Introduction
Hybridoma technology is a foundational method for the production of monoclonal antibodies (mAbs), combining antibody-producing B cells with immortal myeloma cells to create hybridomas. This process—involving fusion agents like PEG, HAT selection, limiting dilution cloning, and screening by ELISA or flow cytometry—yields specific, high-affinity IgG antibodies for diagnostics, therapeutics, and research. Key steps include choosing HGPRT-deficient myeloma lines, using feeder cells, stabilizing clones, and options for humanization or recombinant expression to reduce immunogenicity. Understanding production, quality control, applications (immunoassays, targeted therapy, imaging), and regulatory considerations is essential for B.Pharm students. Now let’s test your knowledge with 30 MCQs on this topic.
Q1. What is hybridoma technology?
- Fusion of a single B cell with another B cell to enhance antibody diversity
- Fusion of an antibody-producing B cell with a myeloma cell to form an immortal antibody-secreting hybrid
- Genetic engineering of bacteria to express antibody fragments
- Chemical synthesis of monoclonal antibodies from peptide sequences
Correct Answer: Fusion of an antibody-producing B cell with a myeloma cell to form an immortal antibody-secreting hybrid
Q2. What is the main purpose of HAT medium in hybridoma production?
- To provide growth factors for unfused myeloma cells
- To selectively allow growth of only fused hybridoma cells while eliminating unfused myeloma and B cells
- To induce antibody secretion from myeloma cells
- To label hybridomas for flow cytometry
Correct Answer: To selectively allow growth of only fused hybridoma cells while eliminating unfused myeloma and B cells
Q3. What role does polyethylene glycol (PEG) play in hybridoma formation?
- Acts as a selection antibiotic in HAT medium
- Promotes fusion of cell membranes to form hybrid cells
- Serves as an adjuvant to boost immune response in mice
- Is used as a cryoprotectant for long-term storage
Correct Answer: Promotes fusion of cell membranes to form hybrid cells
Q4. Which characteristic is essential for a myeloma fusion partner used in hybridoma technology?
- HGPRT-positive and secretes high levels of endogenous immunoglobulin
- HGPRT-deficient, immortal, and non-immunoglobulin-secreting
- Normal lifespan and secretes polyclonal antibodies
- Primary B cell origin with high affinity for antigen
Correct Answer: HGPRT-deficient, immortal, and non-immunoglobulin-secreting
Q5. Why are feeder cells used after fusion?
- To provide antigens for hybridoma selection
- To supply growth factors and support the initial growth of hybridomas
- To directly secrete the monoclonal antibody of interest
- To remove unfused myeloma cells by phagocytosis
Correct Answer: To supply growth factors and support the initial growth of hybridomas
Q6. Which screening method is most commonly used to identify antigen-specific hybridoma clones?
- Western blot for heavy-chain sequencing
- ELISA to detect secreted antigen-specific antibodies
- Gram staining of culture supernatant
- Mass spectrometry of secreted proteins
Correct Answer: ELISA to detect secreted antigen-specific antibodies
Q7. What is the purpose of limiting dilution cloning in hybridoma technology?
- To increase fusion efficiency between cells
- To isolate single hybridoma cells and derive monoclonal populations
- To label antibodies with enzymes for detection
- To convert polyclonal sera into monoclonal antibodies
Correct Answer: To isolate single hybridoma cells and derive monoclonal populations
Q8. What is a major disadvantage of ascites production of monoclonal antibodies?
- It yields antibodies with perfect human glycosylation patterns
- It is ethically problematic and can introduce host contaminants such as serum proteins
- It produces very low antibody titers compared to in vitro methods
- It cannot be used for IgG isotypes
Correct Answer: It is ethically problematic and can introduce host contaminants such as serum proteins
Q9. Which antibody isotype is most commonly produced by mouse hybridomas for therapeutic or diagnostic use?
- IgA
- IgM
- IgG
- IgE
Correct Answer: IgG
Q10. What is the main goal of humanizing a murine monoclonal antibody?
