T.P. Whitehead

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 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.

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.

Investigation of a novel solid-phase chemiluminescent analytical system, incorporating photographic detection, for the measurement of glucose

A method has been developed for the rapid determination of substances by use of solid-phase reagents and a luminescence indicator reaction coupled with photographic detection of the light. The viability of the assay has been demonstrated for glucose estimations. The method uses small sample sizes (5-20 mul) and shows good sensitivity, e.g., detection of glucose down to 28 nmole.

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).

Iron Deficiency in a Northern Thai Population: The Effects of Iron Supplements Studied by Means of Plasma Ferritin Estimations

Iron deficiency is a common problem, particularly in developing countries, but traditional laboratory methods of detecting this condition are unreliable. The prevalence of iron deficiency in a Northern Thai population (pre-school, school children, adult women) has been assessed by means of plasma ferritin concentrations. The results were compared with prevalences based on blood haemoglobin concentrations. Estimations of prevalences based on plasma ferritin values were 10–24% in non-vegetarian and 49–71% in vegetarian groups, whilst those based on blood haemoglobin were 11–21% (non-vegetarian) and 24–50% (vegetarian). Dietary supplementation with iron produced dramatic rises in plasma ferritin in all of the groups studied. The effects on blood haemoglobin concentration and haematocrit were less marked. These results highlight the extent of iron deficiency in a Thai population and demonstrate the sensitivity of plasma ferritin as a test for detecting this condition and assessing the response to dietary supplementation.

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.

Metabolic Profiling Using Reversed Phase High Performance Liquid Chromatography: Analysis of Urine from Patients with Rheumatoid Arthritis

Abstract Optimal conditions are described for the reversed phase HPLC separation of UV absorbing constituents including peptides and proteins in urine. Metabolic profiles have been obtained for urinary constituents in the molecular weight range > 1000, 1000–10,000, and > 10,000 for healthy controls, patients with rheumatoid arthritis and osteoarthritis. A computer program was employed to compare individual and groups of profiles. Considerable variation was encountered in the patterns of constituents and comparison of the metabolic profiles revealed no diagnostically significant differences between the various groups of subjects.

Influence of 8-anilinonaphthalene sulphonic acid and salicylate on peroxidase activity

Abstract 8-Anilino-l-naphthalenesulphonic acid inhibits the reaction of horse radish peroxidase with 2,2′-azinobis(3-ethylbenzothiazoline-6-sulphonate)diammonium salt and various oxidants. The possible significance of this observation for competitive enzyme immunoassays with peroxidase labels has been investigated using solid-phase immunoassays based on either plastic tubes or polystyrene beads coated with thyroxine antiserum.