Thomas Patterson Whitehead

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.

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.

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.

Sensitive Sandwich Enzyme Immunoassay for Serum Ferritin on Microtitre Plates

A solid-phase sandwich enzyme immunoassay for serum ferritin is described. The assay procedure was simple, showed good precision, and was suitable for the routine determination of serum ferritin concentration in a clinical laboratory. The assay compared well with a conventional radioimmunoassay for ferritin. The assay was extremely sensitive and had a detection limit of 2·2 pg.

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.

External Quality Assessment of Serum Enzyme Activity Assays and the Effect of Calibration on Interlaboratory Concordance

The design and results of a UK external quality assessment scheme for six enzymes are described, from 21 surveys over a period of 7 years. Improvements in interlaboratory agreement and the adoption of reliable methods are documented. The potential of enzyme calibration materials in further improving numerical concordance between laboratories using different assay conditions, including temperature, is demonstrated and discussed.

Matrix effects in clinical analysis: commutability of control materials between the Ektachem, Beckman and SMA 1260 glucose and urea methods

The effects of the protein matrices of selected human and animal based control materials on the Ektachem, Beckman and SMA 12/60 glucose and urea methods have been investigated by means of commutability studies. The commutability of control materials between the Ektachem and SMA 12/60 was greater than that between the Ektachem and Beckman methods and was similar to that reported for conventional methods. No trends were apparent amongst control sera from different species nor with different total protein levels. Some variation in commutability was found with different batches of Ektachem glucose and urea slides. In addition the effect of lyophilisation of fresh human serum on its analytical behaviour with different experimental batches of Ektachem glucose/urea slides was studied and found to be negligible.

Characterisation of the protein matrix of quality control sera by a high resolution two-dimensional electrophoresis technique

A high resolution two-dimensional electrophoresis technique (Iso-Dalt) has been employed to characterise the protein components of freshly drawn human serum and various human- and animal-based quality control sera. This technique allows a direct comparison to be made between the protein components of different materials. Similarities have been demonstrated between the protein components (protein matrix) of freshly drawn human serum and human-, equine- and bovine-based control sera, though some differences existed between sera from these three sources, mainly in the acidic high molecular weight quadrant and the lipoprotein and haptoglobin regions. The Iso-Dalt technique also revealed differences in the protein matrices of the various human-based quality control sera tested. Differences attributable to manufacturing technique were also discernible by inspection of the two-dimensional maps of the protein matrices. Although characterisation and comparison of protein components of the matrix of serum is difficult, the Iso-Dalt technique has proved a valuable tool in this characterisation and the subsequent assessment of the similarity of quality control sera to human serum. This type of information is valuable when considering the suitability of human- or animal-based sera for use in internal and external quality control procedures.

Evaluation of the Kodak Ektachem glucose and urea methods

An evaluation of the Kodak Ektachem glucose and urea methods is described. Aspects evaluated included precision, linearity, accuracy, correlation with routine methods, interferences (haemoglobin, bilirubin, protein, dextran, lipaemia, ethylene-diamine tetraacetic acid, fluoride/oxalate, and heparin), and carryover. Stability and batch-to-batch variation in glucose and urea reagents were also investigated. The performance of the Ektachem glucose and urea methods was shown to be as satisfactory as conventional analytical methods. The requirement to reconstitute control serum samples in a bicarbonate diluent in order to obtain accurate results presents problems for the analysis of lyophilised specimens circulated by external quality assessment schemes. The complex calibration model and the significance of variation in the calibration parameters need further explanation. The Ektachem methods are designed specifically for use with human serum. However, the methods performed satisfactorily with cerebrospinal fluid, pleural effusions, and animal serum but not with urine.