Immunoassays: RIA, ELISA, luminescence assays — principles and quantification MCQs With Answer

Introduction

This quiz collection focuses on immunoassays — Radioimmunoassay (RIA), Enzyme‑Linked Immunosorbent Assay (ELISA), and luminescence‑based assays — emphasizing core principles and quantification methods relevant to M.Pharm students. Questions probe assay formats (competitive, sandwich), labels (125I, HRP, AP, luciferase), detection modes (colorimetric, chemiluminescent, time‑resolved fluorescence), calibration and curve fitting (4‑parameter logistic), analytical figures of merit (LOD, LOQ, linear range, hook effect), and assay validation (precision, accuracy, cross‑reactivity). The items are designed to deepen conceptual understanding and practical interpretation needed for designing, optimizing, and validating immunoassays in pharmaceutical analysis.

Q1. Which characteristic best explains why 125I is commonly used as a tracer in RIA?

• High beta emission energy that allows scintillation counting
• Short half‑life minimizing radioactive waste
• Gamma emission suitable for high‑sensitivity detection and stable labeling of proteins
• Inherent chemiluminescent properties for easy signal generation

Correct Answer: Gamma emission suitable for high‑sensitivity detection and stable labeling of proteins

Q2. In a competitive ELISA for a small drug molecule, how does increasing sample analyte concentration affect the measured signal when using an enzyme‑labeled antigen tracer?

• The signal increases proportionally with analyte concentration
• The signal decreases because free analyte competes with labeled antigen for antibody binding
• The signal remains unchanged because competition does not affect enzyme activity
• The signal first increases then decreases due to hook effect

Correct Answer: The signal decreases because free analyte competes with labeled antigen for antibody binding

Q3. Which ELISA format is most appropriate for quantifying large proteins where two non‑overlapping epitopes are available?

• Competitive ELISA using labeled antigen
• Indirect ELISA with secondary antibody detection
• Sandwich (capture) ELISA using capture and detection antibodies
• Direct ELISA employing only labeled primary antibody

Correct Answer: Sandwich (capture) ELISA using capture and detection antibodies

Q4. What is the major analytical advantage of time‑resolved fluorescence (TRF) over conventional fluorescence detection in immunoassays?

• Shorter measurement time due to faster decay of lanthanide labels
• Lower background by time‑gating measurements because lanthanide labels have long emission lifetimes
• Elimination of the need for calibration curves
• Compatibility only with enzymatic substrates such as TMB

Correct Answer: Lower background by time‑gating measurements because lanthanide labels have long emission lifetimes

Q5. Which substrate–enzyme pair is correctly matched for a colorimetric ELISA read at 450 nm after stop solution?

• Alkaline phosphatase (AP) with luminol
• Horseradish peroxidase (HRP) with TMB (stopped with acid)
• HRP with p‑nitrophenyl phosphate (pNPP)
• Luciferase with TMB

Correct Answer: Horseradish peroxidase (HRP) with TMB (stopped with acid)

Q6. Which statement best describes the high‑dose “hook” effect in sandwich immunoassays?

• Very high analyte concentrations give falsely high signals due to enzyme saturation
• Very high analyte concentrations give falsely low signals because capture and detection antibodies are saturated separately, preventing sandwich formation
• High doses increase nonspecific binding that enhances signal linearly
• Hook effect occurs only in competitive assays and leads to plateauing of the signal

Correct Answer: Very high analyte concentrations give falsely low signals because capture and detection antibodies are saturated separately, preventing sandwich formation

Q7. Which parameter is most appropriate to describe the lowest analyte concentration that can be reliably distinguished from a blank in immunoassay validation?

• Linearity range
• Limit of detection (LOD)
• Limit of quantification (LOQ)
• Dynamic range

Correct Answer: Limit of detection (LOD)

Q8. When fitting ELISA calibration data for a typical sigmoidal dose–response, which mathematical model is most commonly employed for accurate interpolation of unknowns?

• Linear regression through the origin
• Four‑parameter logistic (4PL) curve
• Exponential decay model
• Simple polynomial of degree 2

Correct Answer: Four‑parameter logistic (4PL) curve

Q9. Which detection method offers the highest analytical sensitivity for immunoassays in routine laboratory practice?

• Colorimetric ELISA with direct measurement at 595 nm
• Chemiluminescent detection using enhanced luminol systems
• Visual endpoint color comparison without instrumentation
• Absorbance measurement of AP/pNPP at 405 nm with plate reader

Correct Answer: Chemiluminescent detection using enhanced luminol systems

Q10. In RIA, what is the primary purpose of performing a bound/free separation step?

