Principles and components of Radio-Immunoassay (RIA) MCQs With Answer

Radio-Immunoassay (RIA) is a highly sensitive analytical method that quantifies trace amounts of hormones, drugs, and biomolecules by competitive binding between radiolabeled tracers and unlabeled analytes for a specific antibody. Principles and components of RIA include antigen–antibody interactions, radiolabeled tracer, separation techniques, detectors, standard curves, and quality control. B. Pharm students should understand radioisotopes (e.g., I-125), tracer specific activity, assay sensitivity, specificity, cross-reactivity, safety and waste handling, and validation parameters relevant to clinical pharmacology, therapeutic drug monitoring, and pharmacokinetics. This focused review prepares students for laboratory practice, data interpretation, and regulatory compliance. Now let’s test your knowledge with 30 MCQs on this topic.

Q1. Which fundamental principle best describes a competitive RIA?

• Unlabeled analyte and radiolabeled tracer compete for limited specific antibody binding sites
• Antibody is radiolabeled and directly measured after binding to antigen
• Tracer binds irreversibly to a solid phase and signal is proportional to tracer amount
• Analyte is measured by enzymatic conversion to a radioactive product

Correct Answer: Unlabeled analyte and radiolabeled tracer compete for limited specific antibody binding sites

Q2. Which component determines RIA specificity most directly?

• Type of radioisotope used for labeling
• Affinity and selectivity of the antibody for the antigen
• Counting efficiency of the detector
• Concentration of the radiolabeled tracer only

Correct Answer: Affinity and selectivity of the antibody for the antigen

Q3. Which radioisotope is most commonly used in classical protein/hormone RIA due to suitable half-life and gamma emission?

• Carbon-14 (C-14)
• Phosphorus-32 (P-32)
• Iodine-125 (I-125)
• Tritium (H-3)

Correct Answer: Iodine-125 (I-125)

Q4. What is meant by “specific activity” of a radiolabeled tracer in RIA?

• Ratio of radiolabel atoms to antibody molecules
• Radioactivity per unit mass of labeled compound
• Total radioactivity added to the assay
• Number of counts detected per minute

Correct Answer: Radioactivity per unit mass of labeled compound

Q5. In a competitive RIA standard curve, what does B/B0 represent?

• Background-subtracted counts divided by zero standard counts
• Bound tracer counts at a given analyte concentration divided by bound tracer counts with zero analyte
• Ratio of bound to free tracer at equilibrium
• Total activity divided by blank activity

Correct Answer: Bound tracer counts at a given analyte concentration divided by bound tracer counts with zero analyte

Q6. Which separation method isolates bound from free tracer using a second antibody or precipitation?

• Solid-phase separation only
• Double-antibody precipitation
• Gel filtration with ultracentrifugation exclusively
• Direct counting without separation

Correct Answer: Double-antibody precipitation

Q7. Which detector is typically used to measure emissions from I-125 in RIA?

• Beta scintillation counter
• Liquid scintillation counter for beta emitters
• Gamma counter calibrated for low-energy photons
• Geiger-Müller tube for alpha particles

Correct Answer: Gamma counter calibrated for low-energy photons

Q8. What is the main advantage of RIA over many classical chemical assays?

• Lower cost of reagents
• Higher throughput with no safety requirements
• Exceptional sensitivity for picogram to femtogram levels
• No need for calibration standards

Correct Answer: Exceptional sensitivity for picogram to femtogram levels

Q9. Which factor most contributes to assay sensitivity in RIA?

• High antibody affinity and low non-specific binding
• Use of high sample volumes regardless of antibody quality
• Counting only the free tracer fraction
• Short incubation times to prevent equilibrium

Correct Answer: High antibody affinity and low non-specific binding

Q10. What is the “hook effect” (prozone effect) in immunoassays such as RIA?

• Loss of signal due to detector saturation by high radioactivity
• Apparent low measured concentration when analyte concentration is extremely high
• Increased background counts from shield leakage
• Decreased antibody affinity at low temperatures

Correct Answer: Apparent low measured concentration when analyte concentration is extremely high

Q11. For valid quantitation, why must RIA include standards and controls?

• Standards provide calibration and controls monitor precision and accuracy
• Controls are optional if instrument sensitivity is high
• Standards are only for method development and not routine runs
• Controls replace the need for blanks and background correction

Correct Answer: Standards provide calibration and controls monitor precision and accuracy

Q12. Which parameter describes how close repeated RIA measurements are to each other?

• Accuracy
• Specificity
• Precision
• Sensitivity

Correct Answer: Precision

Q13. Cross-reactivity in RIA refers to:

• Interference by nonradioactive contaminants that quench counts
• Non-specific binding of tracer to assay tubes
• Recognition and binding of structurally related compounds by the antibody
• Loss of radioactivity due to decay

Correct Answer: Recognition and binding of structurally related compounds by the antibody

Q14. Which sample preparation step is commonly required for steroid measurement by RIA?

