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Thursday, 21 July 2016 17:16

More Zika Colors

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Zika virus-infected Vero cells stained with DAPI (blue) to indicate the cell nucleus, and flavivirus antibody 4G2 (green). Photograph by Amy Rosenfeld. Zika virus-infected Vero cells stained with DAPI (blue) to indicate the cell nucleus, and flavivirus antibody 4G2 (green). Photograph by Amy Rosenfeld.
In the last Zika Diaries article I lamented that one of the antibodies that we use to detect Zika virus infection within cells was out of stock at the supplier. Here’s how we got around that little problem.
 
Antibody 4G2 is a mouse monoclonal antibody originally produced against dengue virus type 2. A monoclonal antibody binds to one part of the antigen - an epitope - typically 5-8 amino acids in length. In contrast, serum contains many different antibodies that recognize different epitopes. 
 
The first step in producing mouse monoclonal antibodies is to immunize mice with the desired antigen - in this case, dengue virus type 2. As the immune response against the antigen develops, B cells arise that recognize different parts of the virus. Each B cell recognizes a single epitope.
 
To obtain B cells that produce monoclonal antibodies, the mouse spleen (which contains many B cells) is removed and cells are placed in a culture dish. To produce B cells that are immortal, the spleen cells are fused with myeloma cells, which are cancer cells derived from antibody-producing plasma cells. After fusion, single cells are isolated and placed in culture. These cells - called ‘hybridomas’ - will secrete monoclonal antibodies, one per cell. The cell culture medium from each hybridoma is checked for the presence of antibody of the desired specificity.
 
In the case of monoclonal antibody 4G2, a hybridoma was identified which produced antibody that binds dengue virus type 2. Subsequently it was found that this monoclonal antibody recognizes the viral E glycoprotein, which is embedded in the viral envelope.
 
What does this have to do with getting antibody for our experiments? When we found that antibody 4G2 was back-ordered, we simply looked to see if the hybridoma cell line which produces the antibody was available. A quick search of the American Type Culture Collection catalog (at atcc.org) for 4G2 revealed that the cell line was available (link).
 
We purchased the 4G2 hybridoma (not cheap at $460 for a non-profit institution) and grew the cells in culture. During growth of the cells, the antibody is secreted into the cell culture medium. We simply took an aliquot of the cell culture medium and used it to stain Vero cells (an African Green Monkey kidney cell line) that had been infected for 72 hours with Zika virus. The results, as shown in the photograph, reveal that we have indeed produced our own lot of monoclonal antibody 4G2 which we can now use in our Zika virus experiments.
 
In producing 4G2 antibody, we didn’t do anything special - the hard part was making the monoclonal antibody, which was done by others (link to paper). We and other virologists are fortunate that the individuals who created antibody 4G2 placed it in the ATCC for all to use. Another example of how sharing reagents moves science forward - a point that I discussed in the second article in this series, Finding Zika Virus (link).
Last modified on Thursday, 21 July 2016 17:35
Vincent Racaniello

Vincent Racaniello is a virologist at Columbia University and science communicator. He is using Zika Diaries to communicate the personal and behind the scenes experiences of his laboratory as it moves from working on poliovirus (for 35 years) to Zika virus.

1 comment

  • Comment Link emily Monday, 25 July 2016 14:59 posted by emily

    You should have asked if anyone had the 4G2 hybridoma cells to share! We have tons of aliquots in our LN2 and would have happily sent you some. Would have saved some time and money :)

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