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

Abstract

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) .