Name: antibody transfection into neurons as a tool to study disease pathogenesis
Uploaded: 2012-09-26T13:00:00
Description: Watch this Scientific Journal Video about Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis at JoVE.com

This work is supported by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs. This study was funded by a VA Merit Review Award (to MCL) and the University of Tennessee Health Science Center Multiple Sclerosis Research Fund.

1. Antibody Labeling (Figure 1)
<ol>
 <li>Make 0.1 M NaHCO3 buffer:
 <ul type="circle">
 <li>8.4 g NaHCO3</li>
 <li>29.2 g NaCl</li>
 <li>1 liter H2O</li>
 </ul>
 </li>
 <li>Bring the buffer up to a pH of 8.4 with this solution (10.6 g Na2CO3, 29.2 g NaCl, 1 liter H20).</li>
 <li>Add 35 &mu;l Atto550 NHS to 70 &mu;l anti-hnRNPA1 and 500 &mu;l NaHCO3 buffer and rotate for 1 hr at room temperature. After the hour-long rotation, inject the mix...

<ol>
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In order to test hypotheses about specific molecules and their expression, genes and other vectors are commonly transfected into cells. The unique aspect of this procedure is the ability to easily transfect antibodies into target cells. We performed this method in five separate experiments each in duplicates. Transfection efficiency results were similar among all experiments. Importantly, the method enables entry of antibodies into the cell without altering antibody structure or function. Further, with the use of fluores...

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>Fluorescent Microscope AxioObserver A1</td>
 <td>Zeiss</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Axiovision version 4.2</td>
 <td>Zeiss</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anti-rhnRNPA1</td>
 <td>Abcam</td>
 <td>Ab4791</td>
 </tr>
 <tr>
 <td>FITC labeled anti-rIgG</td>
 <td>OZ Biosciences</td>
 <td>AI20100</td>
 </tr>
 <tr>
 <td>Atto550 NHS ester</td>
 <td>Sigma</td>
 <td>92835</td>
 </tr>
 <tr>
 <td>Ab-DeliverIN</td>
 <td>OZ Biosciences</td>
 <td>AI20100</td>
 </tr>
 <tr>
 <td>DMEM/F12 +10% FBS+1%antibiotics</td>
 <td>Gibco</td>
 <td>11320-033 </td>
 </tr>
 <tr>
 <td>Poly-D-Lysine 8-well Culture Slides </td>
 <td>BD</td>
 <td>354632 </td>
 </tr>
 <tr>
 <td>Multi-Purpose Rotator</td>
 <td>Scientific Industries Inc</td>
 <td>Model 151</td>
 </tr>
 <tr>
 <td>DAPI Mounting Media</td>
 <td>Millipore</td>
 <td>S7113</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Ambion</td>
 <td>9626</td>
 </tr>
 <tr>
 <td>Dialyzer</td>
 <td>Thermo Fisher Scientific</td>
 <td>66380</td>
 </tr>
 <tr>
 <td>Amicon Ultra-0.5</td>
 <td>Millipore</td>
 <td>UFC501096</td>
 </tr>
 <tr>
 <td>Paraformaldehyde (4% solution)</td>
 <td>Electron Microscopy Sciences</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Nano-Drop</td>
 <td>Thermo Fisher Scientific</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

antibody transfection into neurons as a tool to study disease pathogenesis

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.

Watch this Scientific Journal Video about Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis at JoVE.com

Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis

A rapid approach to investigate interactions and effects on molecular mechanisms related to the presence of antibodies in an intracellular environment is described. The method involves transfection of antibodies into live cells using a non-covalent complex formation based on a lipid formulation. The technique is adaptable to immortalized cell lines and primary cells.

Antibodies provide the ability to gain novel insight into various events taking place in living systems.  The ability to produce highly specific ...

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