A rapid luminescently monitored enzyme immunoassay for IgE.

A novel halophenol-enhanced luminol-peroxide luminescent detection method for horseradish peroxidase has been tested in an enzyme immunoassay for IgE. The luminescent enzyme immunoassay was reproducible (within-batch CV, 3.9-13.2%) and values obtained on serum samples showed good agreement with those obtained by colorimetric enzyme immunoassay. The major advantages of the luminescent detection method are that it is rapid, 30 s compared with 30 min for the colorimetric assay using o-phenylenediamine, and the luminescent signal is intense and stable for several minutes.

Enhanced chemiluminescent reactions catalyzed by horseradish peroxidase.

Enhancement of light emission from the horseradish peroxidase-catalyzed oxidation of diacyl hydrazides on addition of 6-hydroxybenzothiazole or phenol derivatives forms the basis of rapid, specific, and sensitive chemiluminescent assays for peroxidase. The advantages and wide applicability of the technique have been demonstrated in a range of ligand-binding assays. Careful selection of chemiluminescent reagents, enhancer, their relative proportions and reaction conditions, and more detailed knowledge of the mechanism of enhancement should enable further improvements in sensitivity and the intensity or constancy of light emission.

Enzyme-labeled antibodies in bioassays.

Antibody-enzyme conjugates are widely utilized in all spheres of specific analyte detection and measurement, and several trends are evident that will sustain, or even extend, this in the coming years. Of principal importance are the trends toward the development of simplified formats for the rapid and sensitive quantitation of a wide range of analytes without expensive or cumbersome instrumentation, and the exploitation of different types of enzyme and antibody molecules. Advances in hybridoma and recombinant genetics are enabling the practical manipulation of the theoretical repertoire of these reagents, facilitating their availability for a myriad of applications.

Enhanced luminescence enzyme immunoassay for factor VIII related antigen.

A sandwich enzyme immunoassay for plasma factor VIII related antigen has been developed which exploits a para-iodophenol enhanced chemiluminescent reaction to detect the horseradish peroxidase label. The assay entailed 15 min incubations with sample and with conjugate and had a detection limit of 0.12 mU. It showed good within batch precision (coefficient of variation = 2.95-5.8%) and results on a series of 57 specimens agreed with results obtained by immunoelectrophoresis (correlation coefficient = 0.97).

A rapid chemiluminescent enzyme-linked immunosorbent assay for cytomegalovirus immunoglobulin G antibodies using instant photographic film.

A rapid and convenient chemiluminescent enzyme-linked immunosorbent assay (ELISA) for IgG antibodies to cytomegalovirus has been developed which uses low cost equipment. Assays were carried out on transparent microtitre plates and used an anti-human IgG horseradish peroxidase conjugate. Bound peroxidase was detected chemiluminescently using a p-iodophenol-luminol-peroxide reagent. Light emission from the wells of the microtitre plate was detected on instant photographic film (ASA 20,000) held in a specially designed shutter type camera. The semi-quantitative technique was tested in a routine laboratory for a period of 7 wk and the results obtained compared well (95.3% agreement) with those obtained by a conventional colorimetric ELISA using an alkaline phosphatase label.

A review of near patient testing in primary care.

AIMS AND OBJECTIVES: The aim was to identify publications relating to near patient testing (NPT), the use of alternative delivery systems between laboratory and general practice, including electronic data interchange (EDI), and computerised diagnostic decision support (CDDS), in the primary care setting to answer the following questions. What is the availability of NPT for primary care? What evidence is available to support the clinical effectiveness of NPT? What evidence is available on the accuracy and reliability of NPT within primary care? What evidence is available on the cost-effectiveness of different NPTs? How may CDDS improve the effectiveness of NPT? What evidence is available that compares NPT and existing laboratory services? What evidence is available on the cost-effectiveness of EDI or alternative delivery systems? HOW THE RESEARCH WAS CONDUCTED: Eight databases were searched, and the bibliographies from relevant publications checked for completeness. Unpublished work and publications not included in the databases were obtained by personal contact with collaborators, and from a postal survey sent to heads of academic departments of general practice and clinical chemistry and to researchers active or interested in the field worldwide. Questionnaires were also sent to 150 commercial organisations. Publications that met agreed definitions and reported original data were included in the systematic review. Of the 1057 publications identified, 102 (92 related to NPT, eight to CDDS, and two to EDI) were passed to the reviewers for appraisal of validity. The limited amount of published research relating to any particular NPT prohibited meta-analysis. Scoring systems to assess the validity of evaluations were also difficult to apply. RESEARCH FINDINGS: A wide variety of NPT systems have been developed. In general, the quality of the methods reported in the literature was poor. The issue of patient convenience and acceptability has not been adequately addressed. No evaluations of alternative delivery systems met the review criteria. No studies have evaluated the telephone or fax machine as a means of reporting results. For EDI, the majority of papers were descriptive. EDI and alternative delivery systems are not a replacement for NPT when the provision of an immediate result might have an impact on the quality of care. EDI may have clinical and cost advantages over traditional means of communication, but this has not been evaluated. The advisory role of the laboratory can be supported by CDDS. The use of CDDS and NPT has not, however, been fully evaluated. Few economic analyses have been conducted, and most were simple cost analyses. There are insufficient data for conclusions to be drawn on the cost-effectiveness of NPT in primary care. RECOMMENDATIONS: FURTHER SYSTEMATIC REVIEWS: Subject-specific systematic reviews are required that include laboratory and secondary care studies, and consider the potential for altering current management and patient acceptability. Priority topics include: biochemistry profiles on desktop analysers; cholesterol testing; urinalysis for the diagnosis of urinary tract infection; anticoagulation control; NPTs for the identification of acute infection. (ABSTRACT TRUNCATED)

