Radioimmunoassay, ELISA and biological standardization MCQs With Answer

Radioimmunoassay, ELISA and biological standardization MCQs With Answer

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This collection of multiple-choice questions focuses on Radioimmunoassay (RIA), Enzyme-Linked Immunosorbent Assay (ELISA), and biological standardization — essential topics in the MPC 201T Advanced Spectral Analysis syllabus for M.Pharm students. The quiz emphasizes theoretical principles and practical considerations: assay formats, labels and detection systems, sensitivity and specificity, sources of error such as matrix and cross-reactivity effects, and approaches to potency estimation and bioassay validation. Questions probe deeper concepts like competitive versus non-competitive designs, tracer selection, calibration and parallel line assay methods, and statistical assumptions behind biological standardization. Use these MCQs to sharpen exam readiness and applied understanding in laboratory assay development and evaluation.

Q1. Which characteristic best explains why 125I is commonly used as a radiolabel in classical radioimmunoassays?

  • High-energy beta emission leading to deep tissue penetration
  • Low gamma energy suitable for sensitive gamma counting and easy labeling of tyrosine residues
  • Short physical half-life reducing radioactive waste concerns
  • Emission of visible light that directly activates photometric detectors

Correct Answer: Low gamma energy suitable for sensitive gamma counting and easy labeling of tyrosine residues

Q2. In a competitive ELISA format for a small hapten drug, what is the main reason a competitive design is chosen over sandwich ELISA?

  • Competitive ELISA provides higher dynamic range for large proteins
  • Sandwich ELISA requires two distinct non-overlapping epitopes which are generally not available on small haptens
  • Competitive ELISA eliminates the need for standard curves
  • Sandwich ELISA cannot use enzyme labels like HRP or AP

Correct Answer: Sandwich ELISA requires two distinct non-overlapping epitopes which are generally not available on small haptens

Q3. During biological standardization by the parallel line assay (PLA), which assumption is critical for valid potency estimation?

  • The dose–response slopes of test and standard are parallel over the chosen response range
  • The test preparation must contain exactly twice the active ingredient of the standard
  • Only binary (yes/no) responses are acceptable for PLA analysis
  • The response must be measured only at a single dose point

Correct Answer: The dose–response slopes of test and standard are parallel over the chosen response range

Q4. Which substrate is most commonly used with horseradish peroxidase (HRP) in ELISA to produce a colorimetric readout for microplate spectrophotometry?

  • p-Nitrophenyl phosphate (pNPP)
  • 3,3′,5,5′-Tetramethylbenzidine (TMB)
  • Radiolabeled iodine (125I)
  • Luminol with alkaline phosphatase

Correct Answer: 3,3′,5,5′-Tetramethylbenzidine (TMB)

Q5. In a RIA measuring insulin, what phenomenon can cause apparent signal suppression when very high analyte concentrations are present and leads to falsely low readings?

  • Hook effect (prozone)
  • Cross-linking enhancement
  • Isotope dilution effect
  • Radiation quenching by the sample matrix

Correct Answer: Hook effect (prozone)

Q6. Which statistical parameter describes the lowest analyte concentration that can be reliably distinguished from zero with defined confidence in immunoassay validation?

  • Limit of Quantification (LOQ)
  • Limit of Detection (LOD)
  • Linear Range Upper Limit
  • Maximum Specificity

Correct Answer: Limit of Detection (LOD)

Q7. Scatchard analysis in RIA is primarily used to:

  • Estimate the half-life of the radioactive tracer
  • Determine binding affinity (Kd) and number of binding sites from equilibrium binding data
  • Measure enzyme turnover in ELISA signal generation
  • Quantify non-specific binding by subtraction of blank values only

Correct Answer: Determine binding affinity (Kd) and number of binding sites from equilibrium binding data

Q8. Which of the following best describes a sandwich ELISA advantage over direct ELISA?

  • Sandwich ELISA requires labeling the antigen which improves specificity
  • Sandwich ELISA offers higher specificity and sensitivity because two antibodies recognize different epitopes of the antigen
  • Sandwich ELISA eliminates the need for blocking steps
  • Sandwich ELISA is only suitable for very small molecules below 1 kDa

Correct Answer: Sandwich ELISA offers higher specificity and sensitivity because two antibodies recognize different epitopes of the antigen

Q9. In biological standardization, the ED50 is defined as:

  • The dose that causes death in 50% of animals
  • The dose required to produce a specified effect in 50% of the maximal response
  • The dose that is 50% of the highest tested concentration
  • The dose that shows 50% cross-reactivity with structurally related compounds

Correct Answer: The dose required to produce a specified effect in 50% of the maximal response

Q10. Which interference is specifically problematic in immunoassays and arises from patient’s endogenous antibodies that bridge assay reagents, causing false signals?

