This quiz collection focuses on fusion methods for hybridoma formation—an essential topic in Immunotechnology for M.Pharm students. It covers the theory and practical parameters of commonly used fusion techniques such as polyethylene glycol (PEG)-mediated fusion, electrofusion, and virus-mediated fusion, together with selection (HAT), feeder cell use, cloning strategies, and troubleshooting. Questions are designed to test deeper understanding of mechanisms, optimization factors, selection markers, and downstream screening methods used in monoclonal antibody generation. Use these MCQs to reinforce lab design decisions, interpret experimental outcomes, and prepare for exams that demand both conceptual insight and applied knowledge in hybridoma technology.
Q1. Which fusogen is most commonly used for chemical-mediated hybridoma formation?
- Polyethylene glycol (PEG)
- Sendai virus (HVJ)
- Electroporation
- Calcium chloride
Correct Answer: Polyethylene glycol (PEG)
Q2. What is the primary purpose of using HAT medium after fusion?
- To select fused hybridoma cells by allowing HGPRT+ hybrids to survive (HAT selection)
- To promote rapid proliferation of unfused myeloma cells
- To induce antibody secretion from myeloma cells
- To inactivate feeder cells
Correct Answer: To select fused hybridoma cells by allowing HGPRT+ hybrids to survive (HAT selection)
Q3. Which characteristic is essential for the myeloma fusion partner in hybridoma production?
- HGPRT-negative and non-immunoglobulin-secreting
- HGPRT-positive and secreting high-affinity antibodies
- Primary B cell with high proliferation rate
- Virus-transformed lymphocyte that secretes IgM
Correct Answer: HGPRT-negative and non-immunoglobulin-secreting
Q4. What is a major advantage of electrofusion compared with PEG-mediated fusion?
- Provides controlled cell alignment and higher reproducibility with less chemical contamination
- Is always faster and cheaper in all laboratory settings
- Does not require optimization of voltage or pulse duration
- Eliminates the need for HAT selection
Correct Answer: Provides controlled cell alignment and higher reproducibility with less chemical contamination
Q5. How does Sendai virus (HVJ) mediate cell fusion for hybridoma formation?
- By promoting membrane merging via viral envelope glycoproteins (hemagglutinin-neuraminidase and fusion protein)
- By introducing PEG-like lipids into the membrane
- By delivering electrical pulses to induce pores
- By activating HGPRT in myeloma cells
Correct Answer: By promoting membrane merging via viral envelope glycoproteins (hemagglutinin-neuraminidase and fusion protein)
Q6. Which range of polyethylene glycol (PEG) molecular weight is commonly used for hybridoma fusion?
- PEG 1500–4000 (commonly PEG 1500 or PEG 3350)
- PEG 50–200
- PEG 8000–20000
- Polyethylene oxide (PEO) only
Correct Answer: PEG 1500–4000 (commonly PEG 1500 or PEG 3350)
Q7. Why are myeloma fusion partners selected to be HGPRT-deficient?
- To prevent survival of unfused myeloma cells in HAT medium unless they fuse with HGPRT+ B cells
- To cause myeloma cells to secrete B-cell growth factors
- To increase baseline antibody secretion from myeloma cells
- To make myeloma cells resistant to aminopterin
Correct Answer: To prevent survival of unfused myeloma cells in HAT medium unless they fuse with HGPRT+ B cells
Q8. What is the commonly used ratio of splenocytes (B cells) to myeloma cells in PEG-mediated fusion?
- 5:1 to 10:1 (splenocytes:myeloma)
- 1:100 (splenocytes:myeloma)
- 1:1 exclusively
- 100:1 (splenocytes:myeloma)
Correct Answer: 5:1 to 10:1 (splenocytes:myeloma)
Q9. What is the principal biophysical action of PEG during chemical fusion?
- It induces membrane dehydration and promotes close membrane apposition leading to lipid mixing and fusion
- It inserts viral fusion proteins into membranes
- It forms transient pores by electrical discharge
- It acts as a nutrient to increase cell division
Correct Answer: It induces membrane dehydration and promotes close membrane apposition leading to lipid mixing and fusion
Q10. What is the main limitation of virus-mediated fusion methods such as Sendai virus?
