The Monoclonal Antibody Faciliy (MAF) has been a part of the Bioexpression and Fermentation Facility since 2012 and can also assist with antigen production and purification.  The MAF works closely with the UGA Office of Animal Care and Use to provide top quality custom antibody development and production.  Following the standards of an ICH Q-10 system, the MAF can provide you with useful tools for research and quality documentation to support your publications, all while keeping your costs to a minimum.  The UGA facilities which house our research animals are fully AAALAC accredited and provide the best quality care for the best results.  Our services include:

  • Immunizing mice
  • Performing fusions
  • Cloning selected cell lines
  • Preserving these selected cell lines in liquid nitrogen
  • Producing and purifying antibodies from cell cultures

For further details please contact Ruth Davis at 706-542-1848 or

Monoclonal antibody discovery - $3,960.00 USD per Project

Services including immunizations, fusions and production of Hybridoma in cell culture. Five mice to be immunized and the highest titer mouse would be used for a fusion. The project includes immunizations, screening by ELISA, 1 fusion, screening of hybridomas and production of 2L of supernatant using the best clone selected.

Turnaround time: 6 months once the project initiated
Standard sample requirements: 2-4 mg of immunogen
Standard deliverables: purified antibody from 2L of culture


The Monoclonal Antibody Facility of the University of Georgia is available for any investigator who has a need for its services. Located in the Davison Life Sciences Complex, the facility is staffed by three full-time personnel.

The facility is equipped to immunize and monitor mice, perform fusions of mouse spleen cells and myelomas to generate antibody-producing hybridomas, isolate and propagate selected cell clones, preserve these cell lines in liquid nitrogen, and produce and purify quantities of antibody. The facility can produce antibodies to synthetic peptides, haptens coupled to carrier proteins, fusion proteins, and more conventional antigens.

The hybridomas resulting from the fusions are screened for antibody production by ELISA. Then, the selected hybridomas are propagated in tissue culture flasks by in vitro techniques rather than using additional mice to produce ascites fluid.


The following is a summary of the procedures performed by the Monoclonal Antibody Facility. More detailed information is available upon request.


Mice are immunized with the antigen of choice to provide a source of antibody-secreting spleen cells. Complete and Incomplete Freund's Adjuvant is routinely used to maximize the chances of a successful result. Mouse serum titers are analyzed to evaluate the success of the immunization series and the effectiveness of the screening assay. Serial dilutions of serum from the immunized mice are tested by ELISA to determine which individual is the best candidate to provide spleen cells for the fusion. 

All needed materials (syringes, common adjuvants, diluents, etc.) are stocked in the facility. Routine immunizations require approximately six months. Some immunogens will require alternative and possibly longer immunization series.


Antibody-secreting spleen cells from the selected immunized mouse are fused with immortalized myeloma cells to produce the hybridoma. The SP2/0 myeloma cell line is used. Proper planning for the fusions is critical. A fusion is performed when the immunized mice have an adequate antibody titer. The screening assay is perfected during serum testing, as it must be developed in advance of the fusion in order to screen the hybridomas produced. 


Because the spleen contains cells that produce a variety of antibodies, the hybridomas resulting from the fusion must be carefully screened to select those producing the antibody of choice. An ELISA test is the most commonly used test for screening hybridomas for antibody production. The Monoclonal Antibody Facility provides this service, but screening may also be performed by the investigator's laboratory.

As hybridoma screening must be done quickly and unambiguously, the screening protocol must be established and agreed upon before the fusion. Hybridoma medium to be tested will be provided early in the morning for those local laboratories performing their own screening. In the case of an ELISA assay, the test samples can be added directly to wells pre-coated with antigen. If an investigator is performing his or her own screening, the test results must be available to the facility by the afternoon following obtaining the samples.

Maintenance of large numbers of hybridomas, once they begin to require individual attention, is laborious and time-consuming. Each of the several hundred cell lines must be cultured independently so that cross-contamination of cell lines does not occur. It is therefore essential that screening be completed before this situation arises. Consequently, screening assays must be performed in a timely fashion. Laboratories performing their own screening assume additional responsibilities since any delays will require assessment of additional fees on an ad hoc basis. Moreover, the longer it takes to identify useful cell lines, the greater the probability that one or more such lines will be lost.

Cloning, Expansion, and Preservation

Once a useful cell line is identified, it is cloned and expanded. During the expansion stage, the cell lines are screened regularly for antigen binding properties. Eight vials of frozen cells will be prepared for long term storage in a liquid nitrogen Dewar. These frozen cells are maintained by the facility. One vial of the frozen cells will be thawed and checked for viability and antibody production. The others will be stored until needed. The facility will typically provide one liter of spent hybridoma medium containing from 10 to 20 g per ml of antibody. 

Concentration and Purification

Spent tissue culture fluid from the hybridomas contains the antibody. These fluids can be further concentrated by precipitating with saturated ammonium sulfate (SAS) and purified by affinity column chromatography. The facility is equipped to perform both SAS precipitation and affinity chromatography with Protein G columns. Isotyping assays and testing of purified antibody can also be performed, for an additional fee.

How The MAF Works

The Monoclonal Antibody Facility uses only female BALB/c mice to make monoclonal antibodies to be used in three general ways: to purify reagents for tests and research, as labeling agents for detection assays, for experimental therapy.

