Selection and screening of hybridoma clones MCQs With Answer

Selection and screening of hybridoma clones is a critical topic for M.Pharm students focusing on therapeutic antibody development and diagnostic reagent production. This quiz collection emphasizes practical and theoretical aspects of hybridoma technology, covering fusion strategies, selective culture conditions, cloning techniques, and analytical assays used to identify high-quality monoclonal antibody–producing cells. Questions probe concepts such as HAT selection, limiting dilution cloning, feeder cell roles, screening by ELISA and functional assays, isotyping, stability assessment, and troubleshooting common failures. These MCQs are designed to deepen understanding, sharpen problem-solving skills, and prepare students for laboratory work and exam questions in immunotechnology and biopharmaceutical research.

Q1. Which selection medium is commonly used after fusion to allow only hybridoma cells to survive while preventing unfused myeloma and B cells from proliferating?

• Neomycin-containing medium
• HAT medium
• LB medium
• Serum-free medium

Correct Answer: HAT medium

Q2. What is the role of aminopterin in HAT selection during hybridoma production?

• It stimulates antibody secretion
• It inhibits de novo nucleotide synthesis forcing salvage pathway dependency
• It acts as a mitogen for B cells
• It selectively kills fused hybridomas

Correct Answer: It inhibits de novo nucleotide synthesis forcing salvage pathway dependency

Q3. Which enzyme deficiency in myeloma parent lines makes HAT selection possible?

• Thymidine kinase deficiency
• HGPRT deficiency
• Topoisomerase I deficiency
• RNA polymerase deficiency

Correct Answer: HGPRT deficiency

Q4. Which screening assay is most commonly used for rapid detection of antibody-secreting hybridoma clones in culture supernatants?

• Polyacrylamide gel electrophoresis
• ELISA
• Flow cytometry with propidium iodide
• Mass spectrometry

Correct Answer: ELISA

Q5. Limiting dilution cloning is performed to achieve what primary objective in hybridoma development?

• Increase antibody affinity
• Isolate single-cell derived clones to ensure monoclonality
• Adapt cells to serum-free conditions
• Introduce selectable markers

Correct Answer: Isolate single-cell derived clones to ensure monoclonality

Q6. Why are feeder cells (e.g., irradiated splenocytes or macrophages) used during early hybridoma cloning?

• They provide selective toxicity against myeloma cells
• They supply growth factors and support low-density hybridoma survival
• They secrete aminopterin to maintain HAT selection
• They act as antigen-presenting cells to increase specificity

Correct Answer: They supply growth factors and support low-density hybridoma survival

Q7. Which method can directly measure antigen-binding affinity of antibodies secreted by hybridoma clones?

• Indirect ELISA endpoint titer only
• Surface plasmon resonance (SPR)
• SDS-PAGE under reducing conditions
• Gram staining

Correct Answer: Surface plasmon resonance (SPR)

Q8. During initial screening of many hybridoma wells, what secondary criterion is important besides binding to antigen to prioritize clones for further evaluation?

• High glucose consumption
• Antibody isotype and subclass
• Cell adherence propensity
• Color change in medium

Correct Answer: Antibody isotype and subclass

Q9. Which technique is best to confirm that a hybridoma-derived mAb recognizes the native conformation of its protein antigen?

• Western blot on denatured protein
• Immunoprecipitation or flow cytometry on intact cells
• DNA sequencing of the heavy chain**
• Gram staining of producing cells

Correct Answer: Immunoprecipitation or flow cytometry on intact cells

Q10. A hybridoma clone shows declining antibody production over time despite continued culture. Which is the most likely cause?

• Loss of antigen in the medium
• Genetic instability or loss of immunoglobulin gene expression
• Excessive antibody affinity maturation
• Contamination with yeast

Correct Answer: Genetic instability or loss of immunoglobulin gene expression

Q11. For subcloning, why are multiple rounds of limiting dilution often necessary?

• To increase secretion rate of antibodies per cell
• To ensure elimination of mixed or oligoclonal wells and obtain true monoclonality
• To change the isotype of the antibody
• To introduce resistance to aminopterin

Correct Answer: To ensure elimination of mixed or oligoclonal wells and obtain true monoclonality

Q12. Which myeloma characteristic is undesirable in fusion partners used for hybridoma production?

• HGPRT deficiency making them HAT-sensitive
• Lack of endogenous immunoglobulin secretion to avoid background
• Secretion of myeloma-derived immunoglobulins that can complicate assays
• Rapid growth to support hybridoma propagation

Correct Answer: Secretion of myeloma-derived immunoglobulins that can complicate assays

Q13. Which assay would you use to determine the neutralizing capability of antibodies from hybridoma supernatants?

• Competitive ELISA only for binding
• In vitro functional neutralization assay (e.g., viral neutralization, toxin neutralization)
• SDS-PAGE molecular weight determination
• Immunohistochemistry on fixed tissue sections

Correct Answer: In vitro functional neutralization assay (e.g., viral neutralization, toxin neutralization)

Q14. Which parameter is most critical to evaluate when selecting a hybridoma for therapeutic antibody development?

• Ability to grow on agar plates
• High specific productivity, stability, and humanization potential
• Resistance to antibiotics used in bacterial cloning
• Color of the culture medium

Correct Answer: High specific productivity, stability, and humanization potential

Q15. How can cross-contamination between hybridoma clones be minimized during screening?

• By using shared tips between cultures
• By practicing aseptic technique, single-use pipette tips, and careful plate handling
• By pooling supernatants from neighboring wells
• By reducing incubation temperatures below physiological levels

Correct Answer: By practicing aseptic technique, single-use pipette tips, and careful plate handling

Q16. Which isotyping method is commonly used to determine heavy chain class (IgG, IgM, etc.) of antibodies from hybridoma clones?

• Isoelectric focusing
• Commercial isotype-specific ELISA or lateral flow isotyping kits
• Southern blot
• DNA fingerprinting

Correct Answer: Commercial isotype-specific ELISA or lateral flow isotyping kits

Q17. What is the benefit of cryopreserving early-passage hybridoma clones once a desirable clone is identified?

• Prevents the need for HAT selection
• Preserves genetic stability and antibody production phenotype for future expansion
• Eliminates the need for further subcloning
• Improves antibody affinity with time

Correct Answer: Preserves genetic stability and antibody production phenotype for future expansion

Q18. In an ELISA-based screening, a hybridoma supernatant shows high signal on antigen-coated wells but also high signal on negative control wells coated with irrelevant protein. What does this indicate?

• High specificity to target antigen
• Non-specific binding or high background; possible cross-reactivity
• Complete absence of antibody in supernatant
• Perfect monoclonality

Correct Answer: Non-specific binding or high background; possible cross-reactivity

Q19. Which molecular method can be used to characterize and preserve the sequence of a promising monoclonal antibody from a hybridoma?

• RT-PCR amplification and sequencing of immunoglobulin heavy and light chain variable regions
• Restriction fragment length polymorphism (RFLP) of genomic DNA
• Gram staining of the culture
• Measuring osmolarity of culture medium

Correct Answer: RT-PCR amplification and sequencing of immunoglobulin heavy and light chain variable regions

Q20. Which screening approach helps prioritize clones that recognize epitopes useful for sandwich ELISA assays?

• Western blot on denatured antigen only
• Pairwise epitope binning or sandwich ELISA pairing tests to find non-competing antibody pairs
• Measuring cell doubling time only
• Testing antibiotic resistance profiles

Correct Answer: Pairwise epitope binning or sandwich ELISA pairing tests to find non-competing antibody pairs

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