Hybridoma Technology

1. Historical Background

2. Principle

3. Step-by-Step Process

Step 1 - Immunization

Step 2 - Selection of Myeloma Partner

• Cannot produce its own antibody (ensuring the only antibody produced by the hybridoma comes from the B cell partner)
• Lacks the enzyme HGPRT (hypoxanthine-guanine phosphoribosyltransferase) - this makes it unable to survive in HAT selection medium

Step 3 - Cell Fusion

• Unfused spleen cells
• Unfused myeloma cells
• Fused hybridoma cells (spleen + myeloma)
• Cell-cell fusions of same type (spleen+spleen, myeloma+myeloma)

Step 4 - HAT Selection (The Key Step)

• Immortality from the myeloma
• HGPRT (and thus HAT-resistance) from the B cell

Step 5 - Cloning and Screening

• ELISA
• Radioimmunoassay
• Immunofluorescence

Step 6 - Monoclonal Antibody Production

• Bulk cell culture - in vitro, large bioreactors
• Ascites fluid - injected into the peritoneal cavity of mice (produces high-titer ascites; less common now due to ethical concerns)

4. Why Monoclonal Antibodies Are Superior to Polyclonal Antisera

• Janeway's Immunobiology 10e

5. Limitations and Evolution of the Technology

Problem: Mouse Origin = Immunogenicity (HAMA)

• Neutralized or rapidly cleared the injected MAb
• Could cause serum sickness

Solution: Engineering More Human Antibodies

• Chimeric antibodies: mouse variable regions + human constant regions
• Humanized antibodies: only mouse CDRs (complementarity-determining regions) grafted onto human IgG framework
• Fully human: generated via phage display libraries or transgenic mice expressing human antibody genes
• Tietz Textbook of Laboratory Medicine, 7th Edition

6. Applications of Monoclonal Antibodies

Diagnostics

• Immunoassays: ELISA, RIA, Western blot, flow cytometry using MAb-based reagents
• Pregnancy tests: detection of hCG
• Blood typing: anti-ABO, anti-Rh reagents
• Tumor markers: detection of PSA, CEA, AFP
• CD marker identification: defining lymphocyte subsets (CD4, CD8 T cells)
• Histopathology: identifying tissue of origin of tumors

Therapeutics (Selected Examples)

• Cellular and Molecular Immunology (Abbas), Katzung's Pharmacology 16e

Research Tools

• Purifying cell populations (cell sorting)
• Blocking or depleting specific molecules/cells to study their function
• Structural studies of receptors and signaling molecules

7. Nomenclature of Monoclonal Antibodies (INN Stem System)

Summary

Hybridoma Technology - Summary

Core Steps

1. Immunize a mouse with the target antigen
2. Harvest spleen cells (B cells secreting specific antibody)
3. Fuse B cells + HGPRT-deficient myeloma cells using PEG
4. Select in HAT medium - only hybridomas survive (B cells die naturally; myeloma cells die without HGPRT)
5. Clone by limiting dilution - one cell per well
6. Screen supernatants for desired antibody specificity
7. Expand positive clones → bulk MAb production

Key Concept: Why HAT Medium Works

Generations of MAbs (Reducing Immunogenicity)

Applications

• Diagnostics: ELISA, pregnancy tests, tumor markers, blood typing, flow cytometry
• Therapeutics: Anti-cancer (rituximab, trastuzumab), anti-inflammatory (adalimumab, infliximab), checkpoint inhibitors (nivolumab, pembrolizumab)
• Research: Cell sorting, receptor studies, functional blocking assays

One-Line Takeaway

A hybridoma = immortal myeloma cell + antibody-producing B cell, selected by HAT medium, cloned, and screened to yield an unlimited supply of one specific antibody.

Hybridoma Technology - Summary with Diagram

Process Diagram

Step-by-Step Summary

Immunize mouse with Antigen X
          ↓
  Harvest spleen B cells
  (some produce anti-X antibody)
          ↓
    Mix with Myeloma cells
  (HGPRT⁻, no antibody secretion)
          ↓
     Fuse using PEG
          ↓
  Culture in HAT medium
          ↓
  ┌───────────────────────────┐
  │ Unfused B cells → DIE     │  (finite lifespan)
  │ Unfused myeloma → DIE     │  (no HGPRT)
  │ Hybridomas → SURVIVE ✓   │  (immortal + HGPRT)
  └───────────────────────────┘
          ↓
  Clone by limiting dilution
  (1 cell per well)
          ↓
  Screen supernatants for anti-X
          ↓
  Expand positive clones
          ↓
  ████ MONOCLONAL ANTIBODY ████

What Each Parent Contributes

HAT Medium - Why It Works

• H (Hypoxanthine) + T (Thymidine) = salvage pathway substrates
• A (Aminopterin) = blocks the main (de novo) DNA synthesis pathway
• Myeloma cells lack HGPRT → cannot use the salvage pathway either → die
• Hybridomas inherit HGPRT from the B cell → use salvage pathway → survive

Generations of Therapeutic MAbs

Murine (-omab)       ████████████████████  0% human
                     e.g. Muromonab

Chimeric (-ximab)    ░░░░░░░████████████  ~65% human
                     e.g. Rituximab, Abciximab

Humanized (-zumab)   ░░████████████████  ~90% human
                     e.g. Trastuzumab, Daclizumab

Fully Human (-umab)  ░█████████████████  >95% human
                     e.g. Adalimumab, Nivolumab

Key Applications at a Glance

One-liner: Hybridoma = B cell specificity + myeloma immortality, selected by HAT, cloned, screened → unlimited identical antibody.