outline the hybridoma method for the production of monoclonal antibodies

Hybridoma Method for Monoclonal Antibody Production – An Extended Cambridge Case Study

1. Why This Topic Belongs in the Cambridge Biology Syllabus

The hybridoma technique is a concrete example that links many of the core concepts in the Cambridge International AS & A Level Biology (9700) syllabus. It draws on:

• Topic 2 – Cell Structure & Organisation: protein structure of immunoglobulins (primary, secondary, tertiary).
• Topic 6 – Gene Expression & Regulation: transcription of antibody genes in B‑cells and myeloma cells.
• Topic 11 – Immunity: primary/secondary immune responses, active vs. passive immunity, artificial immunity, and vaccination principles.
• Practical Skills (Paper 3/5): use of selective media (HAT), cell‑fusion techniques, ELISA, and data analysis – all assessed under AO3.

Understanding hybridoma production therefore reinforces knowledge from several other syllabus sections while providing a real‑world laboratory context.

2. Antibody Structure & Function (≈150 words)

An immunoglobulin G (IgG) molecule is a Y‑shaped protein composed of two identical heavy (H) chains and two identical light (L) chains linked by disulphide bonds. The variable regions (V_H and V_L) form the tips of the Y (Fab fragments) and create a unique antigen‑binding site for each B‑cell clone. The constant regions (C_H and C_L) make up the stem (Fc fragment), determining the antibody class and mediating effector functions such as complement activation and binding to Fc receptors. In a hybridoma, every antibody molecule has the same V‑region (identical specificity) and the same Fc region (identical biological activity), giving a true monoclonal antibody.

3. The Immune Response Context

Primary vs. Secondary Responses

• Primary response: The first encounter with an antigen activates naïve B‑cells, which differentiate into plasma cells (antibody‑secreting) and memory B‑cells. Antibody titres rise slowly (≈5–7 days) and are mainly IgM, later switching to IgG.
• Secondary (memory) response: Re‑exposure to the same antigen triggers rapid proliferation of memory B‑cells, producing high‑affinity IgG within 1–2 days. This is the basis of vaccination.

Vaccination & Synthetic Antibodies

Vaccines present antigen (often with an adjuvant) to mimic a primary infection, establishing memory B‑cells without causing disease. Monoclonal antibodies derived from hybridomas can be used as synthetic vaccines – for example, a neutralising mAb can be administered prophylactically to block viral entry, or mAbs can be employed in neutralisation assays to evaluate vaccine‑induced immunity (e.g., anti‑SARS‑CoV‑2 spike mAbs used to test serum from vaccinated donors).

4. Step‑by‑Step Hybridoma Procedure

1. Immunisation of the Host

   • Inject purified antigen (often conjugated to a carrier protein such as KLH) into a mouse subcutaneously or intraperitoneally.
   • Give 2–3 booster injections at 2‑week intervals to expand the pool of antigen‑specific B‑cells.
   • 10–14 days after the final boost, the spleen contains a high frequency of Ig‑producing B‑cells (peak primary response).

2. Spleen Cell Harvesting

   • Euthanise the mouse ethically; aseptically remove the spleen.
   • Pass the spleen through a sterile cell strainer into RPMI‑1640 + 10 % heat‑inactivated foetal calf serum (FCS) + antibiotics.
   • Obtain a single‑cell suspension and keep on ice (≤4 °C) until fusion.

3. Fusion with Myeloma Cells

   • Use a non‑secreting, HGPRT‑deficient myeloma line (e.g., SP2/0‑Ag14, NS0, P3X63Ag8.653).
   • Mix spleen cells : myeloma cells at 5 : 1 to 10 : 1 in a 15 mL conical tube.
   • Add polyethylene glycol (PEG 1500, 50 % v/v) dropwise while gently stirring; PEG promotes membrane fusion.
   • After 30–60 min, dilute slowly with RPMI‑1640 to stop the reaction.

4. Selection in HAT Medium

   • Plate fused cells into 96‑well plates (≈200 µL/well) containing HAT medium:

     • Hypoxanthine 0.1 mM
     • Aminopterin 0.025 mM – blocks de‑novo purine/pyrimidine synthesis
     • Thymidine 0.1 mM – provides substrate for the salvage pathway

   • Only hybridomas (B‑cell + myeloma) retain functional HGPRT and survive; unfused myeloma (HGPRT‑) die, unfused B‑cells undergo apoptosis after 3–4 days.

5. Screening for Antibody Production

   • 7–10 days after plating, collect 50 µL supernatant from each well.
   • Test for antigen‑specific binding by ELISA (or immunofluorescence/lateral‑flow).
   • Record wells with a strong, specific signal as positive clones.

6. Cloning of Positive Hybridomas

   • Perform limiting‑dilution cloning (0.5 cell/well) in fresh HAT medium to isolate a single hybridoma cell.
   • Re‑screen the sub‑clones to confirm monoclonality and continued antibody production.

7. Expansion & Harvest

   • Scale‑up the confirmed clone in larger flasks or a bioreactor using serum‑free or low‑serum medium.
   • Harvest culture supernatant; typical yields 10–100 mg mAb L⁻¹.
   • Purify by protein A or protein G affinity chromatography, then dialyse into a storage buffer.
   • Store purified mAb at 4 °C (short term) or –20 °C (long term) with 0.02 % sodium azide as preservative.

5. Cambridge‑Style Practical Checklist (AO3 Mapping)

6. Risk Assessment (Biosafety & Chemical Hazards)

7. Important Considerations & Troubleshooting

• Myeloma line choice – must be HGPRT‑negative, EBV‑negative, and preferably non‑secreting (e.g., SP2/0‑Ag14, NS0, P3X63Ag8.653).
• Aseptic technique – laminar flow cabinet, sterile reagents, regular glove changes to avoid bacterial or mycoplasma contamination.
• HAT rationale – aminopterin blocks de‑novo nucleotide synthesis; only hybridomas with functional HGPRT can survive via the salvage pathway.
• Quality control – determine isotype, affinity (e.g., surface plasmon resonance), and endotoxin level before downstream use.
• Common problems

  • No hybridoma growth – check PEG concentration, verify myeloma HGPRT status, ensure HAT medium is fresh.
  • High ELISA background – increase washing steps, use 5 % milk or BSA blocking, confirm secondary antibody specificity.
  • Loss of production after cloning – re‑clone from an earlier passage, or screen multiple sub‑clones.

8. Applications of Monoclonal Antibodies

• Diagnostic – Pregnancy test

  • Monoclonal anti‑hCG antibodies are immobilised on a nitrocellulose strip.
  • Urine hCG binds the capture antibody; a labelled anti‑hCG mAb provides a visible line (lateral‑flow immunoassay).

• Therapeutic – Trastuzumab (Herceptin)

  • Humanised IgG1 mAb that binds the extracellular domain of HER2 on breast‑cancer cells.
  • Blocks receptor signalling and recruits immune effector cells via its Fc region, leading to tumour cell death.

• Research – Neutralisation assay

  • Anti‑SARS‑CoV‑2 spike mAb used to quantify neutralising activity of serum from vaccinated individuals.

9. Linking to the Cambridge Immunity Syllabus (Outcome 11.2)

10. Summary Flowchart (Suggested Diagram)