Cloning of hybrids

2.6 Cloning of hybrids

Antibody-secreting hybrid cells from positive culture wells must be cloned to ensure that the antibody is homogeneous and monospecific. Cloning is necessary to ensure that non-producers, arising either in the original fusion wells or as spontaneous variants, do not outgrow the antibody-secreting hybrids. If continuous growth of a hybrid line is required, it will be necessary to repeat the cloning and positive selection procedure at regular intervals.

Alternatively: prepare a large batch of cryopreserved cloned cells, and discard and replace growing lines at regular intervals. Cloning the initiation of a cell line from a single progenitor may be achieved either (a) in soft agar, (b) by limiting dilution, or (c) by using the continuous- flow cytometer (see also Section 8.5), but obviously the finer points of the technique are appara- tus-dependent.

2.6.1 Cloning in soft agar

This is the original method used by Köhler and Milstein but usually limiting dilution is now the method of choice.

Preparation of soft agar stock solution

MATERIALS Agarose (Sigma Type 1X-A)

Water, twice distilled

METHOD

1 Prepare a 2% w/v solution of agarose in twice-distilled water and dispense into glass bottles.

2 Autoclave at 120°C for 15 min and store at 4°C for use.

Cloning technique

MATERIALS AND EQUIPMENT Hybrid cells Agarose solution, 2% w/v, as above Tissue culture medium, double strength, with 20% serum 24-well culture plates Water bath at 44°C Microwave oven

METHOD

1 Melt the agarose in the microwave oven and allow it to equilibrate in a 44°C water bath. Similarly, equilibrate the tissue culture medium to 44°C.

2 Mix equal volumes of agarose and tissue culture medium, and return the mixture to the water bath. The agarose will solidify if this is not done rapidly.

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2.6CLONING OF HYBRIDS

3 Dispense 1 ml of the agarose tissue culture medium into each well of the tissue culture plate and allow it to solidify (subbed wells). Allow two cloning wells for each positive hybrid culture.

4 Count the hybrid cells and prepare suspensions at 2 × 10 3 cells/ml and 1 × 10 3 cells/ml.

5 For each cell suspension: mix 0.5 ml of cells with 1.0 ml of the agarose–tissue culture medium mixture.

6 Add 0.6 ml of the cell–agarose mixture to each of two subbed wells.

7 Repeat for all cells to be cloned.

8 Allow the agarose to solidify and incubate the plate in a humid incubator gassed with 5%

CO 2 in air. Cell colonies will grow within 1–2 weeks and will be visible as white spots in the agarose; each discrete spot represents an individual clone.

9 Pick off 10 discrete colonies per well using sterile Pasteur pipettes and transfer to separate 200 µl microcultures.

TECHNICAL NOTES • The underlay agarose is used to ensure that the cell colonies grow away from the well bottom.

This aids manipulation of clones during isolation. • Only discrete cell colonies must be isolated. • The cloning efficiency of this technique is usually between 20 and 70% (percentage of original

cells that grow as colonies). If growth is poor, a feeder layer of macrophages may be used, under the agarose.

• Not all of the colonies isolated will grow to produce lines of antibody-secreting cells. It is necessary therefore to screen and select for antibody activity. If the screening assay can be designed around the agar cloning plate, for example a modification of the Jerne plaque assay (see Section 9.1.1), it is possible to select antibody-secreting clones directly.

• The precision of cloning can be greatly enhanced by the direct selection of single hybrid cells from colonies growing in a primary fusion well detected as antibody positive. Cells can be picked by means of a micromanipulator and grown up either in agarose, as here, or in macrophage-supplemented microcultures.

2.6.2 Cloning by limiting dilution

The direct counterpart of the limiting dilution technique used to estimate the frequency of antigen-reactive lymphocytes. This is based on the same principle of random dispersion of rare elements, in this case hybrid cells, according to the Poisson distribution (see also Section 9.11).

Preparation in advance Prepare macrophage feeder layers in 96-well, flat-bottomed microculture plates. Allow one plate

for each positive hybrid well to be cloned. MATERIALS AND EQUIPMENT

Hybrid cells for cloning Microculture plates with macrophage feeder layers

Incubator, 37°C, humidified and gassed with 5% CO 2 in air

54 C H A P T E R 2: Monoclonal antibodies: production, purification and enzymatic fragmentation

METHOD

For each positive hybrid well:

1 Harvest and count the cells.

2 Prepare cell suspensions at 10 and 5 cells/ml.

3 Add 100 µl aliquots of the 10 cells/ml suspension to each of 48 wells. Repeat into remaining wells for the suspension at 5 cells/ml.

4 Incubate the plates in a humid 37°C incubator gassed with 5% CO 2 in air. Colonies should

be visible after 1–2 weeks.

5 Test supernatants for antibody activity and select positive wells for culture.

TECHNICAL NOTES • The initial distribution of cells per well follows Poisson statistics, thus although about 40% of

the wells will receive only one cell (and therefore initiate a true clone), a significant proportion will receive two or more cells. Cloning must be repeated to ensure the homogeneity of any interesting hybrid line.

• It is advisable to reclone both the plasmacytoma and hybridoma lines at regular intervals. This

will eliminate any variant cells, especially spontaneous non-secreting hybridoma variants, before they overgrow the culture.