Aptamer BC 007 - A broad spectrum neutralizer of pathogenic autoantibodies against G-protein-coupled receptors.

The effect of autoantibodies on G-protein coupled receptors in the pathogenesis of diseases, especially of the heart and vascular system, is an increasingly accepted fact today. Dilated cardiomyopathy (DCM) is the most intensively investigated pathological situation of these. With DCM, autoantibodies against the ß1-adrenoceptor and the muscarinic M2-receptor have been found in high percentage of investigated patients. Immunoadsorption for autoantibody removal has already shown a long-term beneficial therapeutic effect, but has remained limited in its application because of the complexity of this method. A new easy applicable treatment strategy has, therefore, been discovered. Because of intra- and inter-loop epitope variability of the ß1-adrenoceptor specific autoantibodies and also the occurrence of further autoantibodies of this class such as the ones against the ß2- and α1-adrenoceptor, the ETA-, proteinase activated-, and the AT1-receptors in different pathological situations, this newly discovered broad-spectrum neutralizer of all these autoantibodies - aptamer BC 007 - is under development. The binding and neutralizing effect was investigated applying a bioassay of spontaneously beating neonatal rat cardiomyocytes and enzyme-linked immunosorbent assay (ELISA) - technology. The usefulness of aptamer BC 007 to specify column technology for the removal of serum autoantibodies was also demonstrated. The presented data suggest that aptamer BC 007 might be an appropriate molecule candidate to support future research about the meaning of G-protein-coupled receptor autoantibodies.

Covalent attachment of functionalized cardiolipin on a biosensor gold surface allows repetitive measurements of anticardiolipin antibodies in serum.

Antiphospholipid antibodies (aPL) are a relevant serological indicator of antiphospholipid syndrome (APS). A solid-state surface with covalently bound ω-amine-functionalized cardiolipin was established and the binding of ß2-glycoprotein I (ß2-GPI) was investigated either by use of surface plasmon resonance (SPR) biosensor, by electrically switchable DNA interfaces (switchSENSE) and by scanning tunneling microscopy (STM). STM could clearly visualize the attachment of ß2-GPI to the cardiolipin surface. Using the switchSENSE sensor, ß2-GPI as specific ligand could be identified by increased hydrodynamic friction. The binding of anti-cardiolipin antibodies (aCL) was detected against the ω-amine-functionalized cardiolipin-modified SPR biosensor (aCL biosensor) using sera from healthy donors, APS patients and syphilis patients. Our results showed that the aCL biosensor is a much more sensitive diagnostic device for APS patients compared to previous methods. The specificity between ß2-GPI-dependent autoimmune- and ß2-GPI-independent infection-associated types of aPLs was also studied and they can be distinguished by the different binding kinetics and patterns.

Steroid binding properties of the 2nd WHO International Standard for sex hormone-binding globulin.

BACKGROUND: Stocks of 95/560, the 1st International Standard for sex hormone-binding globulin (SHBG), are running out, and 08/266 has been prepared as a replacement. We compared the steroid binding capacities of 95/560 and 08/266. METHODS: 95/560 and 08/266 stored at -20 °C, and accelerated thermal degradation samples of 08/266 were subjected to gelfiltration. Binding of the SHBG-containing fractions to a dihydrotestosterone derivative was then investigated using a surface plasmon resonance biosensor. The SHBG content of the gelfiltration fractions was determined with an immunoassay. Steroid binding capacity of each standard was then calculated as the mean of the ratio of biosensor binding/SHBG concentration. Affinity constants for the SHBG steroid interaction were calculated. RESULTS: Maximum achievable biomolecular interactions were lower for 95/560 than for the 08/266 preparations. 95/560 exhibited steroid binding capacity only half as high as any of the novel standards, as well as a lower affinity constant for the SHBG steroid interaction. No significant differences could be observed between the samples of 08/266 stored at different temperatures. CONCLUSIONS: The steroid binding capacity of 08/266 is two times higher than that of 95/560. Steroid binding of lyophilized 08/266 is preserved at storage temperatures of up to 45 °C for at least 6 months.

Point-of-care testing (POCT): Current techniques and future perspectives.

Point-of-care testing (POCT) is a laboratory-medicine discipline that is evolving rapidly in analytical scope and clinical application. In this review, we first describe the state of the art of medical-laboratory tests that can be performed near the patient. At present, POCT ranges from basic blood-glucose measurement to complex viscoelastic coagulation assays. POCT shortens the time to clinical decision-making about additional testing or therapy, as delays are no longer caused by transport and preparation of clinical samples, and biochemical-test results are rapidly available at the point of care. Improved medical outcome and lower costs may ensue. Recent, evolving technological advances enable the development of novel POCT instruments. We review the underlying analytical techniques. If new instruments are not yet in practical use, it is often hard to decide whether the underlying analytical principle has real advantage over former methods. However, future utilization of POCT also depends on health-care trends and new areas of application. But, even today, it can be assumed that, for certain applications, near-patient testing is a useful complement to conventional laboratory analyses.

Standardized antigen preparation to achieve comparability of anti-beta2-glycoprotein I assays.

INTRODUCTION: The Sydney classification for diagnosis of the antiphospholipid syndrome (APS) first introduced the determination of anti-beta2-glycoprotein I (anti-beta2-GPI)-antibodies in serum as laboratory criteria. In this context, widely differing results of anti-beta2-GPI assays are a concerning issue. Considerable efforts have been made to optimize ELISAs, however little attention was hitherto spent to the antigen preparation. We evaluated the influence of different beta2-GPI preparations on the ability to separate ill and healthy patients and on the comparability of anti-beta2-GPI-assays. MATERIALS AND METHODS: Microplates were coated with various beta2-GPI preparations and anti-beta2-GPI IgG- and IgM-ELISAs were performed for 21 APS patients and 21 controls using the monoclonal calibrators HCAL and EY2C9. Subsequently, by use of a surface plasmon resonance (SPR) biosensor, affinity constants for the HCAL- and EY2C9-interaction with each beta2-GPI preparation were determined and antigen binding of sera of APS patients and controls was studied. RESULTS: All ss2-GPI preparations showed good discrimination ability ill vs. healthy but poor inter-assay comparability in the ELISAs. Affinity constants for HCAL and EY2C9 were independent of the beta2-GPI variant (K(A) 0.105 - 0.200 and 0.449 - 1.04 x 10(10)M(-1); K(D) 50.0 - 95.5 and 9.61 - 22.3 x 10(-11)M, respectively). In the biosensor, reactivity to the different beta2-GPIs was negligible for the controls and varied considerably for patients. CONCLUSION: Inter-assay comparability of anti-beta2-GPI ELISAs is highly dependent upon the beta2-GPI preparation. Only agreement on one common beta2-GPI preparation will improve the requested inter-assay comparability.