- Biosafety concerns and potential viral contamination or immunogenicity
- They cannot fuse mammalian cells at all
- They give higher cloning efficiency without need for HAT
- They eliminate the requirement for feeder cells
Correct Answer: Biosafety concerns and potential viral contamination or immunogenicity
Q11. In electrofusion protocols, what is the purpose of applying an alternating current (AC) field before the DC pulse?
- To align cells into pearl-chain formations facilitating cell–cell contact before pulsing
- To immediately create permanent membrane pores
- To heat the suspension for increased fusion
- To sterilize the cells prior to fusion
Correct Answer: To align cells into pearl-chain formations facilitating cell–cell contact before pulsing
Q12. At which temperature are PEG-mediated fusions typically performed to maximize viability?
- 37°C
- 4°C
- 0°C (on ice)
- 50°C
Correct Answer: 37°C
Q13. Why are feeder cells (irradiated splenocytes or peritoneal exudate cells) used after fusion?
- To provide growth factors and support that enhance survival and outgrowth of nascent hybridomas
- To fuse with hybridomas and increase antibody diversity
- To metabolize aminopterin in HAT medium
- To act as the primary source of antibody production
Correct Answer: To provide growth factors and support that enhance survival and outgrowth of nascent hybridomas
Q14. Which screening method is most commonly used to identify antibody-producing hybridoma clones?
- Enzyme-linked immunosorbent assay (ELISA)
- Gram staining
- DNA fingerprinting
- Patch-clamp electrophysiology
Correct Answer: Enzyme-linked immunosorbent assay (ELISA)
Q15. Mechanistically, how does PEG concentration and exposure time affect fusion outcome?
- Higher PEG concentration and longer exposure increase fusion but can reduce viability if excessive
- PEG concentration has no measurable effect on fusion
- Longer exposure to PEG always improves viability and fusion indefinitely
- Lower PEG concentrations produce more multinucleated hybrids
Correct Answer: Higher PEG concentration and longer exposure increase fusion but can reduce viability if excessive
Q16. Which cloning technique is routinely used to ensure monoclonality of antibody-producing hybridomas?
- Limiting dilution cloning
- Gel electrophoresis cloning
- CRISPR-mediated cloning
- Southern blot cloning
Correct Answer: Limiting dilution cloning
Q17. What is a heterokaryon in the context of cell fusion?
- A fused cell that contains two or more distinct nuclei from the original cells
- A cell that lacks any nuclei after fusion
- A viral particle used for fusion
- A specialized feeder cell
Correct Answer: A fused cell that contains two or more distinct nuclei from the original cells
Q18. In the HAT selection system, why is aminopterin included in the medium?
- To block de novo nucleotide synthesis so cells must use the salvage pathway (requiring HGPRT)
- To directly stimulate antibody secretion
- To act as a chromogenic substrate for ELISA
- To encourage myeloma overgrowth
Correct Answer: To block de novo nucleotide synthesis so cells must use the salvage pathway (requiring HGPRT)
Q19. Which factors most strongly increase fusion efficiency in practical hybridoma protocols?
- High cell viability, optimal PEG concentration/exposure time (or optimized electrofusion parameters), and correct cell ratio
- Using only frozen cells without recovery
- Irradiating cells immediately before PEG addition
- Omitting HAT selection entirely
Correct Answer: High cell viability, optimal PEG concentration/exposure time (or optimized electrofusion parameters), and correct cell ratio
Q20. After obtaining HAT-resistant antibody-producing clones, what downstream analyses confirm antibody isotype and specificity?
- Isotyping assays combined with specificity testing such as ELISA or Western blot
- Gram staining and motility tests
- Measuring HGPRT activity alone
- Sequencing mitochondrial DNA only
Correct Answer: Isotyping assays combined with specificity testing such as ELISA or Western blot
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