Antibodies are powerful tools in immunology, with diverse applications in diagnostics and therapeutics. Polyclonal antibodies recognize multiple epitopes, while monoclonal antibodies target specific ones. Understanding their production methods and characteristics is crucial for their effective use.

Humanized monoclonal antibodies have revolutionized medicine by reducing immunogenicity and improving compatibility with the human immune system. These engineered antibodies are used in cancer immunotherapy, autoimmune disease treatment, and infectious disease prevention, showcasing their versatility in modern healthcare.

Antibody Production and Applications

Monoclonal vs polyclonal antibodies

• Polyclonal antibodies
  • Produced by multiple B cell clones in response to an antigen
  • Recognize multiple epitopes on the same antigen
  • Production methods involve immunizing an animal (rabbit, goat, or horse) with the antigen and collecting serum containing the antibodies
  • Applications include immunohistochemistry, ELISA (Enzyme-Linked Immunosorbent Assay), and Western blotting
• Monoclonal antibodies
  • Produced by a single B cell clone recognizing a specific epitope on an antigen
  • Production methods use hybridoma technology by immunizing a mouse with the antigen, fusing B cells from the mouse's spleen with myeloma cells, and selecting and culturing the hybridoma cells that produce the desired antibody
  • Applications include diagnostic tests (pregnancy tests, cancer biomarker detection), targeted cancer therapies (rituximab, trastuzumab), and treatment of autoimmune diseases (infliximab for rheumatoid arthritis)

Antibody cross-reactivity in diagnostics

• Cross-reactivity occurs when an antibody recognizes and binds to an epitope on an antigen other than its intended target due to structural similarities between epitopes on different antigens
• Implications for diagnostic testing include
  • False-positive results where cross-reactive antibodies bind to non-target antigens, leading to incorrect positive test results and potentially causing unnecessary treatment or further testing
  • False-negative results where cross-reactive antibodies bind to the target antigen with lower affinity, reducing the sensitivity of the test and potentially leading to missed diagnoses and delayed treatment
  • Reduced specificity (ability to correctly identify individuals without the condition) and sensitivity (ability to correctly identify individuals with the condition) of diagnostic tests

Production of humanized monoclonal antibodies

• Humanized monoclonal antibodies are engineered to contain more human protein sequences to reduce immunogenicity and improve compatibility with the human immune system
• Production process involves
  1. Identifying the complementarity-determining regions (CDRs) of a mouse monoclonal antibody
  2. Grafting the CDRs onto a human antibody framework
  3. Expressing the humanized antibody in a mammalian cell line
  4. Purifying and characterizing the humanized antibody
• Clinical uses include
  • Cancer immunotherapy by targeting specific tumor antigens (PD-1, CTLA-4) and enhancing the immune system's ability to recognize and destroy cancer cells
  • Treatment of inflammatory and autoimmune diseases by neutralizing pro-inflammatory cytokines (TNF-α, IL-6) and modulating the immune response to reduce tissue damage
  • Infectious disease prevention and treatment by neutralizing viral or bacterial pathogens (respiratory syncytial virus, anthrax toxin) and providing passive immunity in high-risk individuals

Antibody characteristics and production techniques

• Antigen: A substance that stimulates an immune response, typically foreign molecules like proteins or polysaccharides
• Immunization: The process of exposing an animal to an antigen to stimulate antibody production
• Affinity: The strength of binding between an antibody and its specific antigen
• Specificity: The ability of an antibody to bind only to its intended target antigen
• Hybridoma: A hybrid cell produced by fusing a B cell with a tumor cell, used in monoclonal antibody production
• Cell fusion: The technique used to create hybridomas by combining B cells with myeloma cells