- To increase mouse-specific epitopes for better immunogenicity
- To graft murine complementarity-determining regions (CDRs) onto human antibody frameworks to reduce immunogenicity in humans
- To convert IgG to IgM format for stronger avidity
- To make the antibody resistant to proteolysis in mouse serum
Correct Answer: To graft murine complementarity-determining regions (CDRs) onto human antibody frameworks to reduce immunogenicity in humans
Q11. Which application commonly uses monoclonal antibodies produced by hybridomas?
- Chemical catalysis in organic synthesis
- Diagnostic immunoassays such as ELISA, immunohistochemistry and flow cytometry
- Primary metabolites production in fermentation
- Replacement of blood plasma in transfusions
Correct Answer: Diagnostic immunoassays such as ELISA, immunohistochemistry and flow cytometry
Q12. How does aminopterin in HAT medium contribute to selection?
- By activating de novo nucleotide synthesis
- By inhibiting the de novo pathway of nucleotide synthesis, forcing reliance on salvage pathway enzymes
- By acting as a nutrient supplement for hybridomas
- By directly killing all hybridoma cells
Correct Answer: By inhibiting the de novo pathway of nucleotide synthesis, forcing reliance on salvage pathway enzymes
Q13. Why are SP2/0 and NS0 cell lines commonly used as myeloma partners?
- They are primary B cells with normal HGPRT activity
- They are HGPRT-deficient, immortal and typically do not secrete their own immunoglobulin
- They secrete high levels of polyclonal antibodies for screening
- They are resistant to PEG-mediated fusion
Correct Answer: They are HGPRT-deficient, immortal and typically do not secrete their own immunoglobulin
Q14. What stability issue can affect hybridoma cultures over time?
- Loss of antigen expression in target cells
- Genetic drift leading to decreased antibody production or altered specificity
- Permanent resistance to HAT medium
- Conversion of IgG to RNA molecules
Correct Answer: Genetic drift leading to decreased antibody production or altered specificity
Q15. What is the difference between affinity and avidity of an antibody?
- Affinity refers to the overall binding strength of multivalent antibodies; avidity is strength of a single binding site
- Affinity is the strength of a single antigen-binding site for an epitope; avidity is the combined strength of multiple interactions
- They are identical terms used interchangeably
- Affinity measures enzymatic activity; avidity measures binding kinetics
Correct Answer: Affinity is the strength of a single antigen-binding site for an epitope; avidity is the combined strength of multiple interactions
Q16. Which assay is commonly used for isotype determination of monoclonal antibodies?
- Isotype-specific ELISA or isotyping kit
- Gram staining of hybridoma culture
- DNA sequencing of the myeloma partner
- High-performance liquid chromatography (HPLC) for antigen
Correct Answer: Isotype-specific ELISA or isotyping kit
Q17. Which label is frequently conjugated to monoclonal antibodies for colorimetric immunoassays?
- Green fluorescent protein (GFP)
- Horseradish peroxidase (HRP)
- Glucose oxidase for metabolic assays
- Calcium phosphate for bone targeting
Correct Answer: Horseradish peroxidase (HRP)
Q18. Which antibody region mediates effector functions like ADCC and complement activation?
- Fab region
- Variable light chain
- Fc region
- Complementarity-determining region (CDR)
Correct Answer: Fc region
Q19. Which of the following is an example of a therapeutic monoclonal antibody developed from hybridoma or derived sequences?
- Penicillin
- Rituximab (anti-CD20)
- Insulin
- Atorvastatin
Correct Answer: Rituximab (anti-CD20)
Q20. What is an advantage of expressing monoclonal antibodies recombinantly (CHO cells) versus traditional hybridoma culture?
- Recombinant expression prevents any need for purification
- Allows scalable production, glycoengineering and sequence optimization for reduced immunogenicity
- Recombinant methods always produce higher-affinity antibodies without modification
- CHO-expressed antibodies do not require validation for clinical use
Correct Answer: Allows scalable production, glycoengineering and sequence optimization for reduced immunogenicity
Q21. Which technique is commonly used for epitope mapping of a monoclonal antibody?
