ترنسفكأيشن الأجسام المضادة إلى الخلايا العصبية كأداة للدراسة إمراض مرض
Summary
ووصف النهج السريع للتحقيق في التفاعلات والآثار على الآليات الجزيئية ذات الصلة وجود الأجسام المضادة في بيئة داخل الخلايا. الأسلوب ينطوي ترنسفكأيشن من الأجسام المضادة في الخلايا الحية باستخدام تشكيل التساهمية غير معقدة تقوم على صياغة المادة الدهنية. هذه التقنية قابلة للتكيف مع خطوط الخلايا والخلايا الأولية خلد.
Abstract
Antibodies provide the ability to gain novel insight into various events taking place in living systems. The ability to produce highly specific antibodies to target proteins has allowed for very precise biological questions to be addressed. Importantly, antibodies have been implicated in the pathogenesis of a number of human diseases including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), paraneoplastic syndromes, multiple sclerosis (MS) and human T-lymphotropic virus type 1 (HTLV-1) associated myelopathy/tropical spastic paraparesis (HAM/TSP) 1-9. How antibodies cause disease is an area of ongoing investigation, and data suggests that interactions between antibodies and various intracellular molecules results in inflammation, altered cellular messaging, and apoptosis 10. It has been shown that patients with MS and HAM/TSP produce autoantibodies to the intracellular RNA binding protein heterogeneous ribonuclear protein A1 (hnRNP A1) 3, 5-7, 9, 11. Recent data indicate that antibodies to both intra-neuronal and surface antigens are pathogenic 3, 5-9, 11. Thus, a procedure that allows for the study of intracellular antibody:protein interactions would lend great insight into disease pathogenesis.
Genes are commonly transfected into primary cells and cell lines in culture, however transfection of antibodies into cells has been hindered by alteration of antibody structure or poor transfection efficiency 12. Other methods of transfection include antibody transfection based on cationic liposomes (consisting of DOTAP/DOPE) and polyethylenimines (PEI); both of which resulted in a ten-fold decrease in antibody transfection compared to controls 12. The method performed in our study is similar to cationic lipid-mediated methods and uses a lipid-based mechanism to form non-covalent complexes with the antibodies through electrostatic and hydrophobic interactions 13. We utilized Ab-DeliverIN reagent, which is a lipid formulation capable of capturing antibodies through non-covalent electrostatic and hydrophobic interactions and delivering them inside cells. Thus chemical and genetic couplings are not necessary for delivery of functional antibodies into living cells. This method has enabled us to perform various antibody tracing and protein localization experiments, as well as the analyses of the molecular consequences of intracellular antibody:protein interactions 9.
In this protocol, we will show how to transfect antibodies into neurons rapidly, reproducibly and with a high degree of transfection efficiency. As an example, we will use anti-hnRNP A1 and anti-IgG antibodies. For easy quantification of transfection efficiency we used anti-hnRNP A1 antibodies labelled with Atto-550-NHS and FITC-labeled IgG. Atto550 NHS is a new label with high molecular absorbtion and quantum yield. Excitation source and fluorescent filters for Atto550 are similar to Cy3 (Ex. 556 Em. 578). In addition, Atto550 has high photostability. FITC-labeled IgG were used as a control to show that this method is versatile and not dye dependent. This approach and the data that is generated will assist in understanding of the role that antibodies to intracellular target antigens might play in the pathogenesis of human diseases.
Protocol
Discussion
من أجل اختبار الفرضيات حول جزيئات معينة والتعبير عنها، وتقاس عادة الجينات وناقلات أخرى في الخلايا. الجانب الفريد من هذا الإجراء هو القدرة على الأجسام المضادة في بالنقل بسهولة الخلايا المستهدفة. أجرينا هذه الطريقة في خمس تجارب منفصلة في كل التكرارات. وكانت النتائج م?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
ويدعم هذا العمل من قبل مكتب البحوث والتنمية، الخدمات الطبية البحوث وزارة شؤون المحاربين القدامى. وقد تم تمويل هذه الدراسة من قبل على جائزة الاستحقاق استعراض VA (لMCL) وجامعة ولاية تينيسي العلوم الصحية مركز التصلب المتعدد صندوق البحث.
