Q.47 HAT (hypoxanthine, aminopterin and thymidine) is used for selecting the hybridomas based on the following
I. Only hybridoma will grow since it inherited the HGPRT genes from B-cells and can synthesize DNA from hypoxanthine.
II. Myeloma cells will not grow in cultures since de novo synthesis is blocked by aminopterin and due to the lack of HGPRT enzyme.
Correct Answer: (C) both I and II are true
HAT medium selects hybridomas because they inherit functional HGPRT from B-cells (I true), enabling salvage pathway nucleotide synthesis when aminopterin blocks de novo synthesis. Myeloma cells lack HGPRT and cannot use either pathway, preventing growth (II true).
Option Analysis
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(A) only I is true: Incorrect. II correctly explains myeloma cell death mechanism.
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(B) only II is true: Incorrect. I accurately describes hybridoma survival via B-cell HGPRT.
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(C) both I and II are true: Correct. Both statements precisely describe HAT selection mechanism.
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(D) I is true and II is false: Incorrect. II true—myeloma HGPRT⁻ blocks salvage pathway under aminopterin block.
HAT medium hybridoma selection via HGPRT enzyme inheritance from B-cells versus myeloma deficiency defines Q.47 core mechanism essential for monoclonal antibody production in biochemical engineering.
HAT Selection Mechanism
De novo pathway: Aminopterin → DHFR inhibition → dNTP block
Salvage pathway: Hypoxanthine + Thymidine → HGPRT/TK required
Cell fates in HAT:
B-cells: HGPRT⁺ but finite lifespan → die naturally
Myeloma: HGPRT⁻ → no salvage pathway → die
Hybridoma: HGPRT⁺ (from B-cell) + immortal → survive
Timeline: Days 1-4 HAT selection → Days 5-7 subcloning → Weeks 2-4 mAb screening.
Pathway Biochemistry
De novo: PRPP → IMP → XMP → GMP (HGPRT bypasses)
Salvage: Hypoxanthine + PRPP → IMP (HGPRT catalysis)
Thymidine: dTMP via thymidine kinase (ubiquitous)
Yield Optimization
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Fusing ratio: 2-5 spleen:myceloma maximizes hybridomas
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Feeder cells: Peritoneal macrophages improve attachment
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HT maintenance: Remove aminopterin post-selection
GATE Biotechnology Integration
Q.47 connects Q.35-45 progression: HAT selection → baculovirus expression (Q.45) → μ_max optimization (Q.44) → Pol III replication (Q.43) → synchronous culture (Q.42) → IPTG systems (Q.41). Essential for bioprocess yield calculations (10⁶ hybridomas/mL → 10-100 μg/mL mAb).
Exam Strategy: Both statements must be individually correct. Recognize myeloma = HGPRT⁻/TK⁻ double mutant; B-cells provide both salvage enzymes. Quantitative: 1/10⁵-10⁶ fusions yield stable clones.
