Application Note

Rapid isolation of antigen-specific clones from hybridoma fusions

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Screening a hybridoma fusion and then subcloning candidate hybrids are time-consuming elements of the monoclonal antibody discovery process. The Genetix ClonePix FL provides a powerful way to fi nd and collect antigen-specifi c clones in a rapid one-step process. The technology works by automatically screening the entire fusion in situ and isolating only antigen-specifi c clones with the desired immunoglobulin isotype. If the fusion has many antigen-positive clones, then ClonePix FL can selectively collect only the best secretors. The gentle nature of the technology ensures high viability of the selected clones (90-100%). By eliminating traditional methods and minimising the man-hours required, ClonePix FL not only shortens the process timeline, but also permits parallel interrogation of multiple antigens.

Plating hybridomas into methylcellulose-based semi-solid medium was fi rst described 25 years ago1 . Using this method, Genetix has developed a technology that traps, detects and measures the relative secretion of monoclonal antibody from the hybridomas (or transfected cells such as myelomas). While the clonal colonies grow suspended in semi-solid medium, the secreted antibody is immobilised in the vicinity of the colony by isotype-specifi c Complex Initiation Factor and specifi c clones are then detected by probing with fl uorescent antigen. The principle is summarised in Figure 1. Up to fi ve fl uorescence wavelengths can be multiplexed with white light images thus allowing multiple probes to be used in a single picking run, or to measure other parameters such as cell viability (using Genetix LiveDetect reagent).

Figure 1. Principle of ClonePix FL technology for detecting antigen-specifi c clones. Single hybridoma cells grow into clonal colonies in semi-solid medium. Secreted monoclonal antibody is trapped in the immediate vicinity of the colony by Complex Initiation Factor (CIF). CIF is specifi c for IgG, so only IgG-secreting clones will develop the secretion complex. Fluorescently conjugated antigen targets only those IgG-secreting clones secreting antigen-specifi c antibody.

Selection of antigen-specifi c clones

Following fusion, it is recommended that the hybridomas are allowed 24 hours to recover before being plated out. The fi rst step is to prepare the semi-solid medium with all components required. For convenience, CloneMedia semi-solid medium containing serum and cloning supplements is available in 100ml quantities, which is ideal for one fusion. Alternatively, semi-solid medium can be made up by adding a 2× medium concentrate of your choice to CloneMatrix. Prior to addition of cells, supplement the medium with any additional components (such as antibiotics and HAT selection agents), and then add 100U/ml Complex Initiation Factor and your fl uorescently-conjugated antigen of interest. The concentration of antigen required is dependent on its properties (molecular weight, dye-protein ratio). As a general guideline, Genetix recommends 0.1-10μg/ml but this should be determined empirically. After gentle but thorough mixing, the whole fusion can be added. The cells can be evenly distributed by rotating the bottle, and then the contents plated out. Genetix PetriWell-6 and PetriWell-1 plates are designed specifi cally for this application to minimise the autofl uorescence and fl aring that occurs when regular culture plates are imaged. The plates also contain reservoirs to place a small volume of sterile water to minimise evaporation during incubation.

After 7-8 days incubation, a plated fusion should generate at least 3000 discrete, HAT-selected hybrid colonies of 100-500 cells each. Using ClonePix FL, the fl uorescing clones (in other words, the clones that are secreting antigen-specifi c IgG; see Figure 2) are automatically targeted for collection and then picked into separate wells of a 96-well destination plate containing the medium of choice for outgrowth (for example, XP Media). The software excludes any clones that are too close to each other (by white light), thereby preventing non-clonality. The user has the option to review and adjust the parameters of the selected clones prior to picking.

If there is concern about the stability of the fl uorescent antigen during the growth period, or if unfused B-cell fl uorescent accumulation is an issue, one or both of the probes can be added after colony growth by using an atomiser applicator 24 h before imaging and picking the colonies.

Figure 2. ClonePix FL selectively detects hybrids secreting antigen-specifi c IgG. Cells were plated and treated as described in Fig. 1 using a FITC-conjugated 60kD bacterial antigen. A. White light image. B. Fluorescent image (excitation 470 +/- 20nm; emission 535 +/- 25nm). Only clones secreting antigen-specifi c IgG accumulate local fl uorescence. Specifi city confi rmed by antigen-specifi c ELISA (not shown).

Selection of highest secreting antigen-specifi c clones

The above method is designed for fi nding rare antigen-positive hybrids. Where a fusion is expected to have many antigen-positive clones, ClonePix FL can selectively collect only the best secretors. In a multi-fl uorescence variation of the above method, Complex Initiation Factor can be replaced by CloneDetect anti-mouse detection agent, which provides a superior indicator of clone production rate. The CloneDetect agent is a fl uorescently-conjugated complex initiation probe specifi c for IgG and is available with a range of fl uorophores. In this case, the fl uorescent antigen will fi nd the antigen positive clones, and the CloneDetect will quantify the amount of IgG secreted.

Selection of highest secreting IgG-specifi c clones

If it is not possible to add a fl uorescently conjugated antigen, for example, if the antigen has poor solubility, the assay can still be run using just CloneDetect reagent. Under these conditions, only clones producing IgG isotype monoclonal will be detected and collected, thus eliminating IgM or non-secretors.

Subcloning

Subcloning is not normally required, but can signifi cantly improve antibody production. Using ClonePix FL, the whole process can be undertaken in one week by transferring the contents of a picked well into a small amount of semi-solid medium (2-4ml) containing CloneDetect reagent and re-plating. After 5-7 days, the subclones can be re-imaged and the highest secretors collected. The collection of a clonal colony rather than a single cell ensures rapid expansion. This simple procedure is also valuable for resurrecting cultures that are failing to secrete (Figure 3).

Figure 3. Recovering a failing hybridoma cell line. A poor producing cell line was screened on ClonePix FL, which showed broad heterogeneity of secretion between subclones at day 5 (A). The highest fl uorescing clones were picked by ClonePix FL, and subsequent re-plating showed good subclone fl uorescence (B).

Conclusions

Using ClonePix FL technology removes the need for serial dilution and overcomes the limitations of cell-sorters by directly detecting and measuring antibody secretion in situ. It enables monoclonal populations of antigen-specifi c, high secreting, IgG isotype hybrids to be established in a much shorter time frame, removing many of the bottlenecks associated with traditional methods. We have validated the technology for a broad range of antigens from a 160kD multimeric protein to a 2.6kD phosphopeptide. The low labour requirement means that more hybridoma fusions can be interrogated in parallel increasing cell line throughput. It is estimated that two scientists using a ClonePix FL should be able to process up to 100 fusions per year.

Reagents and consumables

To support this application, Genetix have developed an extensive range of optimised media, fl uorescently conjugated detection agents and cell culture plates.

For further information please contact the Genetix team at info@genetix.com.

References

1. Davis, J.M., Pennington, J.E., Kubler, A.M. & Conscience, J.F. A simple, single-step technique for selecting and cloning hybridomas for the production of monoclonal antibodies. J. Immunol. Methods (1982) 50, 161–171.

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