Gary Harold Gregory Henry Thorpe

Enhanced chemiluminescent assay for antioxidant capacity in biological fluids

Abstract Enhanced chemiluminescence techniques have been used for several years in the field of diagnostic immunoassays. We report the use of an enhanced chemiluminescent reaction involving horseradish peroxidase and luminol for the detection of antioxidants and measurements of antioxidants capacity (T.P. Whitehead and G.H.G. Thorpe, UK Patent Application, Pub. no. GB2245062, 199 ). The addition of solutions of known antioxidants such as ascorbate, urate or vitamin E (or biological fluids containing them) to a glowing chemiluminescent reaction temporarily interrupts light output. Light emission is restored after an interval that is linearly related to the molar quantity of antioxidant added. In this way we have been able to quantify total antioxidant capacity in a variety of biological fluids. A normal range for antioxidant capacity in human serum has been identified and a range of clinical conditions, in which free radical activity is implicated, have been studied.

[29] Enhanced chemiluminescent reactions catalyzed by horseradish peroxidase

Abstract 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.

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≥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 (μmol/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 periodontitis (P) group [175 (53)μmol/L] than in the nonperiodontitis (NP) group [254 (110)μmol/L1: P<0·01], as were saliva:serum AO ratios [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.

Enhancement of the horseradish peroxidase-catalyzed chemiluminescent oxidation of cyclic diacyl hydrazides by 6-hydroxybenzothiazoles

6-Hydroxybenzothiazole, 2-cyano-6-hydroxybenzothiazole, and 2-(6-hydroxy-2-benzothiazolyl)thiazole-4-carboxylic acid (dehydroluciferin) dramatically enhance light emission from the horseradish peroxidase conjugate catalyzed oxidation of luminol, isoluminol, N-(6-aminobutyl)-N-ethyl isoluminol, and 7-dimethylaminonaphthalene-1,2-dicarboxylic acid hydrazide by either peroxide or perborate. Light emission is enhanced by up to 1000-fold, which is an improvement over the enhancement previously observed using firefly luciferin (4,5-dihydro-2-(6-hydroxy-2-benzothiazolyl)thiazole-4-carboxylic acid). Enhancement is influenced by enhancer concentration and pH. Spectral scans of light emitted in enhanced and unenhanced reactions are similar, suggesting that aminophthalate products, and not the enhancers, are the emitters.

[33] Photographic detection of chemiluminescent and bioluminescent reactions

Publisher Summary In chemiluminescent and bioluminescent assays, light emission is usually measured with a photomultiplier tube (PMT) or a silicon photodiode. A less expensive and simpler alternative is photographic film. The combination of photography and either chemiluminescence or bioluminescence provides a simple and convenient system for assaying a wide range of substances. The kinetics of light emission—that is, a flash versus a prolonged emission—is important factors in the design of instrumentation for photographic assays. In reactions that produce a prolonged emission, the use of film poses no major problems as the reactions can be initiated prior to exposure to the film. In their simplest form, the results of photographic assays are assessed by visual comparison of exposures (black through grey to white) produced by samples and standards. Further quantitation can be achieved if the reaction vessel is held in a cylindrical cavity above the film. The area of film exposed beneath the cavity is dependent upon the intensity of light emission, and intensity is proportional to the diameter of the exposed zone. Photographic results can also be quantitated using a densitometer. This method is employed to determine allergen-specific IgE levels from photographic prints obtained in MAST chemiluminescent enzyme immunoassays.

Enhanced luminescence determination of horseradish peroxidase conjugates: Application of Benzothiazole Derivatives as Enhancers in Luminescence Assays on Microtitre Plates

Abstract The benzothiazole derivatives, 2-cyano-6-hydroxybenzothiazole, 6-hydroxybenzothiazole and dehydroluciferin, enhance light emission from the horseradish peroxidase-catalysed oxidation of cyclic diacylhydrazides such as luminol. The relatively intense and prolonged light emission from reactions enhanced by benzothioazole derivatives is easily detected and is utilised in a rapid assay for specific antibody against cytomegalovirus done on black polystyrene microtitre plates. Rapid measurements are possible when a prototype manually-operated microtitre plate reader is used. Light emission from individual wells was quantified by an end-window photomultiplier tube positioned either just above the microtitre plate surface, or some distance away, the light being collected through a fibre optic light guide. The assay was also done on transparent (poly(vinyl chloride) microtitre plates with simultaneous measurement of light emission from several wells; this was achieved with simple instrumentation and a 20 000-ASA Polaroid instant photographic film.

Enhanced luminescent enzyme immunoassays for rubella antibody, immunoglobulin e and digoxin

A novel firefly luciferin- enhanced luminescent procedure for the quantitation of horseradish peroxidase labels has been directly incorporated into established enzyme immunoassays. The procedure is rapid and sensitive and uses readily available reagents. Light emission from the enhanced reaction is high and relatively constant and thus easily measured. The luminescence procedure has been successfully incorporated into immunometric assays for rubella antibody and human IgE and into a competitive immunoassay for digoxin.

Enhanced chemiluminescence enzyme immunoassay

Exploitation of light-emitting reactions (chemiluminescent or bioluminescent) in immunoassay falls into two main areas. Components of a chemiluminescent (e.g., luminol) or bioluminescent reaction (e.g., a luciferase, aequorin) can be used as a label, or a chemiluminescent or bioluminescent reaction can be used to monitor an enzyme label or its products. Table 1 lists some representative examples of immunoassays which have end-points based on light emission. Recently, considerable attention has focussed on the chemiluminescent assay of horseradish peroxidase labels following the discovery of a new enhanced chemiluminescent end-point for peroxidase labels (ref. 1).

Camera luminometer for use with luminescent assays

An instrument, a camera luminometer, has been developed that detects light emission from luminescent reactions using high-speed instant photographic film (ASA 20000). The luminometer simultaneously monitors light emission from 63 reaction vessels in the form of a 7 × 9 array. It also incorporates a multiple pipette, which enables luminescent reactions to be simultaneously initiated directly above the instant film. Photographic results are interpreted visually and semi-quantitative assessment is possible. This may be improved by interposing a stepped neutral density filter between each reaction vessel and the film. The application of this instrument is illustrated by an enhanced luminescent enzyme immunoassay for serum ferritin and chemiluminescent assays for Co(II), Cr(III), peroxide and luminol. The film has a limited exposure latitude and so the assays operate as threshold tests with the following detection limits: ferritin, 1 ng; Co(II), 5 pmol; Cr(III), 500 fmol; peroxide, 500 pmol; and luminol, 500 fmol.

Immobilized enzymes in analysis

Abstract The sensitivity and specificity of enzyme reactions has led to the widespread use of enzymes as reagents in analysis. This article surveys the applications of immobilized enzymes in clinical analysis with particular emphasis on bioluminescent and chemiluminescent assays and test devices intended for use outside the main clinical laboratory.