Materials
| Name of Reagent | Company | Catalogue Number |
| Fluorescent Microscope AxioObserver A1 | Zeiss | |
| Axiovision version 4.2 | Zeiss | |
| Anti-rhnRNPA1 | Abcam | Ab4791 |
| FITC labeled anti-rIgG | OZ Biosciences | AI20100 |
| Atto550 NHS ester | Sigma | 92835 |
| Ab-DeliverIN | OZ Biosciences | AI20100 |
| DMEM/F12 +10% FBS+1%antibiotics | Gibco | 11320-033 |
| Poly-D-Lysine 8-well Culture Slides | BD | 354632 |
| Multi-Purpose Rotator | Scientific Industries Inc | Model 151 |
| DAPI Mounting Media | Millipore | S7113 |
| PBS | Ambion | 9626 |
| Dialyzer | Thermo Fisher Scientific | 66380 |
| Amicon Ultra-0.5 | Millipore | UFC501096 |
| Paraformaldehyde (4% solution) | Electron Microscopy Sciences | |
| Nano-Drop | Thermo Fisher Scientific |
References
- Burkhardt, H. Epitope-specific recognition of type II collagen by rheumatoid arthritis antibodies is shared with recognition by antibodies that are arthritogenic in collagen-induced arthritis in the mouse. Arthritis Rheum. 46, 2339-2348 (2002).
- Chester, K. A. Lambert-Eaton syndrome antibodies: reaction with membranes from a small cell lung cancer xenograft. J. Neuroimmunol. 18, 97-104 (1988).
- Lee, S. M. HTLV-1 induced molecular mimicry in neurological disease. Curr. Top Microbiol. Immunol. 29, 125-136 (2005).
- Reeves, W. H. Systemic lupus erythematosus. Antibodies to DNA, DNA-binding proteins, and histones. Rheum. Dis. Clin. North Am. 20, 1-28 (1994).
- Levin, M. C. Autoimmunity due to molecular mimicry as a cause of neurological disease. Nat. Med. 8, 509-513 (2002).
- Lee, S. M. Autoantibodies that recognize functional domains of hnRNPA1 implicate molecular mimicry in the pathogenesis of neurological disease. Neurosci. Lett. 401, 188-193 (2006).
- Kalume, F. Molecular mimicry: cross-reactive antibodies from patients with immune-mediated neurologic disease inhibit neuronal firing. J. Neurosci. Res. 77, 82-89 (2004).
- Levin, M. C. Cross-reactivity between immunodominant human T lymphotropic virus type I tax and neurons: implications for molecular mimicry. J. Infect. Dis. 186, 1514-1517 (2002).
- Lee, S. A potential link between autoimmunity and neurodegeneration in immune-mediated neurological disease. J. Neuroimmunol. 235, 56-69 (2011).
- Smeenk, R. J. Antinuclear antibodies: cause of disease or caused by disease. Rheumatology (Oxford). 39, 581-584 (2000).
- Lee, S. Post-translational glycosylation of target proteins implicate molecular mimicry in the pathogenesis of HTLV-1 associated neurological disease. J. Neuroimmunol. 204, 140-148 (2008).
- Reisinger, H. Serum-free transfection of CHO cells with chemically defined transfection systems and investigation of their potential for transient and stable transfection. Cytotechnology. 60, 115-123 (2009).
- Weill, C. O., Biri, S., Erbacher, P. Cationic lipid-mediated intracellular delivery of antibodies into live cells. Biotechniques. 44, Pvii-Pxi (2008).
- Radic, M. Z. Heterogeneous nuclear ribonucleoprotein P2 is an autoantibody target in mice deficient for Mer, Axl, and Tyro3 receptor tyrosine kinases. J. Immunol. 176, 68-74 (2006).
- Vincent, A. Successful ‘passive transfer’ of paraneoplastic stiff person syndrome with antibodies to an intracellular antigen. Brain. 133, 3164-3165 (2010).
- Vincent, A. Stiff, twitchy or wobbly: are GAD antibodies pathogenic. Brain. 131 (Pt 10), 2536-2537 (2008).
- Magrys, A. The role of anti-alpha-enolase autoantibodies in pathogenicity of autoimmune-mediated retinopathy. J. Clin. Immunol. 27, 181-192 (2007).
- Geis, C. Stiff person syndrome-associated autoantibodies to amphiphysin mediate reduced GABAergic inhibition. Brain. 133, 3166-3180 (2010).
