit that had lost an extracellular domain. Both forms of the enzyme occurred in freshly solubilized membrane preparations as well.

5'-Nucleotidase↗
PubMed1984

Immunoaffinity purification of intact, metabolically active, cholinergic nerve terminals from mammalian brain.

A method for the immunoaffinity purification of cholinergic nerve terminals from mammalian brain was developed. A sheep antiserum to Torpedo electric-organ synaptic membranes, previously shown to be specific for cholinergic terminals in mammalian brain, was incubated with crude mitochondrial fractions prepared from rat brain. Cholinergic nerve terminals sensitized by this serum were purified from the mitochondrial fractions on a high-capacity cellulose immunoadsorbent bearing a mouse monoclonal anti-(sheep immunoglobulin G) antibody. Adsorption of nerve terminals on to the immunoadsorbent was assessed by using a variety of enzyme markers and gave a maximum yield of 24% of choline acetyltransferase, whereas non-specific binding was less than 1.0% for all of the enzymes measured. Cholinergic terminals were purified 26-fold from rat caudate nucleus, 30-fold from rat hippocampus and 38-fold from rat cerebral cortex. The terminals were shown to be intact, osmotically sensitive and metabolically active.

Animals↗
PubMed1984

Proinsulin in human serum: problems in measurement and interpretation.

The immunoreactivity of an extracted pancreatic human proinsulin standard in an indirect immunoradiometric assay was found to be at least one hundred times higher than that of biosynthetic human proinsulin. Limited tryptic digestion of the biosynthetic proinsulin increased its immunoreactivity in the assay and this was attributed to the production of partially cleaved proinsulin molecules which still retained the C-peptide moeity. This inference was confirmed by the finding that pure samples of 65/A1 and 32/33 split proinsulins reacted in the assay very similarly to the pancreatic proinsulin standard. The implications of these results are that the immunoassays of proinsulin using antisera to C-peptide may recognise the intact proinsulin molecule very poorly, if at all, and that the 'proinsulin' measured by such assays of human serum may be largely if not entirely intermediates of proinsulin cleavage.

C-Peptide↗
PubMed1984

A high sensitivity immunochemiluminometric assay for human thyrotrophin.

A two-site immunochemiluminometric assay (ICMA) has been developed for the measurement of human thyrotrophin (TSH). The procedure involves reaction of serum samples with monoclonal antibodies to TSH which have been labelled with a chemiluminescent acridinium ester followed by reaction with solid-phase polyclonal antibodies to TSH. The total reaction time is 2 h and bound labelled antibody is quantified luminometrically. The assay has an absolute sensitivity of 0.004 mU/l and a linear dose-response range up to 60 mU/l. Circulating TSH concentrations in 84 normal subjects were in the range 0.4 to 4.0 mU/l and in 16 hyperthyroid patients were less than 0.03 mU/l. This assay should thus prove useful as a first line test of thyroid function.

Antibodies, Monoclonal↗
PubMed1984

Immunolocalization of complement component C9 on necrotic and non-necrotic muscle fibres in myositis using monoclonal antibodies: a primary role of complement in autoimmune cell damage.

Monoclonal antibodies to human complement component C9 were used to localise the membrane attack complex (MAC) of human complement on muscle fibres from patients with autoimmune myositis. A pigeon erythrocyte 'model' system was used to screen the available monoclonal antibodies for their ability to bind C9 after its insertion into the MAC. Three of the five antibodies tested were demonstrated, using radioiodine- or rhodamine-labelled second antibody, to bind. The antibody giving maximum binding in this system was used to demonstrate that MACs were present on histologically normal as well as on necrotic fibres in myositis. No binding was seen in muscle from control subjects. These results suggest a primary role of complement in the pathogenesis of muscle fibre necrosis in myositis.

Adult↗
PubMed1984

New separation method for monoclonal immunoradiometric assays and its application to assays for thyrotropin and human choriogonadotropin.

We describe a novel separation procedure for immunoradiometric assays involving monoclonal antibodies in which both radiolabeled and capture antibodies are used in solution, the capture antibody being labeled with fluorescein isothiocyanate (FITC). Separation is achieved by incubation with anti-FITC antibodies on magnetic particles. This technique enhances reaction kinetics relative to those of assays in which a solid-phase capture antibody is used, thus allowing faster reaction times and more economic use of the monoclonal antibodies. The use of anti-FITC magnetic solid phase produces an assay having a highly specific separation method, minimal nonspecific binding, and high sensitivity. The method is illustrated by application to assays for thyrotropin and human choriogonadotropin.

Antibodies, Monoclonal↗
PubMed1984

Two-site monoclonal antibody assays for human heart- and brain-type creatine kinase.

Monoclonal antibodies have been raised against human heart- and brain-type creatine kinase (CK-MB and CK-BB). We used a low-affinity monoclonal antibody to develop a simple two-step immunoaffinity purification procedure for native CK-MB. Antibodies of higher affinity were used to construct specific two-site immunoradiometric assays for CK-MB and CK-BB. In the assay for CK-MB we used an 125I-labeled B subunit-specific antibody with an immobilized anti-M subunit antibody--adding either simultaneously, for a 1-h assay in which between 5 and 1000 ng of CK-MB per milliliter could be measured with an intra-assay CV of 4% to 20%, or sequentially, for a 2-h magnetic separation assay in which between 0.5 and 1000 ng/mL could be measured with an intra-assay CV of 14 to 19%. In both versions CK-BB up to 100 ng/mL and CK-MM up to 5000 ng/mL did not interfere. In the assay for CK-BB we used an 125I-labeled, reduced, and alkylated monoclonal antibody specific for the B subunit of CK-BB, and removed bound isoenzyme with a second immobilized monoclonal antibody specific for a different epitope on the B subunit. Total incubation time for this assay was 5 h. Intra-assay CV was 7.5 to 20% between 0.1 and 1000 ng/mL. CK-MB up to 1000 ng/mL and CK-MM up to 100 000 ng/mL did not interfere. Inter-assay CVs in all three assays varied from 9 to 21%.

Antibodies, Monoclonal↗