Specific Antibodies to Theophylline for Use in a Homogeneous: Enzyme Immunoassay

Singh, Prithipal; Hu, Mae W.; Gushaw, James B.; Ullman, Edwin F.

doi:10.1080/01971528008058474

Cited by 4 publications

(2 citation statements)

References 11 publications

(2 reference statements)

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“…Subsequent experiments revealed that the optimum pH for coupling NHS-activated 3carboxymethylriboflavin to G6PDH is slightly acidic pH 6.5 rather than the pH 9.0-9.5 frequently reported in the literature for ligand-enzyme conjugation reactions involving NHS methods (31,32). bis-Tris propane was utilized to prepare several different conjugation reaction buffers since it provides a wide pH range (pH 6.3 to 9.5) and has no primary amino group.…”

Section: Resultsmentioning

confidence: 99%

Solid phase enzyme-linked competitive binding assay for riboflavin

Sig

Meyerhoff

1988

Analytical Biochemistry

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A new solid-phase enzyme-linked assay for riboflavin (vitamin B2) is described. The assay is based on the competition between analyte vitamin molecules and a glucose-6-phosphate dehydrogenase-3-carboxymethylriboflavin conjugate for a limited number of riboflavin-binding protein sites immobilized on Sepharose particles. Significant improvements in conjugate catalytic activity and thus detectability are achieved by optimizing the reaction conditions used to covalently link 3-carboxymethylriboflavin to the enzyme. Optimization experiments include studying the effects of reaction pH and organic solvent composition. Final assay detection limits and the sensitivity of the dose-response curves are dependent on the ratio of conjugate to binding protein sites utilized in an equilibrium assay protocol. Selectivity of the method correlates well with that predicted based on the known association constants of riboflavin-binding protein with flavin analogs. The assay is shown to offer adequate detection limits and selectivity for direct measurement of riboflavin in urine, infant formula, and vitamin capsules.

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Section: Resultsmentioning

confidence: 99%

Solid phase enzyme-linked competitive binding assay for riboflavin

Sig

Meyerhoff

1988

Analytical Biochemistry

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“…This provided a quick, simple immunogen with the enzyme linked via the 3-position. The coupling of a carrier protein in this position has been shown by Singh et al 9 to produce specific antibodies for theophylline. They hypothesised that the methyl group at the 3-position was of limited importance to precise molecular differentiation between the possible cross-reacting compounds and theophylline, and that by using a 3-carboxyalkyl linkage the electronic and steric characteristics at this site would be retained.…”

Section: Discussionmentioning

confidence: 99%

Direct Determination of Theophylline in Serum by Fluoroimmunoassay Using Highly Specific Antibodies

et al. 1985

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SUMMARY. Described is the development of a fluoroimmunoassay for theophylline using a fluorescein labelled derivative of theophylline as tracer and antibodies coupled to magnetisable solid-phase particles. Three approaches are described for the preparation of antibodies for theophylline, of which one produced highly specific, high titre antibodies. The fluoroimmunoassay using these antibodies required a 10 ul, sample, reached equilibrium within 5 min, and the results . correlated closely with those of an established enzymoimmunoassay method. Potentially interfering endogenous fluorophores from the serum sample were reliably removed at the separation step of the bound and free fractions. There was no significant cross-reactivity with all other structurally related compounds.Theophylline (1,3-dimethylxanthine) is considered the drug of choice in the prevention and treatment of asthmatic symptoms in children and adults! and is also used for the treatment of apnea and bradycardia in infants.f It has a narrow therapeutic range and a wide variation in biological half-life, so the monitoring of serum theophylline levels is considered necessary for safe and effective use of the drug.3--{j Various methods are available, as reviewed recently.P: 7 These include: spectrophotometry; gas chromatography; high pressure liquid chromatography; and radio and enzymoimmunoassay. The non-immunological methods all have disadvantages; they may require large sample volumes, and perhaps use technically complex and time consuming sample preparations. Immunoassays are simple, permit direct analysis with small sample volumes and allow large throughput. A theophylline immunoassay, however, requires highly-specific antibodies. This is due to the potential cross-reactivity of the major metabolites of the drug and the naturally occurring xanthines, caffeine (1,3,7-trimethylxanthine) and theobromine (3,7-dimethylxanthine) .

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