- Peptide library scanning or mutational analysis of the antigen
- Gram staining of the antigen sample
- Measuring antibody half-life in serum only
- Using HAT medium to select epitopes
Correct Answer: Peptide library scanning or mutational analysis of the antigen
Q22. In HAT selection, what is the role of hypoxanthine?
- It acts as an antibiotic to kill bacteria in culture
- It is a substrate for the salvage pathway enzyme HGPRT, allowing DNA synthesis in HGPRT-positive cells
- It denatures un-fused cells by osmotic shock
- It fluorescently labels hybridomas for sorting
Correct Answer: It is a substrate for the salvage pathway enzyme HGPRT, allowing DNA synthesis in HGPRT-positive cells
Q23. Which culture system is typically used for large-scale in vitro monoclonal antibody production?
- Shake flasks only
- Bioreactors, such as stirred-tank or perfusion systems
- Plate-based microcultures exclusively
- Bacterial fermenters without eukaryotic cells
Correct Answer: Bioreactors, such as stirred-tank or perfusion systems
Q24. How do monoclonal antibodies differ from polyclonal antibodies?
- Monoclonal antibodies recognize multiple epitopes; polyclonal recognize a single epitope
- Monoclonal antibodies are identical and recognize a single epitope; polyclonal are heterogeneous and recognize multiple epitopes
- They are the same; the terms are interchangeable
- Polyclonal antibodies are derived from a single hybridoma clone
Correct Answer: Monoclonal antibodies are identical and recognize a single epitope; polyclonal are heterogeneous and recognize multiple epitopes
Q25. The limiting dilution cloning method relies on which statistical principle to isolate single cells?
- Benford’s law
- Poisson distribution to dilute cells so wells contain either zero or one cell statistically
- Central limit theorem applied to antibody affinity
- Hardy-Weinberg equilibrium for genetic stability
Correct Answer: Poisson distribution to dilute cells so wells contain either zero or one cell statistically
Q26. A common contaminant in ascites-derived monoclonal antibody preparations is:
- Host mouse serum proteins including polyclonal IgG
- Synthetic PEG residues embedded in the antibody
- Plant-derived lectins
- Human red blood cells
Correct Answer: Host mouse serum proteins including polyclonal IgG
Q27. How is flow cytometry useful in hybridoma or antibody development?
- To directly fuse B cells with myeloma cells
- To analyze and sort cells based on antigen binding and to measure antibody binding to cell-surface antigens
- To purify antibodies from culture supernatant
- To sterilize cultures before fusion
Correct Answer: To analyze and sort cells based on antigen binding and to measure antibody binding to cell-surface antigens
Q28. What defines a chimeric monoclonal antibody?
- Fully murine variable and constant regions
- Murine variable regions fused to human constant regions
- Completely human antibody produced by mice
- Antibody conjugated to a small molecule drug
Correct Answer: Murine variable regions fused to human constant regions
Q29. What is the biochemical function of HGPRT relevant to HAT selection?
- It degrades amino acids in the culture medium
- It catalyzes the salvage pathway converting hypoxanthine/guanine to nucleotides required for DNA synthesis
- It phosphorylates aminopterin to an active toxic form
- It is a membrane receptor mediating cell fusion
Correct Answer: It catalyzes the salvage pathway converting hypoxanthine/guanine to nucleotides required for DNA synthesis
Q30. Which quality tests are essential for therapeutic monoclonal antibodies produced from hybridomas?
- Mycoplasma testing, endotoxin assay, sterility, and potency/affinity characterization
- Only pH measurement is required
- Gram staining and carbohydrate fermentation tests
- Testing for bacterial plasmid retention
Correct Answer: Mycoplasma testing, endotoxin assay, sterility, and potency/affinity characterization
I am a Registered Pharmacist under the Pharmacy Act, 1948, and the founder of PharmacyFreak.com. I hold a Bachelor of Pharmacy degree from Rungta College of Pharmaceutical Science and Research. With a strong academic foundation and practical knowledge, I am committed to providing accurate, easy-to-understand content to support pharmacy students and professionals. My aim is to make complex pharmaceutical concepts accessible and useful for real-world application.
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