Direct measurement of low density lipoprotein in whole blood.
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Biomedical subjects
Publications and source records attributed to B F Rocks.
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A competitive silver-enhanced gold-labelled immunoassay has been developed for the direct measurement of low density lipoprotein (LDL) in whole blood. Immobilized LDL and sample LDL compete for added antibody. Quantitation of the bound antibody/antigen complex is achieved by the addition of gold-labelled anti-immunoglobulin G followed by enhancement of absorbance by addition of silver ions. Whole-blood samples from fasting patients were assayed directly for LDL by the procedure and the corresponding plasma samples were assayed for total cholesterol, high density lipoprotein and triglycerides followed by the indirect calculation of LDL cholesterol. The correlation between the two methods was good (r = 0.82) and the SEGLISA exhibited good precision.
A silver enhanced, gold labelled, immunosorbent assay (SEGLISA) for the detection of IgG antibodies to the rubella virus in human serum was developed. Pre-coated microtitre wells are used as the immobilised base of rubella antigens on to which any rubella antibodies from patient samples will bind. This antigen/antibody complex is then visualised firstly by gold labelled anti-immunoglobulin G, which binds to any human IgG that may be present, and then by silver amplification, resulting in a black permanent deposit on the microtitre well surface. Patient samples (n = 121) were screened using a commercially available enzyme linked immunosorbent assay (ELISA) and an equivalent SEGLISA. Results were comparable but the SEGLISA does not have the disadvantages associated with enzyme labels. The silver deposit may also be read visually or the dried plate may be stored for future reference.
A silver-enhanced gold-labelled immunosorbent assay (SEGLISA) for the detection of antibodies to the immunodeficiency virus (HIV) in whole-blood samples is described. This new non-isotopic, non-enzymic immunoassay incorporates use of solid phase viral antigens which bind any HIV antibodies present in the test sample. The antigen/antibody complex is then detected by gold-labelled anti human immunoglobulin G (IgG) followed by silver amplification. We found that whole blood samples give false positives when using a horseradish peroxidase label, whereas the SEGLISA correctly identified 50 HIV antibody positive samples and 50 HIV antibody negative samples when using whole blood. The use of whole blood collected on filter paper is also described. The SEGLISA has good precision (CV = 7.5%) and sensitivity.
Attempts to increase the sensitivity of fluorescein-based fluorescence immunoassays by using multiple labelling have generally been unsuccessful because of concentration quenching. We have labelled antibodies to human immunoglobulin G with multiple fluorescein fluorophores attached by means of a disulphide linkage: this linkage can be rapidly and easily broken by treatment with dithiothreitol, allowing fluorescein to be released from the antibody and measured in free solution. Application of this technique to a fluorescence labelled immunosorbent assay for antibodies to the human immunodeficiency virus gave an approximately 20-fold increase in signal compared with an equivalent assay using fluorescein isothiocyanate.
We describe a new immunoassay for the detection of antibodies to the human immunodeficiency virus. The method is based on a silver enhanced gold-labelled immunosorbent assay (SEGLISA). Test sera are incubated in microtitre wells on which antigens have been coated. If present in the test sera, antibodies to the human immunodeficiency virus bind to the solid-phase antigens. Bound antibodies are quantitated with anti-human immunoglobulin labelled with gold. Positive specimens produce a faint pink deposit which is better visualised by silver enhancement which gives an intense black colour. The intensity of the colour is proportional to the bound antibody concentration. All the reagents are stable and the silver enhancement takes place under ambient light conditions. The assay has many of the advantages of micro enzyme-linked immunosorbent assays but does not suffer from the drawbacks associated with the use of an enzyme label. It requires fewer manipulations and is quicker to carry out than an equivalent enzyme-linked test. As the silver layer is permanent dried wells may be stored for future reading or checking.
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Human immunoglobulin G, human serum albumin and testosterone were labelled with the 4-aminosalicylic acid derivative of diethylenetriaminepentaacetic acid complexed with terbium ions. An exceptionally large amount of label, of the order of a few hundred moles of complex per mole of analyte, could be conjugated to the compounds tested by the use of poly-L-lysine. Self-quenching appears to be minimal, even with this high local concentration of fluorophores. The tracers were stable at 4 degrees C, and gave competitive calibration graphs at physiological concentrations.
The development and assessment of a fluorescence-labelled immunosorbent assay for the detection of antibodies to the human immunodeficiency virus is described. Test serum is incubated in microtitre wells on which antigens have been coated. If present in the test serum, antibodies to the human immunodeficiency virus bind to the solid-phase antigens. In turn the antibodies are quantified with anti-human immunoglobulin labelled with fluorescein. Positive samples produce an intense fluorescence which is measured in a spectrofluorimeter. When used to test a panel consisting of normal serum and antibody-positive serum from infected patients the assay proved to be 100% specific and to have a sensitivity of 100%. The assay has many of the advantages of micro enzyme-linked immunosorbent assays, but does not suffer from the drawbacks associated with the use of an enzyme label. It requires fewer manipulations and is quicker to carry out than an equivalent enzyme-linked test.
Cytidine deaminase is an enzyme of nucleic acid metabolism, the measurement of which has been proposed as a useful test for the early detection of pre-eclamptic toxaemia in pregnancy. The enzyme converts the nucleoside cytidine to uridine, with the release of ammonia, and it is the measurement of this latter compound that forms the basis of the conventional methods for the assay of cytidine deaminase. The low activity of the enzyme requires long incubation times, which in turn increase the possibility of contamination by exogenous ammonia. We have developed a new method for determining cytidine deaminase activity, utilising high performance liquid chromatography to measure the production of uridine. This method uses much shorter incubation times and is unaffected by ammonia contamination. This paper describes the development of the method and its comparison with the established assay. The relative merits of each are discussed. Finally, the adaptation of incubation and chromatographic conditions, in order to measure other enzymes of nucleic acid metabolism which are of clinical interest, is briefly mentioned.
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The range of plasma concentrations in 177 and erythrocyte magnesium concentrations in 104 elderly patients was found to be similar to that for healthy adults. No significant difference was observed between the sexes or between patients taking diuretics and those not receiving diuretic therapy. The latter finding is contrary to some previously reported studies.
The osmolality of plasma and heparinised whole blood samples collected from hospital patients was estimated using measurement of the depression of freezing point. There was no clinically significant difference between osmolality measurement made on either whole blood, or plasma taken from the same patient. Neither cell volume nor haemolysis was found to affect the measurement. The reproducibility of whole blood measurements was similar to that for determinations carried out on plasma. Measurement of osmolality on whole blood is quicker and cheaper and needs a smaller specimen than if serum or plasma is used.
Plasma and erythrocyte magnesium were measured in 105 patients with premenstrual syndrome (PMS) using a simple atomic absorption spectroscopy method. The erythrocyte magnesium concentration for the patients with PMS was significantly lower than that of a normal population. The plasma magnesium did not show this difference. The significance of this apparent cellular deficiency of magnesium is discussed.
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Flow-injection analysis is a precise, elegant, and economical technique, but its most troublesome feature is the mode of injection of the sample slug. We describe an alternative approach in which the sample is aspirated by the sample probe. In the simplest version the probe normally rests in reagent; when sampling is to take place, the pump is stopped and the probe is transferred to the sample container. The pump makes a predetermined angular movement, the probe is returned to reagent, and the pump is restarted. In more advanced versions the same approach is combined with the merging zone technique. The system is economical, precise, and capable of full automation in a multichannel discretionary analyzer.