Below is an outline of the process and procedures taken at MAF

What In the World Is a Monoclonal Antibody?

Mono: One
Clone: A strain of cells descended from a single cell
Antibody: A molecule of animal origin that has immunological activity only against the antigen to which it was made

OK, so what’s an ANTIGEN?

Antigens are soluble substances
They may be toxins or foreign proteins
They may be whole cells OR just parts of a cell, like a component of the cell wall
Proteins usually make the best antigens
Very small proteins (peptides) can be coupled (attached) to carriers so they make better antigens

How does the MAF make a monoclonal antibody?

Immunize the mice - Housing

Our lab uses only female BALB/c mice to make monoclonals.
The mice are housed in a laminar flow hood to minimize their exposure to other antigens.
Each box contains mice for only one project.

Immunize the mice - Identification

Each mouse has a microchip implanted to identify it.
Each time the mouse is inoculated or bled, the chip is checked.

Immunize the mice - Inoculation

The mice are aseptically inoculated with the antigen combined with an adjuvant.
Inoculations are done either subcutaneously or intra-peritoneally.
Normal dose per mouse is between 50 and 100 micrograms of protein.
Inoculations are performed every 21 to 28 days.


Test the serum - Bleeding the mice

A capillary tube is applied to nicked vein.
Blood is drawn into the tube.

Test the serum - In the Lab

Whole blood from the immunized mice is transported to the MAF lab.
The whole blood is spun down in a centrifuge to separate the serum.
The serum is diluted in a series from 1:300 to 1:300,000.

Test the serum - ELISA

Diluted serum is tested by an ELISA (Enzyme Linked ImmunoSorbent Assay)
Color change in the ELISA substrate indicates the amount of antibody present in the diluted serum.

Test the serum - Decision time

When the serum titer of the mice has reached a plateau, an additional ELISA test is performed to determine the predominant isotype present. The two isotypes that are most common in mouse serum are IgG and IgM.
A fusion is done when the IgG level is high and the IgM level is low.
Sometimes additional testing is done (Western blots, immunofluorescence) to determine whether the serum response is specific for the selected antigen.
The mouse with the strongest, most specific response is chosen for the fusion.


Perform a fusion - Prepare spleen cells

Anesthetize the best responder among the immunized mice.
Perform a cardiac puncture and withdraw whole blood from the mouse.
Euthanize the mouse.
Aseptically open the mouse’s abdomen.
Dissect away the peritoneal membrane to expose the spleen.
Carefully remove the spleen to a sterile petri dish.
Make a single cell suspension of the spleen.

Perform a fusion - Hybridomas

Dilute the spleen cells.
A special line of tumor cells (SP 2/0) has already been grown in a flask.
Mix the spleen cells with the tumor cells.
Add a chemical agent to the mixture to “soften” the cell membranes.
Place the now-fused spleen/tumor cells, called hybridomas, on top of the feeder cell layer in the 96-well plate.

Perform a fusion - Growing the cells (hybridomas)

Fusions are performed in a biological safety cabinet.
Cells are grown in 96-well plates.

Perform a fusion - Growing the cells (hybridomas)

Cells are grown in a 37o C incubator.
Cells are kept in an atmosphere of about 6% CO2.
The cells are fed after 7 days of incubation.
The cells are checked for growth after 10 days of incubation.


Screen the fusion for the right cells

When growth is detected in one of the wells of the 96-well plate, cell supernatant from that well is tested by ELISA.
If the well tests positive, cells from it are removed and diluted. The diluted cells are plated into new 96-well plates at a dilution that will contain only one cell per well.
The wells are tested again and positive wells are re-diluted. This process is called cloning.
The object of cloning is to obtain a population of identical cells all producing the same antibody - monoclonal cells.


Grow the hybridomas - 24-well plates

Once a monoclonal population of cells is obtained, they are expanded to produce the desired amount of antibody.
The antibody is produced by the cells and released into the cell supernatant.
First, the monoclonal cells are expanded into 24-well plates.

Grow the hybridomas - Flasks

Then, when the cells in the 24-well plates are growing well and look healthy, they are transferred into flasks.
Cells in the flasks are expanded, as needed.
Some cells are preserved for future use by freezing.

Harvest the antibody

The antibody is produced by the cells and released into the cell supernatant.
Media is added to the flasks until the desired volume of antibody-containing supernatant is obtained.
When the desired volume is reached, the supernatant is refrigerated and tested by ELISA.
If the testing is successful, the supernatant can be used by the investigator, or concentrated and purified.

Concentrate and Purify the Product

The antibody can be concentrated by precipitation with a solution of saturated ammonium sulfate.
Concentrated antibody can then be purified by passing it through a column.

So, why make monoclonal antibodies anyhow?

Monoclonal antibodies can be used in three general ways:

  1. to purify reagents for tests and research
  2. as labeling agents for detection assays
  3. for experimental therapy.

More specifically, monoclonal antibodies can:

  • Be attached to a color agent of fluorescent chemical for immunological staining.
  • Be attached to a medium in a column to purify other substances that will bind to them (affinity purification).
  • Coat plates for ELISA testing, as for a diagnostic test.
  • Be used as therapy by attaching to a particular target cell and marking it for lysis by natural killer cells or medicines
  • Be radiolabeled (attached to an isotope) for diagnostic imaging such as PET (Positron Emission Technology)