outline the hybridoma method for the production of monoclonal antibodies

Hybridoma Method for Monoclonal Antibody Production

1. Overview

The hybridoma technique, developed by Köhler and Milstein, allows the production of large quantities of a single type of antibody (monoclonal antibody). It combines the antibody‑producing capacity of B‑lymphocytes with the immortality of myeloma cells.

2. Key Steps

1. Immunisation of the Host Animal

   • Inject the antigen of interest into a mouse (or other suitable species).
   • Administer booster injections to enhance the antibody response.
   • After 10–14 days, the spleen contains a high number of antigen‑specific B‑cells.

2. Spleen Cell Harvesting

   • Euthanise the animal and remove the spleen aseptically.
   • Prepare a single‑cell suspension in a suitable medium (e.g. RPMI‑1640 with 10 % FCS).

3. Fusion with Myeloma Cells

   • Choose a myeloma cell line that is deficient in hypoxanthine‑guanine phosphoribosyltransferase (HGPRT‑).
   • Mix spleen cells and myeloma cells at a ratio of 5:1 to 10:1.
   • Add polyethylene glycol (PEG) to induce membrane fusion.
   • Incubate for 30–60 min, then dilute with medium to stop fusion.

4. Selection of Hybridomas

   • Plate the fused cells in HAT medium (hypoxanthine‑aminopterin‑thymidine).
   • Only hybrid cells (HGPRT‑ myeloma + HGPRT+ B‑cell) can survive.
   • Non‑fused myeloma cells die; non‑fused B‑cells undergo apoptosis.

5. Screening for Antibody Production

   • After 7–10 days, collect supernatants from each well.
   • Test for specific antibody activity using ELISA or other binding assays.
   • Identify wells producing the desired antibody.

6. Cloning of Positive Hybridomas

   • Perform limiting‑dilution cloning to isolate a single hybridoma clone.
   • Confirm monoclonality by re‑screening supernatants.

7. Expansion and Harvest

   • Grow the selected clone in larger flasks or bioreactors.
   • Collect culture supernatant containing the monoclonal antibody.
   • Purify the antibody by protein A/G affinity chromatography.

3. Important Considerations

• Use of an appropriate myeloma line (e.g. SP2/0, NS0) that is HGPRT‑ and non‑immunogenic.
• Maintain aseptic technique throughout to avoid contamination.
• Regularly test for mycoplasma and other pathogens.
• Store purified antibodies at 4 °C for short term or –20 °C for long term.

4. Summary Table

5. Suggested Diagram