  • Matrix effect from lipemia
  • Heterophile antibodies or human anti-animal antibodies (HAAAs)
  • Enzyme inhibitor contamination from sample collection tubes
  • Photobleaching of chromogenic substrates

Correct Answer: Heterophile antibodies or human anti-animal antibodies (HAAAs)

Q11. Which label would be least appropriate for a homogeneous (no separation) immunoassay that relies on changes in fluorescence upon binding?

  • Fluorescent donor–acceptor pair for FRET
  • Luminescent chelate for time-resolved fluorescence
  • Radioisotope 125I
  • Fluorescent dye attached to antigen

Correct Answer: Radioisotope 125I

Q12. For potency determination of a biological drug using the slope-ratio method, what is directly compared between standard and test preparation?

  • The absolute absorbance at a single concentration
  • The slopes of the log-dose versus response linear segments, assuming common slope if applicable
  • The radioactive counts per minute at zero dose
  • The concentration of impurities measured by HPLC

Correct Answer: The slopes of the log-dose versus response linear segments, assuming common slope if applicable

Q13. Which blocking agent is commonly used in ELISA to reduce non-specific binding to polystyrene plates?

  • Hydrochloric acid
  • Bovine serum albumin (BSA) or non-fat dry milk proteins
  • Radioactive iodine
  • Sodium dodecyl sulfate at 10% (w/v)

Correct Answer: Bovine serum albumin (BSA) or non-fat dry milk proteins

Q14. In RIA equilibrium binding assays, what is the primary purpose of separating bound from free tracer?

  • To measure total radioactivity of the sample without discrimination
  • To quantify specifically the fraction of tracer that is antibody-bound, enabling calculation of analyte concentration
  • To sterilize the sample before counting
  • To induce enzymatic conversion of the tracer for colorimetric readout

Correct Answer: To quantify specifically the fraction of tracer that is antibody-bound, enabling calculation of analyte concentration

Q15. Matrix effects in immunoassays refer to:

  • Only the dilution factor used when preparing standards
  • Alteration of assay signal due to endogenous sample components like lipids, bilirubin, or heterophile antibodies
  • The designed selectivity of the antibody for its epitope
  • The plate coating concentration variability between batches

Correct Answer: Alteration of assay signal due to endogenous sample components like lipids, bilirubin, or heterophile antibodies

Q16. When validating an ELISA for clinical use, which parameter assesses the closeness of measured values to the true value across the assay range?

  • Precision (repeatability)
  • Accuracy (trueness)
  • Specificity
  • Robustness

Correct Answer: Accuracy (trueness)

Q17. Which of the following best describes an advantage of using enzyme labels (e.g., HRP, AP) over radioisotopes in immunoassays?

  • Enzyme labels always yield higher sensitivity than radioisotopes
  • Enzyme labels avoid radiation hazards and typically enable signal amplification with stable, safe reagents
  • Enzyme labels do not require calibration or standard curves
  • Enzyme labels do not suffer from background or matrix interference

Correct Answer: Enzyme labels avoid radiation hazards and typically enable signal amplification with stable, safe reagents

Q18. In potency assays for vaccines using in vivo challenge models, which ethical and scientific consideration has prompted the development of alternative in vitro assays?

  • In vitro assays are always cheaper irrespective of validation costs
  • The 3Rs principle (Replacement, Reduction, Refinement) and desire for more reproducible, mechanistic endpoints to reduce animal use
  • In vivo assays cannot measure potency reliably
  • Regulators prohibit any in vivo testing for potency

Correct Answer: The 3Rs principle (Replacement, Reduction, Refinement) and desire for more reproducible, mechanistic endpoints to reduce animal use

Q19. Which approach is most appropriate to detect and compensate for non-parallelism between the standard and test preparations in a bioassay?

  • Ignore the non-parallelism and report relative potency anyway
  • Dilute samples into the assay’s linear range and investigate causes; consider alternative assay formats or matrix matching if non-parallelism persists
  • Use only the highest concentration point to estimate potency
  • Replace the standard with an unrelated molecule to force parallelism

Correct Answer: Dilute samples into the assay’s linear range and investigate causes; consider alternative assay formats or matrix matching if non-parallelism persists

Q20. Which quality control practice is essential in RIA to account for tracer degradation over time?

  • Assume constant tracer activity and run assays without controls
  • Include time-zero background only
  • Monitor specific activity and include internal standards and control samples in each run to detect tracer decay or altered binding characteristics
  • Only measure samples immediately after adding tracer without incubation

Correct Answer: Monitor specific activity and include internal standards and control samples in each run to detect tracer decay or altered binding characteristics

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