Bulk Antibody Production by Ascites

A convenient way to obtain a high concentration of the desired monoclonal antibody (mAb) is to inoculate mouse ascites with suitable hybridoma cells. As a leading developer of antibody development, Creative Biolabs has years of experience in antibody discovery and bulk production, our scientific team can help you with every step of the development process, from design to validation. Our ascites production service will help you obtain high-quality antibodies with short turnaround timeline.

Ascites Production

Ascites are characterized by fluid that fills the abdominal cavity due to inflammation. The hybridoma cells are injected into the abdominal cavity to form ascites containing the desired antibody. The titer of mAb in ascites is usually more than 100 times higher than the culture supernatant, so it can be significantly diluted. Creative Biolabs has established a developed Hyperdoma™ platform, which is a high-throughput hybridoma generation and screening platform. With years of experience and professional technology, now we can provide the ascites production service for antibody bulk production. Our service mainly includes the following steps:

• Phase 1: Priming the Mouse

The mouse’s peritoneal cavity needs to be primed before hybridoma cells are injected. This promotes growth and antibody production in the hybridoma cells. Pristine is often used to induce immune reactions and prevent fluid from leaking out of the peritoneum. The mouse is then primed for two weeks prior to hybridoma cells being injected.

• Phase 2: Injecting the Hybridoma Cells

Hybridoma cells are grown in tissue culture prior to injection into a mouse for ascites preparation. The process is timed such that the cells are in the growth phase in tissue culture at the time during which they are injected. It is also important that the hybridoma is in the same genetic background as the host mouse to avoid host rejection. The number of cells that are injected ranges from hundreds of thousands to millions.

Fig.1 Historical methods of mAb production.1

• Phase 3: Incubation Time

Ascites fluid is allowed to accumulate for about one to two weeks before any is taken out. Care is taken to observe the condition of the mice daily. The ascites fluid is removed from the peritoneal cavity.

• Phase 3: Ascites Isolation

After extracting the ascites from the mice, it is spun down in a centrifuge. This process separates the lipid compartment from the cellular compartment to the bottom. In the middle is the aqueous compartment namely ascites. Fluid containing the antibody of interest can be collected over a series of draws ranging from 1 mg-30 mg per mouse, but more commonly 2 mg-10 mg depending on the hybridoma cell line

Scientific Needs for Mouse Ascites Production of mAb

Although in vitro techniques can be used for more than 90% of mAb production, it should be recognized that there are situations in which in vitro methods will be ineffective. Since hybridoma characteristics vary and mAb production needs are diverse, in vitro techniques are not suitable in all situations, and requiring their use might impede research.

1) Some hybridoma cell lines do not adapt well to in vitro conditions. Under such circumstances, the rising concentration of antibody might adversely affect hybridoma growth or secretion. MAb from mouse ascitic fluids might be essential for experiments in which mAb are used in mice. The need for the mouse ascites method arises when small volumes of concentrated antibody are needed for rapid screening in mice in order to select hybridomas with the desired bioreactivity.

2) Rat hybridoma cell lines do not generate ascites efficiently in rats, usually adapt poorly to in vitro conditions but generate ascites in immunocompromised mice.

3) Downstream purification can lead to protein denaturation and decreased antibody activity.

4) When a pure product is not necessary for research goals but maintenance of high affinity and biologic activity is necessary, the mouse ascites method often offers the best option.

5) Serum-free or low-serum conditions cannot provide sufficient amounts of mAb for some purposes.

6) Culture methods sometimes yield populations of IgG mAb that are glycosylated at positions different from those harvested from mouse ascites fluid, thereby influencing antigen-binding capacity and important biologic functions.

7) When hybridoma cells producing mAb are contaminated with infectious agents, such as yeasts or fungi, the cells often must be passed through mice.

Ascites production service at Creative Biolabs is available in nude and BALB/c mice, with a turnaround time of just 5-7 weeks. Our customer service representatives are available 24 hours a day. Please contact us for more information

Reference

1. Groff, Katherine, Jeffrey Brown, and Amy J. Clippinger. "Modern affinity reagents: Recombinant antibodies and aptamers." Biotechnology advances 33.8 (2015): 1787-1798.

All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.