What is Ascites?

 

Ascites is the term that describes the fluid that fills the peritoneal cavity due to inflammation. For in vivo monoclonal antibody production, hybridoma cells are injected into the peritoneal cavity to resulting in ascites fluid containing the desired antibody.

Ascites Production

One advantage of making ascites antibodies is that it is quick and an alternative when in vitro is not an option. One disadvantage is that it is “dirty,” because cells, cellular debris, lipids, and other proteins are also secreted into the intraperitoneal cavity. To overcome this limitation, more downstream processing is required. Raw ascites fluid is spun down by centrifugation to isolate the aqueous proteins from the lipids and the cell debris and purification.

How Much Antibody is in Ascites?

Ascites fluid can contain antibodies at concentrations that range from 1-3mg/ml. This range is higher than what is secreted by hybridoma cells that do not express well in an in-vitro system. However, since ascites also contains antibodies that were produced in the mouse that bind other antigens, the titer for each batch of ascites should be determined.

What Applications are Ascites Antibodies Used For?

Ascites antibodies are useful for the same assays as antibodies produced through animal-free methods. Routine applications include western blotting, immunohistochemistry, immunocytochemistry, and ELISA. Since ascites contains proteases and would degrade; purification is highly recommended before proceeding with any assay. In addition, because ascites antibodies are a mixture of the desired antibodies and nonspecific antibodies from the host, low specificity may be an issue. To improve signaling, we recommend extra blocking and washes steps and incubating the sample at 4°C for 12-16 hours.

What are the Disadvantages of Ascites Antibodies?

The disadvantage of making antibodies through the ascites is that it requires animals. The mice need daily observation and must be kept in a clean facility otherwise they risk of contamination from other biological agents such as viruses, bacteria, or mycoplasma.