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Biomedical subjects

C H Self

Publications and source records attributed to C H Self.

At least 37 records · Page 2Linked to original sources
PubMed1993

Colorimetric assay of glycoprotein glycation free of interference from glycosylation residues.

We have developed a colorimetric assay for determining the degree of glycation of serum proteins that is unaffected by glycosylation residues. This was accomplished by reducing the proteins with sodium borohydride prior to periodate oxidation. Previous periodate-based methods, which offer several advantages over other glycation assays, cannot determine glycoprotein glycation because interference from sialic residues in the glycan chain can lead to overestimation of the amount of glycation products. Without reduction, glycation of fetuin was double that of asialofetuin glycated under identical conditions. We found that borohydride reduction before periodate oxidation increases the amount of formaldehyde released in proportion to the extent of glycation, irrespective of the degree of glycosylation. Using two glycoproteins and an unglycosylated protein, we showed how measurement of the formaldehyde increase enables the extent of glycoprotein glycation to be determined without removal of interfering sugars.

Animals↗
PubMed1993

Determination of one thousandth of an attomole (1 zeptomole) of alkaline phosphatase: application in an immunoassay of proinsulin.

Enzyme amplification has proved to be a highly sensitive quantification technique for immunoassays. We have shown that by using a fluorescent end-point, even more sensitive enzyme amplification assays can be generated than hitherto reported. We describe some general properties of this system and demonstrate its application in an assay for human proinsulin in plasma. The detection system can be used to measure less than one thousandth of an attomole (1 zeptomole) of alkaline phosphatase, equivalent to about 350 molecules of alkaline phosphatase per well of a microtiter plate. We have used this system to construct a proinsulin assay with a sensitivity of 0.017 pmol/L.

Alkaline Phosphatase↗
PubMed1992

Electrochemical sensors for direct reagentless measurement of superoxide production by human neutrophils.

Electrochemical sensors based on immobilised cytochrome c or superoxide dismutase for the measurement of superoxide radical production by stimulated neutrophils are described. Cytochrome c was immobilised covalently at a surface-modified gold electrode and by passive adsorption to novel platinised activated carbon electrodes (PACE). The reoxidation of cytochrome c at the electrode surface upon reduction by superoxide was monitored using both xanthine/xanthine oxidase and stimulated neutrophils as sources of the free radical. In addition, bovine Cu/Zn superoxide dismutase was immobilised to PACE by passive adsorption and superoxide, generated by xanthine/xanthine oxidase, detected by oxidation of hydrogen peroxide produced by the enzymic dismutation of the superoxide radical. A biopsy needle probe electrode based on cytochrome c immobilised at PACE and suitable for continuous monitoring of free radical production was constructed and characterised.

Biopsy↗
PubMed1992

Amplified enzyme-linked immunoassay of human proinsulin in serum (detection limit: 0.1 pmol/L).

We describe an amplified enzyme-linked immunoassay of human proinsulin in serum that detects intact proinsulin and both the 32/33 and 65/66 split forms. The method uses the IgG fraction of a polyclonal antibody raised in a guinea pig against intact proinsulin, which we used to coat plastic microtiter plates. A sandwich was formed with proinsulin by using a monoclonal antibody against C-peptide labeled with alkaline phosphatase. We quantified the reaction by using the enzyme amplification procedure, which detected as little intact proinsulin as 0.1 pmol/L. We found no cross-reactivity with C-peptide in the assay, and decreased recovery attributable to the presence of insulin could be demonstrated only with a 30-fold excess of this hormone over proinsulin.

Alkaline Phosphatase↗
PubMed1990

Production and utilization of monoclonal antibodies to human/rat corticotrophin-releasing factor-41.

Murine monoclonal antibodies against human/rat corticotrophin-releasing factor-41 (CRF-41) were produced and characterized for use in the immunological and biological characterization of CRF-41. Spleen cells from BALB/c mice immunized with CRF-41 conjugated to bovine gamma-globulin were fused with a BALB/c-derived non-secretor X-63 myeloma line. Hybridomas were selected for CRF antibody production by enzyme-linked immunosorbent assay, and positive hybridomas cloned twice. Three monoclonal antibodies were obtained (KCHMB001, KCHMB002 and KCHMB003) and characterized as IgG1, IgG1 and IgG2a isotypes respectively, with affinity constants for rat CRF-41 of 30, 53 and 34 nmol/l respectively. All three monoclonal antibodies recognize an epitope contained between residues 34 and 41 of the human/rat sequence. The antibodies were able to neutralize the ACTH-releasing activity of rat CRF-41, applied to rat pituitary fragments in vitro, in a dose-dependent manner. Isoelectric focusing showed that KCHMB003 detected bands of synthetic rat CRF-41 and rat [Met(O)21,38]-CRF-41 at pH 7.1 and 6.8 respectively. Use of KCHMB003 in a two-site enzyme-amplified immunoassay showed that this antibody recognizes both synthetic rat CRF-41 and immunoreactive CRF-41 in rat hypothalamic tissue extracts.

