Hybridoma technique fundamentals MCQs With Answer

Hybridoma Technique Fundamentals MCQs With Answer

This set of multiple-choice questions focuses on the core principles and practical aspects of the hybridoma technique, tailored for M.Pharm students. It covers immunization strategies, cell fusion methods (PEG and electrofusion), HAT selection mechanics, choice of myeloma cell lines, feeder cell utilities, cloning and subcloning approaches, screening and characterization assays, antibody isotyping, cryopreservation, and common stability or production issues. The questions are designed to deepen conceptual understanding and support exam preparation by linking theory to laboratory practice and troubleshooting. Answers are provided after each question to facilitate rapid self-assessment and targeted revision.

Q1. Which cell types are fused together in the classical hybridoma technique to produce monoclonal antibody-secreting hybridomas?

• Spleen B lymphocytes and myeloma cells
• Fibroblasts and T lymphocytes
• Macrophages and erythrocytes
• HeLa cells and NK cells

Correct Answer: Spleen B lymphocytes and myeloma cells

Q2. What is the principal reason HAT medium selects for hybridoma cells after fusion?

• Aminopterin blocks de novo purine synthesis, so only HGPRT-positive hybridomas can use the salvage pathway and survive
• HAT provides growth factors specifically used only by hybridomas
• Thymidine in HAT induces differentiation of myeloma cells into hybridomas
• Hypoxanthine in HAT kills myeloma cells directly

Correct Answer: Aminopterin blocks de novo purine synthesis, so only HGPRT-positive hybridomas can use the salvage pathway and survive

Q3. What is the primary function of polyethylene glycol (PEG) in hybridoma production?

• Act as an antibiotic to prevent contamination
• Mediate fusion of cell membranes between lymphocytes and myeloma cells
• Stain hybridomas for microscopy
• Inhibit apoptosis of unfused lymphocytes

Correct Answer: Mediate fusion of cell membranes between lymphocytes and myeloma cells

Q4. Which myeloma cell line is commonly used because it is HGPRT-deficient and thus suitable for HAT selection?

• SP2/0-Ag14 (HPRT-negative myeloma cell line)
• CHO-K1
• HEK293
• Jurkat

Correct Answer: SP2/0-Ag14 (HPRT-negative myeloma cell line)

Q5. What is the purpose of performing limiting dilution cloning on newly formed hybridoma cultures?

• To enrich for feeder cells in the culture
• To isolate wells derived from a single hybridoma cell and ensure monoclonality (≈0.5 cell/well)
• To increase the HAT concentration for selection
• To select for higher molecular weight antibodies

Correct Answer: To isolate wells derived from a single hybridoma cell and ensure monoclonality (≈0.5 cell/well)

Q6. What is the principal role of feeder cells when culturing hybridomas after fusion?

• Provide extracellular matrix for hybridoma adhesion only
• Secrete growth factors and cytokines that support the initial growth of hybridomas
• Convert aminopterin into an inactive form
• Produce antibodies to stimulate hybridoma maturation

Correct Answer: Secrete growth factors and cytokines that support the initial growth of hybridomas

Q7. Compared to PEG-mediated fusion, what is a main advantage of electrofusion for hybridoma generation?

• Electrofusion never requires any downstream screening
• Electrofusion uses electric pulses to align and fuse cells, offering greater control and often higher fusion efficiency
• Electrofusion only fuses identical cell types and thus eliminates heterokaryons
• Electrofusion is effective without any equipment or buffers

Correct Answer: Electrofusion uses electric pulses to align and fuse cells, offering greater control and often higher fusion efficiency

Q8. Which high-throughput assay is most appropriate for initial screening of hybridoma supernatants for antigen-specific antibody production?

• ELISA (enzyme-linked immunosorbent assay)
• Southern blot
• Electron microscopy of cells
• Chromatography of culture medium

Correct Answer: ELISA (enzyme-linked immunosorbent assay)

Q9. Why is ascites-based monoclonal antibody production discouraged in contemporary laboratory practice?

• It consistently produces antibodies of lower affinity
• It raises ethical concerns due to animal welfare issues and gives variable glycosylation/contamination
• It is prohibitively expensive compared with in vitro culture
• Ascites-derived antibodies cannot be purified by Protein A/G

Correct Answer: It raises ethical concerns due to animal welfare issues and gives variable glycosylation/contamination

Q10. Which method is commonly used to determine the heavy-chain isotype of a monoclonal antibody produced by a hybridoma?

