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Development of monoclonal antibodies

5 female BALB/c mice (6-8 weeks old) are immunized intraperitoneally 5 times with 50-100mg of antigen followed by boosting. For weaker immunogens muramyldipeptide is used as adjuvant. (We have also experience with different kind of in vitro immunization procedures).

Fusion with Sp2/0 myeloma cells is performed by simple procedure. The myeloma cells (content of one 75 cm2 cell culture flask) and cells from immunized mouse spleen are washed 3 times in 50 ml conical tube by centrifugation (250 g for 10 min ) with PBS. The final pellet is mixed by tapping the tube and 1 ml of 50% PEG 4000 (Merck) is added over 1 min with gentle shaking. The cells are centrifugated 100 g for 5 min, the PEG solution is decanted and the cells are gently resuspended in HAT medium and seeded into 96-well cell culture plates (200 ml of cell suspension into each well). The plates are placed in a humid 37oC incubator gassed with 5,6% CO2 in air for 9-10 days. The cell culture medium is RPMI 1640, containing 15% FCS, 2% mouse macrophage cell line condensed medium, bicarbonate, and 30mM HEPES, tylosintartrate, b-mercaptoethanol and HAT (10-4 M hypoxantine, 1,6 x 10-5 M tymidine and 4 x 10-7 M aminopterin).

The clones are tested usually 9-10 days after fusion. The testing depends on which kind of antibodies you need . We can do ELISA screening of hybridomas both in solution, and on solid phase. If you wish to get monoclonals against a cellular antigen we can do screening both on different permeabilized and unpermeabilized cells ( skin fiboblasts, amniotic cells, neural cells differentiated from stem cells, etc.).

The positive hybridomas are cloned usually by limiting dilution in 96-well plates, and clones stored in liquid nitrogen.

To get larger quantities of purified monoclonal antibodies ascitic fluid and/or in vitro supernatant production is used.

Purification from ascitic fluid is performed by ammonium sulfate precipitation, ion-exchange chromatography with blue DEAE-Toyopearl 650S using Pharmacia FPLC system, and non-specific IgG2a and IgG2b antibodies are adsorbed to Protein-A Sepharose (Pharmacia). Specific antibodies are concentrated by ammonium sulfate precipitation and dialysed overnight against 0,9% NaCl.