In a previous blog post, we provided An Introduction to Ascites Production. Here we are going to expand on that post by discussing some of the reasons you would chose ascites production in mice for production of monoclonal antibodies. Briefly, after an immunogen is injected into a mouse, a cell line is harvested from the spleen which is capable of producing a single antibody. This cell line is fused with a tumor cell line and thus immortalized, creating a hybridoma.

Picture of a human melanoma cell line growing in tissue culture

Unfortunately, no method of creating hybridomas or monoclonal antibodies is completely void of using an animal. However, hybridomas are only capable of producing antibodies. To accomplish this, you have to use an in-vitro or in-vivo expression system. In-vitro production of monoclonal antibodies is very good for most cases, but there are times where in-vivo monoclonal antibody production, or production of a monoclonal via ascites is necessary. This is what we will outline here.

There are some hybridomas which do not express well in an in-vitro system, and thus need to be used in ascites production for successful antibody expression. This is well documented [1,2] and there are many hypotheses as to why this is the case. For these cell lines, the only method of creating the monoclonal antibody is to use ascites production in mice. Antibodies which are produced for vaccines against infectious organisms cannot be raised in high concentrations with cultures that are serum-free or have low-serum. With many cell lines, as the serum levels are decreased, the amount of antibody produced decrease dramatically as well [3].

Also, mouse ascites fluid is preferable when high affinity and/or biological functionality is important, but the monoclonal antibody can have some other proteins present. Both in-vitro and ascites production yields antibodies which are not pure and need to be purified, ascites produces antibodies at a very high concentration. Because of this, the highly concentrated antibody can often be used without downstream purifications which can lead to denaturing of the antibody and decreased antibody reactivity. Purification of in-vitro produced antibodies can result in decreased antibody binding efficiency [4,5]. A great example of this is monoclonal antibodies raised against OKT3, which has vast therapeutic implications, cannot be expressed in-vitro and maintain antigen binding after purifications, so it must be produced using ascites fluid [6].

With regards to structure and function, antibodies which are produced via in-vitro or ascites can also have very different glycosolation patterns, resulting antibody-antigen binding and function [7]. Sometimes this change can be of no effect. However, in one case an IgG monoclonal antibody was documented to contain terminal mannoses at all glycosolation sites [7].

Additionally, ascites production may be necessary because of how the antibodies will be used in downstream experiments. Monoclonal antibodies from ascites might be required if the downstream experiments will be performed in mice. This is especially true for rapid screening of mouse for hybridoma selection, where a very small volume of very high antibody concentration is required. Also, if you are using a rat hybridoma cell line, you cannot produce ascites in rats. Rat hybridomas must be used in immunocompromised mice for ascites production [8].

Abcore recently finished expanding our Southern California laboratory by constructing a state of the art, brand new ascites production facility. We can handle individual projects using just a few mice, or we have a capacity of over 4,000 mice for larger industrial projects. Contact ustoday for more information on development of your hybridoma cell lines using ascites fluid in mice.

 

  1. Varmus, H. Response to the petition requesting the National Institutes of Health to prohibit the use of animals and implement non-animal alternatives in the production and use of monoclonal antibodies. Appendix C. Sept. 18, 1997; Bethesda, MD.
  2. Hendriksen, C., J. Rozing, M. VanderKamp, W. deLeeuw. The production of monoclonal antibodies: Are animals still needed? ATLA 1996 24:109–110.
  3. Chandler, J. In Testimony Before the National Research Council’s Committee on Monoclonal Antibody Production. November 10, 1998: Washington, DC.
  4. Underwood PA, Bean PA. The influence of methods of production, purification and storage of monoclonal antibodies upon their observed specificities. J Immunol Methods. 1985 Jun 25;80(2):189-97. PubMed PMID: 4008939.
  5. Lüllau E, Heyse S, Vogel H, Marison I, von Stockar U, Kraehenbuhl JP, Corthésy B. Antigen binding properties of purified immunoglobulin A and reconstituted secretory immunoglobulin A antibodies. J Biol Chem. 1996 Jul 5;271(27):16300-9. PubMed PMID: 8663142.
  6. Stein, K. In Testimony Before the National Research Council’s Committee on Monoclonal Antibody Production. November 10, 1998; Washington, D.C.
  7. Leibiger H, Hansen A, Schoenherr G, Seifert M, Wüstner D, Stigler R, Marx U. Glycosylation analysis of a polyreactive human monoclonal IgG antibody derived from a human-mouse heterohybridoma. Mol Immunol. 1995 Jun;32(8):595-602. PubMed PMID: 7609736.
  8. Wolf, M. CL6-well experimental screening device application: Murine and rat hybridoma. 1998 CELLine Technical Report IV.