• To remove radioactive decay products from the tracer
• To separate antibody‑bound labeled tracer from unbound labeled tracer so that signal correlates with analyte concentration
• To increase the half‑life of the isotope used
• To convert gamma counts into luminescent units

Correct Answer: To separate antibody‑bound labeled tracer from unbound labeled tracer so that signal correlates with analyte concentration

Q11. How is percent cross‑reactivity of an interfering compound with the assay analyte most practically assessed?

• By comparing molecular weights of analyte and interferent
• By calculating ratio of concentrations producing equal assay response (IC50 or ED50 comparison) and expressing as percentage
• By measuring color intensity of each compound at 450 nm
• By determining half‑life of both compounds in plasma

Correct Answer: By calculating ratio of concentrations producing equal assay response (IC50 or ED50 comparison) and expressing as percentage

Q12. Which of the following assay adjustments most directly reduces nonspecific binding in an ELISA?

• Increasing incubation temperature to denature nonspecific proteins
• Using an appropriate blocking buffer (e.g., BSA or skim milk) and optimized wash steps
• Using a higher concentration of tracer antigen to outcompete nonspecific interactions
• Eliminating the wash steps to preserve weak interactions

Correct Answer: Using an appropriate blocking buffer (e.g., BSA or skim milk) and optimized wash steps

Q13. In kinetic (rate) ELISA readouts, which quantity is measured to infer analyte concentration?

• Total accumulated color after an indefinite incubation time
• Initial reaction rate (slope) of substrate turnover per unit time
• Absolute enzyme concentration in the sample independent of antibody binding
• Endpoint luminescence intensity after substrate depletion

Correct Answer: Initial reaction rate (slope) of substrate turnover per unit time

Q14. Which luminescence assay type relies on an enzyme that oxidizes luciferin in the presence of ATP to emit light?

• Electrochemiluminescence using ruthenium complexes
• Bioluminescence using luciferase
• Chemiluminescence using luminol and HRP
• Time‑resolved fluorescence using europium chelates

Correct Answer: Bioluminescence using luciferase

Q15. During ELISA validation, which metric expresses repeatability as the ratio of standard deviation to mean, typically reported as a percentage?

• Bias percentage
• Recovery percentage
• Coefficient of variation (CV%)
• Signal‑to‑noise ratio

Correct Answer: Coefficient of variation (CV%)

Q16. Which approach is best to extend the dynamic range of an ELISA without compromising accuracy at low concentrations?

• Always use a single undiluted standard for all samples
• Prepare serial dilutions of samples and use parallel calibration curves or fitting using 4PL
• Increase incubation times uniformly for all standards and samples
• Omit blocking to increase signal for low concentrations

Correct Answer: Prepare serial dilutions of samples and use parallel calibration curves or fitting using 4PL

Q17. What is the principal drawback of using RIA compared with non‑radioactive immunoassays in modern pharmaceutical analysis?

• Lower sensitivity compared with colorimetric ELISA
• Regulatory, safety, and disposal issues associated with radioactive materials
• Inability to measure small molecules
• Absence of commercially available antibodies for RIA

Correct Answer: Regulatory, safety, and disposal issues associated with radioactive materials

Q18. Which calibration strategy is most suitable when matrix effects significantly influence immunoassay signal?

• Use buffer calibration standards prepared in distilled water only
• Use matrix‑matched calibration standards prepared in the same biological matrix as samples or use standard addition
• Rely solely on manufacturer kit standards without further verification
• Calibrate using arbitrary units derived from internal enzyme activity

Correct Answer: Use matrix‑matched calibration standards prepared in the same biological matrix as samples or use standard addition

Q19. What is the mechanistic reason chemiluminescent assays can achieve lower background than conventional absorbance ELISA?

• Chemiluminescent reactions require very high temperatures that reduce background
• Signal generation is an emission reaction with virtually no ambient light‑absorbing matrix signal, enabling high signal‑to‑noise ratios
• Catalytic turnover is absent so only single events are counted
• Chemiluminescent substrates produce colored products that are read at noninterfering wavelengths

Correct Answer: Signal generation is an emission reaction with virtually no ambient light‑absorbing matrix signal, enabling high signal‑to‑noise ratios

Q20. In a competitive RIA, the measured bound radioactivity is inversely proportional to analyte concentration because:

• Antibody affinity decreases with analyte presence
• Free analyte displaces labeled tracer from antibody binding sites, reducing the bound fraction of tracer
• Radiation quenching increases in presence of analyte
• Tracer is enzymatically degraded by analyte

Correct Answer: Free analyte displaces labeled tracer from antibody binding sites, reducing the bound fraction of tracer

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