Cancer Antigen Discovery Method Slide

2015-01-13

azim58 - Cancer Antigen Discovery Method Slide
Antigen Discovery Method

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some powerpoints this slide is found in
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General Meeting Presentation\General CIM Lab Meeting Presentation 2
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Source of Diagram: http://www.azim58.wikispaces.com/Antigen+Discovery+Method+Diagram+12-4-11

Here is the method I plan to use to discover tumor specific antigens.  The first step is to construct a cDNA library from the tumor of a mouse.  RNA is extracted from the tumor, reverse transcribed to DNA, and cloned into plasmids.  These plasmids can then be transformed into bacteria, and these bacteria will then produce proteins found in the original tumor.  At this stage, I then want to find out which tumor proteins the immune system of that mouse can recognize.  To do this I can take antibodies found in the blood of the mouse, and determine whether they bind any tumor proteins in my library.  
     There are also some additional steps that could be taken in order to enhance this discovery process.  For example instead of just using antibodies, I could create a phage library from the RNA of the B cells which make the antibodies.  Phage are viruses that infect bacteria.  When I put the antibody genes into the phage, they can express antibodies or mini forms of antibodies on their surface.  The advantage of doing this is that I can produce as much antibody as I would like.  Another additional step which may help me discover tumor specific antigens is to purify the antibody libraries using the random peptide arrays.  When I screen my cDNA library with a smaller set of antibodies, I may obtain fewer false positives.
     Once a tumor specific protein is found, this can be vaccinated into a mouse and tested for immunogenicity.

Alternative explanation for antigen discovery method
My main project has been to discover tumor specific antigens targeted by the antibodies of the immune system.  The primary method for accomplishing that goal was to screen a tumor cDNA library with antibody containing sera.  However, some other methods which may complement the primary method have also been pursued.  Phage antibody libraries allow for the amplification of antibodies as well as allow for the antibody sequence to be easily determined by sequencing the DNA within the genome of the phage.  Additionally, random peptide arrays may allow for the purification of specific antibodies so that a smaller subset of antibodies can be used to probe biological material such as a cDNA library.

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