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ePaper created 2016-03-17, 15:06:18 | version 1.45.01
70 Pyrogen detection using immunoreceptors According to global estimates, about 18 million patients die annually from the consequences of sepsis. This complication is caused by pyrogens – bacteria, viruses or fungi or their resi- dues that enter a patient´s blood circulation via contaminated surgical instruments or drugs. The pyrogens, also known as Pathogen-Associated Molecular Patterns (PAMPs) [1], are recognized by receptors of the innate immune system, the Pattern Recognition Receptors (PRRs), which initiate produc- tion of fever-inducing messenger substances. In order to prevent pyrogens from entering the blood via medicinal devices or pharmaceuticals, these must be checked for the presence of pyrogens. EU and FDA regulations cur- rently approve four commercial detection systems [2, 3] but these are either very time-consuming procedures or limited to certain pyrogens. In order to avoid the limitations of conventional tests, for several years Fraunhofer IGB has been developing alternative in vitro test systems based on the use of PRRs [4]. The toll-like receptors (TLRs) represent the largest and best known group of PRRs. The PAMP assay patented by Fraunhofer IGB [5, 6] is a sensitive and universally adaptable in vitro method suitable for all pyrogens. However, this cell-based test method also re- quires a well-equipped laboratory with experience in handling cell cultures. ImmuStick – A “test strip” for pyrogens With the ImmuStick, we developed an unconventional, inno- vative detection system for pyrogens using PRRs as biosensors without the use of animal testing or time-consuming and device-intensive tests. Pyrogens are detected using test strips based on the immunochromatographic principle. Immobilized receptor domains of individual PRRs serve as binding mol- ecules for the corresponding pyrogen. If an applied sample contains the corresponding pyrogen, a color-labeled ligand will be released, indicating the presence of the pyrogen (Fig. 1). Detection of the pyrogen is based on a classical competitive immunoassay. After the test strips are wetted with the analyte solution, the pyrogen contained in it (TLR4 ligand lipopolysac- charides (LPS); yellow) displaces the weaker binding labeled ligand black-red in the biosensor area ia capillary flow, these migrate together with the control molecules (blue-red) to the result window where specific antigens are immobili ed as capture molecules (Y). If the antibodies bind their antigen (the labeled ligand or the control molecule), application of a pyrogen-containing sample solution will result in bi-colored test lines in the result window. The control line appears only after sufficient application of sample solution to the Im- muStick, thus verifying that the test has functioned properly (Fig. 4). ig shows fluorescence microscope images of T R bound to agarose beads. The images show the displacement of a ligand bound weakly to TLR4 (TAMRA, red) by LPS (Alexa Fluor488, green) and the quantitative analysis of this binding study (Fig. 3). IMMUSTICK – THE INNATE IMMUNE SYSTEM ON A TEST STRIP Christina Kohl, Anke Burger-Kentischer 1 MEDICINE 2