Amino Acid Sequence↗
PubMed1990

Development and performance of a highly sensitive and specific two-site immunometric assay of calcitonin gene-related peptide.

Calcitonin gene-related peptide (CGRP), a potent endogenous circulating vasodilator, is produced by the alternative splicing of the calcitonin/CGRP gene and is expressed mainly in neural and cardiovascular tissues. We recently reported a highly sensitive radioimmunoassay of CGRP, based on an antiserum recognizing the C-terminus of the molecule. We have also found that circulating immunoreactive CGRP is heterogeneous; thus we are unable to measure selectively the intact molecule with our one-site competitive approach. We therefore attempted to construct a two-site immunometric assay involving two antibodies, one that detects the C-terminus and another that recognizes the midregion of the molecule. To enhance assay sensitivity, we applied a colorimetric amplification system to this assay. This rapid, robust, and reproducible assay provides more nearly accurate estimates of circulating CGRP and offers a sensitive and more specific alternative to the radioimmunoassay, with advantages in speed, simplicity, and convenience.

Amino Acid Sequence↗
PubMed1989

The development of a two-site enzyme immunometric assay (EIA) for calcitonin and its application in the measurement of the hormone in normal subjects, MTC-patients and post-menopausal women.

We have developed a sensitive, two-site enzyme immunometric assay (EIA) using high affinity monoclonal antibodies for the measurement of immunoreactive human calcitonin (i-hCT) and compared the performance of the assay with that of the conventional radioimmunoassay (RIA). The sensitivity of an overnight EIA (2 pmol/l) was comparable with the long incubation (7 days) RIA. Both, the EIA and the RIA have been applied for the measurement of i-hCT in three groups: normal volunteers (n = 53), post-menopausal women (n = 25) and patients with MTC (n = 20). The results showed that in all three groups, the level of i-hCT was somewhat lower than that measured by RIA due to its greater specificity. The EIA can provide an attractive alternative to the conventional RIA for both routine diagnostic work and physiological studies.

Antibodies, Monoclonal↗
PubMed1988

A highly specific and sensitive enzyme-immunometric assay for calcitonin gene-related peptide based on enzyme amplification.

Calcitonin gene-related peptide (CGRP) is an important member of the peptide family encoded by the calcitonin gene. It has been found to be a potent vasodilator in man and a major circulating gene product (Girgis et al., 1985). The present study reports the development of a sensitive and rapid two-site immunoassay for CGRP based on enzyme amplification (Self, 1985). The assay has been easy to construct, taking advantage of available antisera raised for other purposes. Nevertheless it has been found to be clearly superior to our previous radioimmunoassay in terms of sensitivity, specificity, speed and convenience.

Animals↗
PubMed1988

Calcitonin determination by a fast and highly sensitive enzyme amplified immunoassay.

A colorimetric enzyme amplification system was used to develop an immunoassay for human calcitonin (hCT) with a sensitivity of 6 pmol/l, and intra- and inter-assay CVs of 12% and 11.8% respectively for the low pool, and 10% and 11.2% for the high pool. The mean recovery of added synthetic hCT (58.5 pmol) from the plasma of 10 patients was 110% (64.4 pmol). The correlation coefficient between radioimmunoassay (RIA) and amplified enzymo-immunoassay was found to be 0.96 (p 0.001). The assay was successfully applied to the measurement of elevated calcitonin levels in plasma from patients with medullary carcinoma of the thyroid (MCT). AEIA offered a reliable and sensitive alternative to RIA for calcitonin determination with the added advantage of convenience as the label employed was much more stable.

Calcitonin↗
PubMed1988

A sensitive and specific two-site enzyme-immunoassay for human calcitonin using monoclonal antibodies.

