प्रस्तुत विधि फैली हुई cardiomyopathy के साथ रोगियों के प्लाज्मा में कार्यात्मक प्रभावी cardiotropic autoantibodies का पता लगाने के लिए, चाहे विशिष्ट प्रतिजन के सेलुलर और पृथक चूहे cardiomyocytes में छोटा करने intracellular कैल्शियम यात्रियों पर अलग रोगी immunoglobulin के प्रभाव का विश्लेषण करने के लिए एक रास्ता प्रदान करता है.
Dilated cardiomyopathy (DCM) is one of the main causes for heart failure in younger adults1. Although genetic disposition and exposition to toxic substances are known causes for this disease in about one third of the patients, the origin of DCM remains largely unclear. In a substantial number of these patients, autoantibodies against cardiac epitopes have been detected and are suspected to play a pivotal role in the onset and progression of the disease2,3. The importance of cardiac autoantibodies is underlined by a hemodynamic improvement observed in DCM patients after elimination of autoantibodies by immunoadsorption3-5. A variety of specific antigens have already been identified2,3 and antibodies against these targets may be detected by immunoassays. However, these assays cannot discriminate between stimulating (and therefore functionally effective) and blocking autoantibodies. There is increasing evidence that this distinction is crucial6,7. It can also be assumed that the targets for a number of cardiotropic antibodies are still unidentified and therefore cannot be detected by immunoassays. Therefore, we established a method for the detection of functionally active cardiotropic antibodies, independent of their respective antigen. The background for the method is the high homology usually observed for functional regions of cardiac proteins in between mammals8,9. This suggests that cardiac antibodies directed against human antigens will cross-react with non-human target cells, which allows testing of IgG from DCM patients on adult rat cardiomyocytes. Our method consists of 3 steps: first, IgG is isolated from patient plasma using sepharose coupled anti-IgG antibodies obtained from immunoadsorption columns (PlasmaSelect, Teterow, Germany). Second, adult cardiomyocytes are isolated by collagenase perfusion in a Langendorff perfusion apparatus using a protocol modified from previous works10,11. The obtained cardiomyocytes are attached to laminin-coated chambered coverglasses and stained with Fura-2, a calcium-selective fluorescent dye which can be easily brought into the cell to observe intracellular calcium (Ca2+) contents12. In the last step, the effect of patient IgG on the cell shortening and Ca2+ transients of field stimulated cardiomyocytes is monitored online using a commercial myocyte calcium and contractility monitoring system (IonOptix, Milton, MA, USA) connected to a standard inverse fluorescent microscope.
प्रस्तुत विधि अस्पष्ट मूल के डीसीएम के साथ रोगियों में कार्यात्मक प्रभावी हृदय autoantibodies का पता लगाने के लिए एक उपयुक्त तरीका प्रदान करता है. अन्य तरीकों की तुलना में, शिविर 6 स्तरों पर उनके प्रभाव से β1 …
The authors have nothing to disclose.
हृदय रोगों में प्रतिरक्षा प्रतिक्रिया (- यह काम Sonderforschungsbereich 19 Transregio (SFB/TR19, C2) ड्यूश Forschungsgemeinschaft (DFG), प्रौद्योगिकी और अर्थशास्त्र परियोजना ZIM-KF 2727801MD0 और अभिनव क्षमता के लिए केंद्र के संघीय मंत्रालय द्वारा समर्थित किया गया zik वृद्धि, शिक्षा और रिसर्च, जर्मनी के संघीय मंत्रालय के BMBF FKZ) 03Z2CN12. आवास और जानवरों के साथ प्रयोगों प्रयोगशाला पशु विज्ञान (Gesellschaft für Versuchstierkunde जीवी SOLAS) और यूरोपीय प्रयोगशाला पशु विज्ञान एसोसिएशन (FELASA) के फेडरेशन के लिए सोसायटी की सिफारिशों के अनुसार में प्रदर्शन किया गया.
Name of the reagent/equipment | Company | Catalogue number | Comments |
Immunosorba preservation solution | Fresenius Medical Care | 903609211 | |
Collagenase type 2 | Cell System | LS004176 | |
BSA, fatty acid free | Sigma-Aldrich | A6003 | |
Laminin | Sigma-Aldrich | L2020 | |
Fura-2 AM | Sigma-Aldrich | F0888 | 1 mg/ml in DMSO |
Econo Pac Chromatography Columns | BioRad | 732-1010 | |
Spectra/Por Biotech Cellulose Ester Dialysis Membrane | Spectrumlabs | 131417 | |
4 well Chambered Coverglass | Nunc | 155383 | |
Myocyte Calcium and Contractility Recording System | IonOptix | ||
IonWizard 6.0 analysis software | IonOptix |