Direct detection of Δ9-tetrahydrocannabinol in aqueous samples using a homogeneous increasing fluorescence immunoassay (HiFi).

The detection of the major active component of cannabis, Δ9-tetrahydrocannabinol (THC), becomes increasingly relevant due to its widespread abuse. For control purposes, some easy-to-use, sensitive and inexpensive test methods are needed. We have developed a fluorescence immunoassay utilising THC-fluorescein conjugate as tracer. Fluorescence spectroscopy of the conjugate revealed an unusual property: The relatively weak fluorescence of a dilute tracer solution was increased by a factor of up to 5 after binding of a THC-specific antibody. Fluorescence lifetime measurements in aqueous solutions suggested two different tracer conformations both associated with quenching of fluorescein fluorescence by the intramolecular THC moiety. After antibody binding, the tracer enters a third conformation in which fluorescence quenching of fluorescein is completely suppressed. Utilising this property, we established a homogeneous competitive immunoassay (homogeneous increasing fluorescence immunoassay) with low detection limits. The test requires only two reagents, the new tracer molecule and an anti-THC antibody. A single test takes only 8 min. The dynamic detection range for THC is 0.5 to 20 ng/mL in buffer, with a limit of detection (LOD) of 0.5 ng/mL. The test also works in diluted saliva samples (1:10 dilution with buffer) with an LOD of 2 ng/mL and a dynamic range of 2-50 ng/mL.

Direct detection of Delta9-tetrahydrocannabinol in saliva using a novel homogeneous competitive immunoassay with fluorescence quenching.

For the detection of the major active component of cannabis, Delta9-tetrahydrocannabinol (THC) in aqueous samples, a homogeneous competitive immunoassay based on fluorescence quenching induced by fluorescence resonance energy transfer (FRET) has been developed. The fluorescence of anti-THC-antibody, labeled with fluorescence dye DY-481XL, can be quenched after its binding to THC-BSA-quencher conjugate (bovine serum albumin coupled with THC and another fluorescence dye, DYQ-661, as quencher). This quenching effect is inhibited when the antibodies bind to free THC in aqueous sample, thus competing for binding sites with the THC-BSA-quencher conjugate. The extent of the inhibition corresponds to the concentration of THC in the samples. The assay principle is simple and the test duration is within 10 min. The detection limit for THC in buffer was 2 ng mL(-1). In pooled saliva samples a detection limit of 50 ng mL(-1) was achieved.

Associations between cat keeping, allergen exposure, allergic sensitization and atopic diseases: results from the Children of Lübeck Allergy and Environment Study (KLAUS).

The role of cat keeping on the promotion of allergies is discussed controversially. We investigated the associations between cat keeping, allergen exposure, allergic sensitization and atopic diseases in pre-school children. A total of 606 children (5- to 6-yr old) were studied in the course of the mandatory school entrance examination. Information on doctor diagnosed asthma and allergic rhinitis, pet keeping and confounders was obtained by questionnaire. The prevalence of atopic eczema was determined by dermatological examination, allergic sensitization was assessed by skin prick test, and the allergen exposure to cat allergen Fel d 1 was measured by a commercial wipe test. Cats were present in 16% of the households and results of the exposure categories (0-III) on cat allergen were 47.2%, 25.5%, 24.3% and 3.0% respectively. The prevalence of cat keeping increased significantly with exposure categories from 0.5% to 61.5% (p(trend) < 0.001). Children (6.3%) were sensitized to cat allergen and sensitization rates increased also significantly with exposure categories from 3.0% to 15.4% (p(trend) < 0.001). Children (9.3%) were diagnosed with atopic eczema and a positive history of asthma/rhinitis was given in 3.6% and 3.9% respectively. Sensitization to cat was associated with atopic eczema (23.3% vs. 7.4%; OR(adj.)= 3.8, CI: 1.4-10.8), asthma (12.5% vs. 3.7%; OR(adj.)= 4.9, CI: 1.1-21.2), allergic rhinitis (6.9% vs. 2.7%; OR(adj.)= 3.1, CI: 0.7-15.2) and any atopic disease (43.5% vs. 16.3%; OR(adj.)= 3.8, CI: 1.5-9.5). The data suggest a promoting effect of cat keeping for atopic diseases.