A highly sensitive, specific and rapid two-site enzyme-immunometric assay (EIA) for the measurement of immunoreactive (ir) human calcitonin (hCT) in human plasma was developed using high-affinity monoclonal antibodies. The assay was validated in terms of sensitivity, specificity and reproducibility and its performance compared with that of a radioimmunoassay (RIA) employing a polyclonal antiserum. The sensitivity of the overnight EIA (2 pmol/l) was comparable with the long-incubation (7 days) RIA. The overnight RIA had a sensitivity of 10 pmol/l. The inter- and intra-assay variations of the EIA were less than 12%. Some related and non-related peptides were compared with synthetic hCT for cross-reactivity in the assay and were found to be negative. The mean recovery of added synthetic hCT from plasma of normal volunteers was 96%. Both RIA and EIA have been applied to the measurement of ir-hCT in normal volunteers and in patients with medullary carcinoma of the thyroid. In both groups, the level of ir-hCT measured by EIA was found to be lower than that measured by RIA, presumably due to the ability of the more specific EIA to detect only the 'mature' form of the hormone. EIA offers an attractive alternative to the more cumbersome and lengthy RIA in current usage, with the added advantage of employing a non-isotopic label.

Alkaline Phosphatase↗
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↗
PubMed1985

Enzyme amplification can enhance both the speed and the sensitivity of immunoassays.

Enzyme immunoassays (EIA) are now used for the quantitation of a wide range of clinically important analytes and have, in many cases, replaced radioimmunoassays, though without improving on the sensitivity of the latter technique. We describe a general enzyme-amplification method which can be used to increase both the speed and the sensitivity of EIA. In this method, the enzyme label is used to catalyse the dephosphorylation of nicotinamide adenine dinucleotide phosphate (NADP+); the NAD+ so formed then catalytically activates an NAD+-specific redox cycle, yielding an intensely coloured formazan dye. The application of this new enzyme detection method has made possible an assay for human thyroid-stimulating hormone (TSH) with a sensitivity of 1 X 10(-5) IU/1 and a progesterone assay which takes only 15 min to complete.

Animals↗
PubMed1985

Calcitonin gene-related peptide: potent vasodilator and major product of calcitonin gene.

In addition to calcitonin and katacalcin, the human calcitonin gene encodes a novel peptide--calcitonin gene-related peptide (CGRP)--a potent vasodilator. A sensitive and specific radioimmunoassay was developed to study plasma levels of CGRP in normal subjects. CGRP circulates at five times the concentration of calcitonin, suggesting that it may be an important physiological regulator of vascular tone and blood flow.

Adult↗
PubMed1985

A fast highly sensitive colorimetric enzyme immunoassay system demonstrating benefits of enzyme amplification in clinical chemistry.

A method for greatly enhancing the sensitivity of assays employing enzyme labels is described which offers advantages in assays for a wide range of analytes. The principle of the new approach is that the enzyme label gives rise to a catalytic activator for a specific secondary detection system, the activity of which is measured and related back to the amount of label present and thus of the analyte it is being used to determine (C.H. Self, Eur. Pat. Appl. 80303478.4, 15.4.81 exclusively licenced to IQ (Bio) Ltd.). The general principle of enzyme amplification is illustrated by using alkaline phosphatase as the labelling enzyme and nicotinamide adenine dinucleotide phosphate (NADP) as its substrate. The nicotinamide adenine dinucleotide (NAD) formed catalytically activates a strictly NAD specific redox cycle which produces a coloured formazan as the end product. The measured absorbance is at least two orders of magnitude greater than that achieved by conventional methods. The application of this method to immunoassay is demonstrated by a sensitive, rapid and precise assay for human prostatic acid phosphatase (PAP). Some of the many other applications of this methodology are discussed.

Acid Phosphatase↗
PubMed1985

Enzyme amplification--a general method applied to provide an immunoassisted assay for placental alkaline phosphatase.

A method is described in which enzyme amplification is used for the quantitative determination of placental alkaline phosphatase. In this particular application, placental alkaline phosphatase is captured on a solid surface by means of a monoclonal antibody. The enzyme is then determined by dephosphorylation of NADP to NAD which activates a strictly NAD-dependent redox cycle leading to the formation of a coloured product.

Alkaline Phosphatase↗
PubMed1976

A general method for the rapid separation and specific detection of antigenic meterial by immunoelectrofiltration using multispecific antisera.

A general technique is described for the separation and detection of specific antigens from complex antigen mixtures, by their electrophoresis through antibody-containing gels. The method does not require purified reagent antigen or fuctionally monospecific antisera and should have a wide applicability in the detection, quantification and characterization of various antigens.

Animals↗