Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis

Summary

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.

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

1. Antibody Labeling (Figure 1)

1. Make 0.1 M NaHCO₃ buffer:
   • 8.4 g NaHCO₃
   • 29.2 g NaCl
   • 1 liter H₂O
2. Bring the buffer up to a pH of 8.4 with this solution (10.6 g Na₂CO₃, 29.2 g NaCl, 1 liter H₂0).
3. Add 35 μl Atto550 NHS to 70 μl anti-hnRNPA1 and 500 μl NaHCO₃ buffer and rotate for 1 hr at room temperature. After the hour-long rotation, inject the mixture into a dialyzer and dialyze in 2 liters of PBS overnight.
4. The next day, concentrate the dialyzed Atto550 NHS anti-hnRNPA1 using a spin column (A....

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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