An investigation of the antioxidant activity of black tea using enhanced chemiluminescence.

Antioxidants are important species which possess the ability to protect the body from damage caused by free radical-induced oxidative stress. A variety of free radical-scavenging antioxidants exist within the body many of which are derived from dietary sources. There is currently much interest in the antioxidant role of flavonoids and other polyphenols found in tea, wine, fruit and vegetables. Enhanced chemiluminescence is a simple technique which can be used as a rapid and sensitive assay for measuring the antioxidant activity of beverages such as green and black tea. This article examines the impact of water temperature, stewing time, leaf concentration and the addition of milk upon the antioxidant activity of black tea solutions. The antioxidant activity of a range of commercially available black and green teas has also been measured.

Antioxidant status in controlled and uncontrolled hypertension and its relationship to endothelial damage.

Hypertension is associated with an increased risk of atherosclerosis. Free radical oxidative damage has been implicated in the atherogenic process. We measured levels of the antioxidants uric acid, thiols, vitamins C, A and E as well as the total antioxidant capacity in 21 normotensive controls, 22 patients whose hypertension was controlled on drugs and 30 patients with uncontrolled hypertension. Mean BPs in the groups were 125/76, 132/80 and 181/98 mmHg, respectively. When compared with controls, both hypertensive groups had significantly lower serum ascorbic acid (54 +/- 5 vs. 37 +/- 6 vs. 38 +/- 5 mumol/l, P < 0.05) and albumin-corrected thiol levels (9.91 +/- 0.18 vs. 8.69 +/- 0.20 vs. 8.92 +/- 0.19 mumol/g, P < 0.05). The levels of the other antioxidants did not differ significantly between the groups. Levels of von Willebrand factor, a marker of endothelial damage, were correlated with SBP but not with antioxidant status. We conclude that hypertensive subjects have lower levels of the antioxidants vitamin C and thiols and this may reflect greater oxidative consumption. The implications for atherogenesis and endothelial function and integrity in hypertension are discussed.

An enhanced chemiluminescence enzyme immunoassay for serum progesterone.

A competitive enhanced luminescent enzyme immunoassay for serum progesterone is described, which is based on a 11 alpha-hydroxyprogesterone 11-hemisuccinyl-horseradish peroxidase conjugate and a black polystyrene microtitre plate sensitised with anti-progesterone IgG. Bound label was determined using a mixture of 4-iodophenol, luminol and peroxide, and the light emitted from the wells of the plate quantitated using a luminescent plate reader. The assay was sensitive (detection limit 0.5 pg), precise (CV 2.7 - 9.0% in the concentration range 4.3-67.7 nM) and showed good correlation (r = 0.99) with a conventional radioimmunoassay.

Enhanced luminescent quantitation of horseradish peroxidase conjugates: application in an enzyme immunoassay for digoxin.

A p-iodophenol-enhanced luminescent end-point has been incorporated into a commercially available heterogeneous competitive enzyme immunoassay for digoxin based on a horseradish peroxidase-digoxin conjugate. The luminescent end-point could be completed in less than 1 min and significantly reduced overall assay time. Results for the assay obtained using enhanced luminescence showed good agreement with those obtained using a colorimetric end-point (correlation coefficient 0.98). Both assays gave acceptable precision within the therapeutic range. The incubation time for the luminescent immunoassay was reduced to 15 min and still gave differentiation between sub-therapeutic, therapeutic and toxic levels of digoxin.

