An Anticaffeine Antibody-Oligonucleotide Conjugate for DNA-Directed Immobilization in Environmental Immunoarrays.

The development of fast and cheap high-throughput platforms for the detection of environmental contaminants is of particular importance to understand the human-related impact on the environment. The application of DNA-directed immobilization (DDI) of IgG molecules is currently limited to the clinical diagnostics scenario, possibly because of the high costs of production of such addressable platforms. We here describe the efficient and specific hybridization of an antibody-oligonucleotide conjugate to a short 12-mer capture probe. The specific antibody used is a monoclonal antibody against caffeine, a stimulant and important anthropogenic marker. With this work, we hope to contribute to broadening the application potential of DDI to environmental markers in order to develop cheaper and more stable high-throughput screening platforms for standard routine analysis of pollutants in a variety of complex matrices.

Caffeine ingestion and antigen-stimulated human lymphocyte activation after prolonged cycling.

This study investigated the effect of caffeine ingestion on antigen-stimulated T- (CD4(+) and CD8(+) ) and natural killer (NK)- (CD3(-) CD56(+) ) cell activation after prolonged, strenuous cycling. In a randomized cross-over design, nine male endurance cyclists (age: 22 ± 3 years, VO(2peak) : 62 ± 4 mL/kg/min, mean ± SD) cycled for 90 min at 70% VO(2peak) 60 min after ingesting 6 mg/kg body mass of caffeine (CAF) or placebo (PLA). Venous blood samples were obtained before supplementation, pre-exercise, immediately post-exercise and 1 h post-exercise. Whole blood was stimulated with Pediacel (five in one) vaccine. At 1 h post-exercise the number of antigen-stimulated CD4(+) cells expressing CD69 decreased on CAF compared with PLA [15 (17) × 10(6) vs 23 (22) × 10(6) cells/L, P<0.05]. In addition, the geometric mean fluorescence intensity (GMFI) of CD69 expression on antigen-stimulated CD8(+) cells decreased on CAF compared with PLA 1 h post-exercise [78 (10)% vs 102 (24)%, P<0.05]. At the same time-point GMFI of CD69 expression on antigen-stimulated CD3(-) CD56(+) cells was increased on CAF compared with PLA [103 (9)% vs 87 (8)%, P<0.05]. These findings suggest that caffeine reduces antigen-stimulated CD69 expression on T cells while at the same time increases NK-cell activation 1 h after intensive cycling.

A highly sensitive caffeine immunoassay based on a monoclonal antibody.

A new immunoassay has been developed based on a commercially available anti-caffeine monoclonal antibody and a de novo synthesized tracer, using horseradish peroxidase and UV-visible detection. Caffeine, which is frequently found in surface waters, can be quantified with a relative error lower than 20% for concentrations above 0.025 microg L(-1) (limit of quantitation, direct analysis). The limit of detection is 0.001 microg L(-1) and can be reduced by solid-phase extraction (SPE). Moreover, with minor adaptations, the assay can be used to quantify caffeine in several beverages, shampoo, and caffeine tablets. The results obtained by ELISA correlate well with those from liquid chromatography-tandem mass spectrometry (LC-MS-MS) for the tested matrices. Several surface waters from Berlin were analysed and all tested positive for caffeine, with concentrations higher than 0.030 microg L(-1). In one run 66 samples can be analysed within 2 h.

Direct determination of theophylline in serum by fluoroimmunoassay using highly specific antibodies.

Described is the development of a fluoroimmunoassay for theophylline using a fluorescein labelled derivative of theophylline as tracer and antibodies coupled to magnetizable 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 microL 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.

Production and characterization of a genetically engineered anti-caffeine camelid antibody and its use in immunoaffinity chromatography.

This work demonstrates the feasibility of using a camelid single domain antibody for immunoaffinity chromatographic separation of small molecules. An anti-caffeine VHH antibody was produced by grafting the complementarity determining sequences of a previously generated antibody onto an anti-RNase A antibody scaffold, followed by expression in E. coli. Analysis of the binding properties of the antibody by ELISA and fluorescence-based thermal shift assays showed that it recognizes not only caffeine, but also theophylline, theobromine, and paraxanthine, albeit with lower affinity. Further investigation of the effect of environmental conditions, i.e., temperature, pH, and ionic strength, on the antibody using these methods provided useful information about potential elution conditions to be used in chromatographic applications. Immobilization of the VHH onto a high flow-through synthetic support material resulted in a stationary phase capable of separating caffeine and its metabolites.

Isolation and characterization of a thermally stable recombinant anti-caffeine heavy-chain antibody fragment.

We have isolated and characterized a caffeine-specific, heavy-chain-only antibody fragment (V(HH)) from llama that is capable of being utilized to analyze caffeine in hot and cold beverages. Camelid species (llama and camel) were selected for immunization because of their potential to make heat-stable, heavy-chain-only antibodies. Llamas and camels were immunized with caffeine covalently linked to keyhole limpet hemocyanin, and recombinant antibody techniques were used to create phage displayed libraries of variable region fragments of the heavy-chain antibodies. Caffeine-specific V(HH) fragments were selected by their ability to bind to caffeine/bovine serum albumin (BSA) and confirmed by a positive reaction in a caffeine enzyme-linked immunosorbent assay (caffeine ELISA). One of these V(HH) fragments (VSA2) was expressed as a soluble protein and shown to recover its reactivity after exposure to temperatures up to 90 degrees C. In addition, VSA2 was able to bind caffeine at 70 degrees C. A competition caffeine ELISA was developed for the measurement of caffeine in beverages, and concentrations of caffeine obtained for coffee, Coca-Cola Classic, and Diet Coke agreed well with high performance liquid chromatography (HPLC) determination and literature values. VSA2 showed minimal cross reactivity with structurally related methylxanthines.

Fever from caffeine.

Since childhood, a 53-year-old women had developed chills, high-grade fever, myalgia, and cephalea after the ingestion of coffee, tea, cola beverages, and some oral "antiflu" compounds. Skin prick tests performed with all the implicated substances were negative. Single-blind oral challenges with both caffeine and theophylline were positive, reproducing exactly the same clinical symptoms and fever. Oral challenge with pentoxifylline was negative. We report a case of caffeine-induced fever in which we have demonstrated cross-reactivity with theophylline, but not with pentoxifylline.

[Immunotropic activity of the cAMP phosphodiesterase inhibitors caffeine and theophylline]. / Issledovanie immunotropnoi aktivnosti ingibitorov tsAMF-fosfodiésterazy kofeina i teofillina.

On mice immunized with ram erythrocytes it was shown that caffeine and theophylline (15 to 240 mg/kg thrice at different time intervals with respect to the antigenic stimulus) possess identical immunotropic activity. High doses of methylxanthines injected a few days before immunization increased and being administered during immunization decreased the indices of humoral immunity. Low doses of the drugs stimulated immunity but only when administered at the time close to the antigenic stimulus application.

Suppressive effect of caffeine on hepatitis and apoptosis induced by tumor necrosis factor-alpha, but not by the anti-Fas antibody, in mice.

Tumor necrosis factor (TNF)-alpha-induced hepatitis and apoptosis, as respectively assessed by serum enzyme activities and hepatic DNA fragmentation were effectively suppressed by a single force-feeding of caffeine (100 mg/kg) 1.5 h before injecting the drug. In contrast, caffeine had no significant effect on anti-Fas antibody-induced hepatitis and apoptosis. These results suggest that caffeine differentially affected TNF-alpha receptor- and Fas-mediated hepatitis and apoptosis.