To test whether the in vivo produced antigens are secreted into the blood stream, we injected mice with RNA-LPX for expression of HIV-1 capsid protein (p24) tagged with the MHC-I secretory peptide. In order to stimulate a strong humoral response, antigens have to be secreted from the transfected cells. After 24 h, expression of GFP was seen in ~6.5% of the dendritic cells isolated from spleens (Fig. The RNA-LPX was then administrated intravenously to BALB/c mice. The RNA copies were then mixed with DOTMA/DOPE liposomes (RNA-LPX) by adjusting the optimal net charge. In our study, we digested a plasmid for expression of secretory GFP and transcribed it in vitro. Uptake of the RNA transcripts will then result in expression of the encoded antigen by dendritic cells 13. DOTMA/DOPE liposomes were previously shown to protect RNA from ribonuclease digestion and mediated efficient uptake of the RNA transcripts mainly by dendritic cells. We then tested whether these transcripts could be transfected in vitro to produce secretory peptides.
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Our in vitro experiments demonstrated that transcription of the digested plasmids resulted in functional transcripts that were translated into peptides at expected sizes. Uptake and translation of RNA transcripts by dendritic cells In vitro translation of the transcripts produced peptide fragments at expected lengths (Fig. Transcripts were capped on their 5′-hydroxyl group. To prepare RNA transcripts for in vivo transfection, the constructs for each antigen were linearized using restriction enzymes and subjected to in vitro transcription. We report a rapid method for generation of monoclonal antibodies with potential applications in research and medicine. In addition, we show that our method can be used for stimulation of humoral immunity. We show that our antibodies could be used for Western blot analysis, suggesting their potential in research. We demonstrate production of monoclonal antibodies using RNA transfection. In our study, we took advantage of this recently developed method for in vivo transfection of mice with RNA transcripts. Expression of the exogenous antigens resulted in activation of T cell and interferon responses 13. showed that using intravenously administered RNA-lipoplexes, dendritic cells can be targeted in vivo. RNA transcripts, however, can be transfected in vivo and lead to production of substantial amounts of peptides. Similarly to monoclonal antibody production, this method requires synthesis and purification of antigenic peptides for stimulation of the humoral response. Peptide vaccines, which immunize patients against certain pathogens or cancer cells, also rely on injection of antigenic peptides 6, 12. The new hybridoma cells are then screened for production of specific antibodies 11. Using this technology, primary B cells from a vaccinated animal are fused with immortal B cells. Development of hybridoma technology was a major advance in producing large amounts of monoclonal antibodies.
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![antibody production antibody production](http://i.ytimg.com/vi/lteHu3zZ7XI/maxresdefault.jpg)
The most common method for antibody production is based on injection of the antigenic peptides to laboratory animals in order to stimulate a humoral response 8, 9, 10. Over the past decades, laboratory animal systems with strong humoral responses have been developed. The wide applications of antibodies in research and medicine demand efficient and rapid methods for antibody production. Stimulation of the immune response and antibody production is also the fundamental basis of peptide vaccines 6, 7.
![antibody production antibody production](https://image.slidesharecdn.com/monoclonalantibodiesbiosep-150827153603-lva1-app6892/95/monoclonal-antibodiesproduction-bioseperartion-6-638.jpg)
In addition, a number of monoclonal antibodies have been approved for medical applications such as cancer therapy 3, 4, 5. Antibodies are essential in many biological techniques such as immunoblotting, immunoprecipitation, immunofluorescence, flow cytometry, ELISA, etc. Currently, commercial antibodies are available in the form of monoclonal (homogenous isotype and antigen specificity) and polyclonal (heterogeneous isotype and antigen specificity) antibodies. Human and mice have 5 antibody isotypes, which are distinguished by immunoglobulin structure 1, 2.