An enhanced chemiluminescence enzyme immunoassay for serum oestradiol.

Oestradiol in serum was determined with a simple enhanced chemiluminescent enzyme immunoassay. The assay is based on oestradiol labelled with horseradish peroxidase and the IgG fraction of an oestradiol antiserum coated on a black polystyrene microtitre plate. The enzyme activity of bound label was determined using a p-hydroxycinnamic acid-enhanced chemiluminescent reaction. The assay was sensitive (1.8 fmol/well), precise (intra- and inter-assay CV 4-10% and 8-12%, respectively for sample concentration in the range 122-1330 pmol/L) and showed good agreement with conventional radioimmunoassays (r = 0.99).

Poor glycaemic control is associated with reduced serum free radical scavenging (antioxidant) activity in non-insulin-dependent diabetes mellitus.

The diabetic patient is at significantly increased risk of developing vascular disease. Its aetiology may involve oxidative damage by free radicals and protection against such damage can be offered by radical-scavenging antioxidants. We investigated whether there was a relationship between glycaemic control as assessed by measurement of glycated haemoglobin (HbA1c) and serum antioxidant status in a population of 118 diabetic outpatients with either insulin-dependent or non-insulin-dependent diabetes. Amongst patients with non-insulin-dependent diabetes mellitus there was a significant inverse correlation between levels of glycated haemoglobin and total free radical scavenging activity (r = -0.456, P < 0.0001). This association resulted primarily because of a similar correlation with uric acid (r = -0.421, P = 0.0003). There was also a weak inverse correlation with vitamin A but no significant association with vitamin C or vitamin E levels. There were no significant associations found amongst the patients with insulin-dependent diabetes. These results indicate that poor diabetic control is associated with reduced serum free radical scavenging (antioxidant) activity in non-insulin-dependent diabetes mellitus. By implication improved glycaemic control may preserve serum antioxidant status in diabetes.

Enhanced chemiluminescent assay for measuring the total antioxidant capacity of serum, saliva and crevicular fluid.

This paper reports the development of an enhanced chemiluminescent (ECL) assay for measuring the total antioxidant (AO) capacity of serum, saliva and a fluid collectable from the gum margin called gingival crevicular fluid (GCF). The theory behind the assay is explained, and the optimum conditions for the assay, and for storage of reagents and clinical samples is described. Calibration lines were linear (R > or = 0.99; P < 0.0001) and the within batch coefficient of variations for a water soluble vitamin E analogue (Trolox), serum and saliva samples were < 5%. In saliva and GCF, a characteristic AO response not seen in serum of the same patients, was identified. Total peripheral (serum) and local (saliva) AO capacities (mumol/L Trolox) were investigated in patients with (n = 18) and without (n = 16) adult periodontitis. Serum AO status did not differ between groups. Salivary total AO concentrations were lower in the peridontitis (P) group [175 (53) mumol/L] than in the non-periodontitis (NP) group [254 (110) mumol/L1: P < 0.01], as were saliva:serum AO ratio's [0.37 (0.11) versus 0.5 (0.18): P < 0.01]. Periodontitis patients may have a reduced salivary AO concentration, which could result from, or predispose to, the damaging effects of reactive oxygen species (ROS). The potential for ROS production in the oral and periodontal environment may explain the presence of a specific antioxidant in oral fluids that is not detectable in serum. The ECL assay described provides a rapid, simple and reproducible method of measuring total antioxidant defence in small volumes of biological fluids.

Comparison of the use of four desktop analysers in six urban general practices.

There is little data on the advantages and disadvantages of using desktop analysers in general practice. This prospective trial compared four of the analysers available in the United Kingdom, in six urban general practices, over a six month period. Of the 2619 tests where the time was noted, 55.8% were performed outside the hours when routine transport to a hospital laboratory was possible (after 12.00 hours). Of the 3530 tests performed the commonest were measurements of cholesterol (14.4 tests per 5000 patients per 30 days), glucose (6.0 tests) and haemoglobin (5.6 tests). Less than 5% of the tests were performed as an emergency despite the speed at which results are available. The main reasons for requesting the tests were screening or case finding (56.9%), with the remainder for monitoring chronic disease, especially diabetes and hypercholesterolaemia. There was evidence that the use of the machines in the four practices reduced requests for hospital laboratory blood tests by 24-40% of pre-study levels. However, there was a considerable increase in testing for cholesterol (three fold) and haemoglobin (eight fold) on the desktop analysers, compared with the number of laboratory tests requested before the study. The cost per test of using such machines is closely related to the level of activity and probably does not compete favourably with hospital testing unless several tests are performed each day. Quality control tests were within the specified limits on at least 98% of occasions, however these tests also identified the need for laboratory back up where a problem was found.

