32.4
Tecnología de hibridoma
La tecnología de hibridoma se utiliza para la producción a gran escala de anticuerpos monoclonales. Los anticuerpos monoclonales se unen a un solo det…
La tecnología de hibridoma se utiliza para producir en masa anticuerpos monoclonales que solo pueden unirse a un solo epítopo, parte de un antígeno que provoca una respuesta inmunitaria.
El proceso comienza con la inyección de un antígeno objetivo en un ratón y luego la recolección de su bazo después de unas semanas para aislar las células B productoras de anticuerpos. Estas células tienen una vida útil limitada y no se pueden cultivar en un laboratorio.
A continuación, las células B se fusionan con las células de mieloma, glóbulos blancos inmortales y cancerosos, mediante pulsos eléctricos o polietilenglicol. Por lo tanto, forman células híbridas inmortales productoras de anticuerpos llamadas hibridomas.
Los hibridomas se seleccionan además en medio de hipoxantina-aminopterina-timidina o HAT en función de la presencia de una enzima funcional de hipoxantina-guanina fosforribosiltransferasa o HGPRT.
En este medio, tanto las células de mieloma HGPRT negativas como las células B HGPRT positivas pero de corta vida no pueden sobrevivir por mucho tiempo. Sin embargo, las células de hibridomas inmortales con la enzima HGPRT pueden proliferar.
Después de la selección, las células de hibridoma que producen anticuerpos monoclonales con una fuerte afinidad por el epítopo objetivo se identifican y se utilizan como células iniciadoras para establecer líneas celulares productoras de anticuerpos.
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Q1: What is the main purpose of hybridoma technology?
Hybridoma technology enables mass production of monoclonal antibodies that bind to a single epitope on an antigen. These antibodies are valuable in research, diagnostics, and disease therapy because they target only one specific antigenic determinant, providing high specificity and consistency for applications requiring precise immune targeting.
Q2: How are B cells and myeloma cells combined in hybridoma creation?
B cells harvested from a mouse spleen are fused with immortal myeloma cells using electric pulses (electroporation) or polyethylene glycol (PEG). This fusion creates hybrid cells called hybridomas that combine the antibody-producing ability of B cells with the unlimited growth capacity of myeloma cells, overcoming the natural limitation that B cells cannot survive long in culture.
Q3: Why is HAT medium used in hybridoma selection?
HAT medium selectively allows only hybridoma cells to survive by blocking default nucleotide synthesis. Myeloma cells lacking the HGPRT enzyme cannot use the salvage pathway and die, while short-lived B cells also perish. Only hybrid cells with functional HGPRT from B cells and immortality from myeloma cells proliferate, ensuring pure hybridoma populations for antibody production.
Q4: What happens during hybridoma screening after HAT selection?
Hybridoma cultures are plated in 96-well plates with one cell per well. Each well is screened using enzyme linked immunosorbent assay to identify cells producing antibodies specific to the target epitope. Positive cells are then grown in larger culture vessels to establish permanent hybridoma cell lines that serve as unlimited sources of monoclonal antibodies.
Q5: Why are monoclonal antibodies more useful than polyclonal antibodies?
Monoclonal antibodies bind to only a single epitope, providing high specificity and consistency. This single-target binding makes them ideal for precise research, diagnostic, and therapeutic applications where cross-reactivity must be minimized. Polyclonal antibodies, by contrast, recognize multiple epitopes and lack this specificity.
Q6: What is the significance of the HGPRT enzyme in HAT medium selection?
The HGPRT enzyme enables cells to synthesize nucleotides via the salvage pathway when aminopterin blocks the default synthesis route. Myeloma cells are HGPRT-negative and cannot survive in HAT medium, while B cells are HGPRT-positive but short-lived. Only hybridomas with functional HGPRT and immortality survive, making this enzyme critical for selecting true hybrid cells.
Q7: How does hybridoma technology overcome the limitations of natural B cells?
Natural B cells produce antibodies but have a limited lifespan and cannot be cultured indefinitely in the laboratory. Hybridoma technology fuses these cells with immortal myeloma cells, creating hybrid cells that retain antibody-producing capability while gaining unlimited growth potential. This overcomes both the finite lifespan and culture limitations of primary B cells.