32.4
Tecnologia de Hibridoma
A tecnologia de hibridoma é utilizada para a produção em larga escala de anticorpos monoclonais. Os anticorpos monoclonais ligam-se apenas a um único…
A tecnologia de hibridoma é usada para produzir anticorpos monoclonais em massa que só podem se ligar a um único epítopo - parte de um antígeno que provoca uma resposta imune.
O processo começa com a injeção de um antígeno alvo em um camundongo e, em seguida, a colheita de seu baço após algumas semanas para isolar as células B produtoras de anticorpos. Essas células têm uma vida útil limitada e não podem ser cultivadas em laboratório.
Em seguida, as células B são fundidas com células de mieloma - glóbulos brancos imortais e cancerígenos, usando pulsos elétricos ou polietilenoglicol. Assim, formando células híbridas imortais produtoras de anticorpos chamadas hibridomas.
Os hibridomas são ainda selecionados em hipoxantina-aminopterina-timidina, ou meio HAT com base na presença de uma hipoxantina-guanina fosforibosiltransferase funcional ou enzima HGPRT.
Nesse meio, tanto as células de mieloma negativo para HGPRT quanto as células B positivas para HGPRT, mas de vida curta, não podem sobreviver por muito tempo. No entanto, as células imortais do hibridoma com a enzima HGPRT podem proliferar.
Após a seleção, as células do hibridoma que produzem anticorpos monoclonais com forte afinidade pelo epítopo alvo são identificadas e usadas como células iniciais para estabelecer linhagens celulares produtoras de anticorpos.
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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.