Reliability and feasibility of a near patient test for C-reactive protein in primary care.

BACKGROUND: The applications of new diagnostic technologies such as near patient tests are relevant to the further development and potential of primary care. Through their use, doctors in the community may increase the accuracy of their diagnoses and improve their ability to monitor disease. A reliable indicator of disease activity in various clinical conditions is C-reactive protein (CRP) and a near patient test for this is now available, although there is little information on its use outside hospitals. AIM: A study was set up to evaluate the feasibility of using a novel near patient test for CRP in primary care to validate the results against the laboratory "gold standard' for CRP (Beckman Array) and to compare results with the usual inflammation test used in general practice. METHOD: Prospective recording of CRP as a near patient test on an "intention to investigate' basis, with validation of results against the Beckman Array system for CRP and hospital laboratory erythrocyte sedimentation rate results, in six general medical practices in Birmingham. Main outcome measures were change in local laboratory usage, characteristics of patients chosen for testing, use of quality control, and comparison of readings with results from the same sample sent to an independent laboratory. RESULTS: Tests of CRP levels were rarely requested before the study was undertaken. During the 3-month study period, 181 near patient tests were carried out, 146 (81%) to establish a diagnosis and the remainder for disease monitoring. Out of the tests, 67% were performed by general practitioners, mostly during the consultation itself. Using a cut-off level of 10 mg I-1, the near patient test and the Beckman Array gave results which agreed in 84% of cases. The sensitivity and specificity of the near patient test results were 97 and 79%, respectively. The predictive value of a positive result was 59% and that of a negative result was 99%. Cohen's Kappa was 62% and the overall mean bias for results in the range of the test was 6.11 mg I-1 (SE = 3.07 mg I-1). Each test took 6 min on average to perform, including all preparations, blood letting, performing the test and averaging the time for quality control estimations. The cost per test averaged pounds 1.72, rising to pounds 4.17 including labour, capital costs, quality controls and consumables (general practitioner performing the assay at average frequency found in this study). CONCLUSIONS: Measurement of CRP is rarely used in primary care and awareness of its value could be raised. This near patient test proved feasible for use by general practitioners and practice nurses. Its reliability compared with a laboratory result was satisfactory overall, and excellent with adequate operator technique.

Rapid assays based on immobilized bioluminescent enzymes and photographic detection of light-emission.

Sensitive assays have been developed for adenosine 5'-triphosphate, the reduced form of nicotinamide adenine dinucleotide, cholyl glycine and alcohol, with immobilized and co-immobilized preparations of bacterial and firefly luciferase as reagents. With high-speed (ASA 20000) instant photographic film as detector, picomole amounts of the various analytes can be detected rapidly. The simplicity and convenience of the analytical combination of co-immobilized bioluminescent enzymes and photographic film for the detection of light make this an ideal technique for rapid screening tests.

Systematic review of near patient test evaluations in primary care.

OBJECTIVE: To identify and qualitatively synthesise the findings from all studies that have examined the performance and effect of near patient tests in the primary care setting. DESIGN: Systematic review of published and unpublished research 1986-99. MAIN OUTCOME MEASURES: Test performance characteristics, measures of effect on clinical practice or patient outcome. RESULTS: 101 relevant publications were identified. The general quality of these papers was low, and consequently only 32 papers were assessed in detail. Although these papers gave some indication of the value of near patient testing in areas such as anticoagulation monitoring and group A beta haemolytic streptococcus testing, the research raised many more questions than it answered. Almost no reports were found of unbiased assessment of the effect of near patient tests in primary care on patient outcomes, organisational outcomes, or cost. CONCLUSIONS: Available research provides little evidence to guide the expansion of use of near patient testing in primary care. Further research is needed in areas of clinical practice where near patient tests might be most beneficial.

Blood glucose measuring systems.

Glucose monitoring and lancing systems have proved useful in a range of patient testing applications in hospitals, general practice and for use by patients at home. Products are becoming easier to use and less technique dependent. It is important to be aware of their specific characteristics and to adhere to manufacturers' and local hospital laboratory guidelines.

Blood lancing systems for skin puncture.

Blood tests increasingly require only a few microlitres of blood. Lancing systems used for the collection of blood have the potential to transmit infection if used improperly. These systems vary in the amount of blood and pain produced.

Selecting blood glucose monitoring systems.

A wide range of blood glucose monitoring systems is available for use in different applications. The systems have varying characteristics which should be taken into consideration during selection. Operator technique can influence the quality of the results.