• Isotyping ELISA or commercial isotyping kit
• HAT selection assay
• Limiting dilution cloning
• PEG fusion efficiency test

Correct Answer: Isotyping ELISA or commercial isotyping kit

Q11. Which technique is typically used to humanize a murine monoclonal antibody while retaining antigen specificity?

• CDR grafting (complementarity-determining region grafting)
• Direct PEGylation of the Fc region
• HAT medium exposure
• Heat inactivation of variable regions

Correct Answer: CDR grafting (complementarity-determining region grafting)

Q12. What is a common molecular cause for a hybridoma to lose antibody productivity over time?

• Chromosomal instability and loss or silencing of heavy or light chain genes
• Immediate resistance to HAT medium
• Excessive antibody glycosylation increasing secretion
• Feeder cell overgrowth replacing hybridomas

Correct Answer: Chromosomal instability and loss or silencing of heavy or light chain genes

Q13. What is the recommended practice for cryopreserving hybridoma cell lines for long-term storage?

• Direct plunge into liquid nitrogen without cryoprotectant
• Freeze in medium containing approx. 10% DMSO using a controlled-rate freezing step, then store in liquid nitrogen vapor phase
• Store at −20°C in saline
• Lyophilize cells at room temperature

Correct Answer: Freeze in medium containing approx. 10% DMSO using a controlled-rate freezing step, then store in liquid nitrogen vapor phase

Q14. How do affinity and avidity of an antibody differ?

• Affinity is the strength of interaction between a single antigen-binding site and its epitope; avidity is the overall combined strength of multivalent interactions
• Affinity refers to valency; avidity refers to antibody isotype
• Affinity is only relevant for polyclonal sera; avidity only applies to monoclonal antibodies
• They are synonymous terms and can be used interchangeably

Correct Answer: Affinity is the strength of interaction between a single antigen-binding site and its epitope; avidity is the overall combined strength of multivalent interactions

Q15. Which enzyme deficiency in commonly used myeloma fusion partners makes HAT selection possible?

• Hypoxanthine-guanine phosphoribosyltransferase (HGPRT/HPRT) deficiency
• Thymidine kinase deficiency
• Glucose-6-phosphate dehydrogenase deficiency
• Adenylyl cyclase deficiency

Correct Answer: Hypoxanthine-guanine phosphoribosyltransferase (HGPRT/HPRT) deficiency

Q16. What ratio of splenocytes (lymphocytes) to myeloma cells is commonly used for PEG-mediated fusion to favor hybridoma formation?

• Approximately 5:1 (lymphocytes:myeloma)
• 1:1 (equal numbers)
• 1:10 (lymphocytes:myeloma)
• 10:0 (lymphocytes only)

Correct Answer: Approximately 5:1 (lymphocytes:myeloma)

Q17. Beyond confirming monoclonality, what is another primary purpose of subcloning hybridoma lines?

• To obtain stable, high-producing clones and eliminate heterogeneity in expression
• To permanently remove HAT sensitivity
• To increase the number of different isotypes produced by a single clone
• To transform hybridomas into primary B cells

Correct Answer: To obtain stable, high-producing clones and eliminate heterogeneity in expression

Q18. Which assay is most informative for determining whether a monoclonal antibody recognizes a linear epitope on a denatured protein?

• Western blot (immunoblot) using denatured antigen
• Native ELISA only
• Limiting dilution cloning
• HAT selection assay

Correct Answer: Western blot (immunoblot) using denatured antigen

Q19. What is a frequent laboratory cause for a hybridoma clone to change specificity or secrete multiple antibodies over time?

• Genetic drift or contamination leading to mixed populations or loss of the original productive heavy/light chain pair
• Excessive incubation in HAT medium increasing specificity
• Switching feeder cell types weekly
• Replacing PEG with another fusogen

Correct Answer: Genetic drift or contamination leading to mixed populations or loss of the original productive heavy/light chain pair

Q20. What are the three components of HAT medium used for hybridoma selection?

• Hypoxanthine, Aminopterin, Thymidine
• Heparin, Albumin, Triton X-100
• Histidine, Asparagine, Tryptophan
• Hydrogen peroxide, Azide, Tween-20

Correct Answer: Hypoxanthine, Aminopterin, Thymidine

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