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Protocols

Hybridoma Production

A hybridoma is a cell line arising from one hybrid cell that is capable of secreting a monoclonal antibody specific to one epitope of your antigen permanently in culture. The hybrid cell is produced through the fusion of specific antibody producing B-cell from an immunized animal (usually a mouse, rat or rabbit) and which has a finite lifespan, with a cell from an “immortal” cultured myeloma cell line (e.g. mouse NS-1 or NS-0).

Production of a mouse hybrid cell

During the fusion process, B cells are isolated from the mouse spleen, mixed with the mouse myeloma cell line and fusion is induced with polyethylene glycol (PEG, see Appendix I). (The relevant myeloma line is used when B cells from other animal species are used). The resulting hybridomas are then cultured in tissue culture medium containing Hypoxathine, Aminopterin, Thymidine (HAT), a step which kills any unfused myeloma cells that might outgrow the other weaker hybridoma cells. Unfused B cells have limited powers of division and will die off naturally in culture. Ten days after the fusion process, culture supernatant is collected and tested for the presence of the desired antibody.

Schematic representation of cell fusion

Equipment needed

Medium and other reagents (See Appendix A for more details)

Before you start (see Appendix A for more details)

Thawing and growth of the myeloma cells

Thaw the myeloma cell line and grow in medium A. Use the following method to thaw and culture the myeloma cell line.

The fusion process

Three days before - Prepare the myeloma cells for the fusion

The myeloma cells need to be in exponential growth phase when you use them and this needs experience. However if you set up two 75 cm2 flasks of your myeloma cells, one at a dilution of 1:40 and one at 1:60 (see below), 3 days before the fusion one of the flasks should be ideal on the day of fusion. (Initially setting up additional flasks at dilutions above and below the ones given here should provide you with the experience necessary judge the growth rate of the myeloma cells for subsequent fusions).

One day before - prepare the medium

The following need to be made and pre-warmed to 37ºC (you can put them in your incubator overnight).

Day of the fusion
Day after the fusion

Appendix I

Culture Medium A:
RPMI 1640 medium with L-Glutamine (bicarbonate buffered) (Lonza Ref. BE12-702F)
+ 10% FBS (Genycell Ref. GCS0101-500)
+ Penicillin (100U/ml)/Streptomycin (100mg/l) (Gibco Ref. 15070-063)

Culture Medium A+:
RPMI 1640 medium with L-Glutamine (bicarbonate buffered) (Lonza Ref. BE12-702F)
+10% FBS (Genycell Ref. GCS0101-500)
+ Penicillin (100U/ml)/Streptomycin (100mg/l) (Gibco Ref. 15070-063)
+ Ultroser G (1%) (Pall Ref. 15950-017)

Culture Medium B (no FBS):
RPMI 1640 Medium with L-Glutamine (Lonza Ref. BE12-702F)
+ Penicillin (100U/ml)/Streptomycin (100mg/l) (Gibco Ref. 15070-063)

Culture Medium C:
 RPMI 1640 Medium with L-Glutamine (Lonza Ref. BE12-702F)
+ 10% FBS (Genycell Ref. GCS0101-500)
+ Penicillin (100U/ml)/Streptomycin (100mg/l) (Gibco Ref. 15070-063)

Culture Medium D:
RPMI 1640 medium with L-Glutamine (bicarbonate buffered) (Lonza Ref. BE12-702F)
+ 10% FBS (Genycell Ref. GCS0101-500)
+ Penicillin (100U/ml)/Streptomycin (100mg/l) (Gibco Ref. 15070-063)
+ Ultroser G (1%) (Pall Ref. 15950-017)
+ HAT (Hypoxathine, Aminopterin, Thymidine) supplement which is usually 50X (dilute 10ml in 500mls of medium) (Gibco Ref. 21060-017)