The Hybridoma Laboratory is a part of the University of Florida Interdisciplinary Center for Biotechnology Research. The main service provided by this laboratory is development of new mouse monoclonal antibodies for investigators at the University of Florida. The laboratory performs ELISA and western blot screening of antibodies. In vitro production and purification services are also of- fered. The purchase of an Octet QK system in December of 2008 has enabled several new services to be added such as antibody quantita- tion, affinity ranking and determination of antigen : antibody binding constants.
Antibody Quantitation Applications
Customers often request hybridoma supernatants for various appli- cations such as ELISA, western blotting and immunohistochemistry. Prior to the acquisition of the Octet system, there wasn’t an ef- ficient way to determine the concentration of mouse antibody in a hybridoma supernatant containing serum. Currently, a two minute quantitation experiment using Anti-Murine IgG Fv biosensors is all that’s needed to generate this important information. Typically an isotype matched saved standard curve is used to analyze the data.
Quantitating the amount of mouse IgG present in supernatants from single-colony wells from hybridoma cloning projects allows for selection of the highest secreting cell lines for final cell banking. When testing culture reagents such as serum supplements or other types of growth factors, quickly analyzing the antibody concentra- tion is an important criterion. Prior to obtaining the Octet system it was necessary to perform small scale affinity purifications of each sample to determine the concentration of antibody.
Most antibody production and purification performed in this core laboratory is ‘research’ scale. Customers typically request 5–20 milligrams of purified antibody. High density Cel-Line flasks are used for most production jobs. The antibody production media is supplemented with low IgG fetal bovine serum. Harvests are obtained from the cell/antibody chamber approximately once per week. The concentration of mouse antibody is determined periodi- cally using the Octet system throughout the run to determine how many harvests are needed to obtain the desired amount of purified antibody. The Cel-Line harvests are pooled prior to purification.
Protein G is the most frequently used affinity matrix in this labora- tory. Octet system quantitation of the pre- and post-purification samples provides critical information regarding the efficiency of the purification process. If a substantial amount of antibody remains in the post-purification sample, it may be run over the Protein G col- umn a second time in order to optimize the yield. Prior to obtaining the Octet system, a second Protein G column run was performed au- tomatically, but often the yield from the second run was negligible.
Often a fusion experiment yields a panel of a dozen or more hybridoma cell lines secreting antibodies that score positive in vari- ous assays. Investigators may only need one or two antibodies for their particular application, and it can be difficult to decide which lines to clone based on antibody screening results that may be very similar for all the cell lines.
Using the Octet system to rank affinities by comparing off rates is a very valuable tool. Biotinylated antigen may be loaded onto a Streptavidin Biosensor or unlabeled antigen may be loaded onto an Amine-Reactive Biosensor. The biosensors can then be dipped into crude hybridoma culture supernatant. Comparing the k dis from the panel of crude supernatants allows investigators to make a more informed choice about which antibody to pursue.
Figure 1 shows results from an affinity ranking experiment. The k assoc is not determined since the analyte concentration is un- known. By comparing the k dis , it is clear that 5C11 and 1E3 have the lowest k dis and therefore are likely higher affinity antibodies. These same hybridoma supernatants were tested on an indirect ELISA. 5C11 gave the strongest signal. The other supernatants also gave strong readings. ELISA readings may be influenced by the antibody concentration in the supernatant: a higher antibody concentration may result in a stronger signal, which may or may not reflect the antibody affinity.
The Octet system is also used to determine K D (binding constants). Once a monoclonal antibody has been developed and purified anti- body is available, a complete kinetics experiment can be performed on the Octet system to determine the antibody:antigen affinity. The antigen can be labeled with biotin and loaded on Streptavidin (SA) biosensors. The biosensors can then be dipped into several different concentrations of purified antibody. The reverse experiment is also possible: loading biotin labeled antibody on the SA biosensors and dipping into several concentrations of purified antigen.
Determining the antibody:antigen affinity allows more complete characterization of antibodies. Figure 2 shows an example of a kinetics experiment using biotinylated antigen-loaded SA biosen- sors dipped in purified antibody. Global fitting of binding data for three concentrations of the antibody measured in duplicate resulted in a measured association rate constant of 2.1E5, dissocia- tion rate constant of 7.65E-5 and affinity constant of 3.64E-10.
Octet Service Delivery
There are several modes that customers can choose from for Octet service in this core laboratory:
Self service: customer pays an instrument fee based on elapsed time the instrument is used (this will help pay for the service con- tract) plus customer pays for the cost of expendables (biosensors, biosensor plate, sample plate, buffers, etc.).
Staff assisted: customer pays for self service charges plus an ad- ditional fee for some staff assistance.
Complete service: customer does not participate in the experi- ment. The complete service charge includes instrument time, expendables and staff time.
To date, several self-service users have been trained to use the Octet system. Most users are able to run their first kinetics experiment as a part of the two-hour training. Typically users are able to run repeat experiments on their own without further assistance. Binding constants are routinely obtained within one day, including the initial experiments which may be required to optimize conditions.
This facility also has a Biacore 3000 which requires more extensive training. The time required to develop optimum conditions is typi- cally longer than with the Octet system. Since the biosensors are so inexpensive, most users choose not to regenerate, which saves a lot of time. Scouting for regeneration conditions on the Biacore is very time consuming.