Lecture 14

Organized Notes: Flow Cytometry & Monoclonal Antibodies

1. Introduction to Flow Cytometry

• Definition & Example: Diagnosis of Immunodeficiency (X-Linked Agammaglobulinemia)

2. History: The Inventor of Flow Cytometry

• Leonard Herzenberg developed flow cytometry in the 1960s to automate cell counting.

• Source: The New York Times, November 11, 2013

3. Measurements Taken with Flow Cytometry

1. Light Scatter

   • Forward Scatter (FSC) → Relative cell size (measured along the light axis)

   • Side Scatter (SSC) → Internal complexity/granularity (measured at a right angle)

2. Fluorescence

   • Excitation & Emission: Absorption of laser light and emission of visible photons

   • FITC Excitation and Emission Spectrum

4. Three Main Systems of Flow Cytometry

1. Fluidics

   • Sheath fluid & sample meet in the flow cell → hydrodynamic focusing (single-file alignment of cells)

2. Optics

   • Lasers (light sources): Violet (405nm), Blue (488nm), Yellow-Green (561nm), Red (633-640nm)

3. Electronics

   • Converts light signals into electronic data

5. Presentation of Flow Cytometry Data

• One-dimensional histograms (intensity measurement)

• Two-dimensional dot plots (light scatter profiles)

• Example: Light scatter profile of lymphocytes

6. Monoclonal Antibodies & Flow Cytometry

• Powerful method for medical discoveries

7. Monoclonal Antibodies: Concept & Uses

• Large-scale production of specific antibodies

• Applications:

  • Neutralizing toxins (e.g., snake venom)

  • Neutralizing pathogens (e.g., HIV, SARS-CoV-2)

  • Neutralizing cytokines (e.g., TNF-α in rheumatoid arthritis)

  • Complement-fixing antibodies for targeting specific cells (e.g., leukemia treatment)

• Discovery: Kohler & Milstein (1975)

8. Epitopes & Paratopes: Antibody-Antigen Interaction

• Epitope (antigenic determinant): Region of antigen recognized by antibody or TCR

• Paratope: Region of antibody binding to the epitope

• "Lock & Key" Fit: Janeway’s Immunobiology (Figures 4-8, 1-13)

9. Monoclonal Antibody Production Process

1. Cloning Antibody-Secreting Cells

   • Immortalization → Screening for specificity → Large-scale production

2. Hybridoma Technology (Kohler & Milstein)

   • B cell (HPRT+, antibody-producing) fused with myeloma (HPRT-, immortal) → Hybridoma

   • Survives in HAT Media (blocks de novo DNA synthesis, forcing salvage pathway use)

10. Traditional Protocol for Monoclonal Antibody Production

1. Immunization → Inject antigen into an animal

2. Fusion → Fuse B cells with myeloma cells

3. Selection → Culture in HAT media

4. Expansion → Grow positive clones

5. Testing → Validate function via assays

11. Assays to Screen for Monoclonal Antibodies

• Hemolytic Plaque Assay (Niels Jerne)

• ELISA (Enzyme-Linked Immunosorbent Assay)

• Western Blot (Immunoblot)

• Flow Cytometry

12. Monoclonal Antibodies as Therapeutics

• Challenges: Mouse-derived antibodies can cause serum sickness (Type III hypersensitivity)

• Solution: Develop humanized monoclonal antibodies (less immunogenic)

• Current Success:

  • 5/10 Bestselling Drugs in 2024 were Humanized Monoclonal Antibodies

13. Humanized Monoclonal Antibodies

• Molecular biology techniques used to replace mouse sequences with human sequences