• Direct dilution into phosphate buffer with no extraction
• Organic solvent extraction or solid-phase extraction to remove binding proteins
• Heating samples to 100°C to denature proteins
• Acid hydrolysis to release conjugates

Correct Answer: Organic solvent extraction or solid-phase extraction to remove binding proteins

Q15. Which quality control metric describes the lowest analyte amount that can be distinguished from zero with a defined confidence?

• Specificity
• Limit of detection (LOD)
• Linearity range
• Precision error percentage

Correct Answer: Limit of detection (LOD)

Q16. In optimizing an RIA, increasing antibody concentration generally causes which change in the standard curve?

• Decrease in maximum bound tracer (B0) and shift of curve to higher signal
• Increase in B0 and a shift making assay less sensitive
• Lower free tracer counts and increased assay sensitivity until saturation effects
• No effect on curve shape or sensitivity

Correct Answer: Lower free tracer counts and increased assay sensitivity until saturation effects

Q17. When preparing a radiolabeled tracer, why is radiochemical purity important?

• Impure tracer reduces specific activity but does not affect binding
• Impurities can alter binding characteristics and increase non-specific signal
• Purity is only relevant for regulatory paperwork, not assay performance
• Radiochemical purity determines the antibody sequence

Correct Answer: Impurities can alter binding characteristics and increase non-specific signal

Q18. What is the primary safety consideration specific to RIA that differs from non-radioactive assays?

• Higher risk of chemical burns from assay buffers
• Regulatory and waste disposal procedures for radioactive materials and operator dose monitoring
• Need for sterile technique to prevent contamination
• No special safety considerations beyond PPE

Correct Answer: Regulatory and waste disposal procedures for radioactive materials and operator dose monitoring

Q19. Which mathematical transformation is commonly used to linearize RIA competitive curves for interpolation?

• Logit-log or log-logit transformation
• Square-root transform only
• No transformation; linear interpolation is always valid
• Reciprocal of counts without log transformation

Correct Answer: Logit-log or log-logit transformation

Q20. Why is decay correction necessary when comparing RIA results over time?

• To adjust for daily changes in sample pH
• To account for reduction in tracer radioactivity due to physical decay affecting counts
• To modify antibody affinity values
• Decay correction is not needed if standards are run each time

Correct Answer: To account for reduction in tracer radioactivity due to physical decay affecting counts

Q21. Which factor can cause matrix effects that distort RIA measurements?

• Presence of heterophilic antibodies, lipids, hemolysis, or binding proteins in the sample
• Using an antibody with very high affinity only
• Counting with a properly calibrated gamma counter
• Strict temperature control during incubation

Correct Answer: Presence of heterophilic antibodies, lipids, hemolysis, or binding proteins in the sample

Q22. In a non-competitive (sandwich) immunoassay, why is RIA less commonly used for this format?

• Radiolabeling antibodies typically generates unstable reagents and safety issues
• Sandwich assays do not require specificity
• RIA cannot detect proteins larger than 10 kDa
• Sandwich format is inherently incompatible with antibody binding

Correct Answer: Radiolabeling antibodies typically generates unstable reagents and safety issues

Q23. What is the role of assay blank in RIA?

• Defines maximum bound activity with no tracer added
• Measures background signal from reagents and tubes for subtraction
• Contains the highest concentration standard for calibration
• Replaces the lowest standard when limits are narrow

Correct Answer: Measures background signal from reagents and tubes for subtraction

Q24. Which best practice reduces non-specific binding in RIA?

• Use of non-ionic detergents, blocking proteins, or appropriate wash steps
• Omitting separation steps to prevent loss of bound fraction
• Increasing incubation temperature without buffer changes
• Using expired tracer to reduce background

Correct Answer: Use of non-ionic detergents, blocking proteins, or appropriate wash steps

Q25. When validating an RIA for a new analyte, which parameter assesses proportional accuracy across the assay range?

• Limit of detection
• Linearity (or linear range and linearity of dilution)
• Specific activity
• Tracer half-life

Correct Answer: Linearity (or linear range and linearity of dilution)

Q26. How does antibody affinity affect the equilibrium time in an RIA?

• Higher affinity antibodies generally reach equilibrium faster and form more stable complexes
• Affinity has no effect on equilibrium; only tracer activity matters
• Lower affinity antibodies always produce lower background and faster equilibrium
• Higher affinity prolongs dissociation so equilibrium is never achieved

Correct Answer: Higher affinity antibodies generally reach equilibrium faster and form more stable complexes

Q27. In RIA kit instructions, the “B0” tube refers to which condition?

• Tube containing only buffer with no tracer or antibody
• Tube with maximum bound tracer in absence of competing unlabeled analyte
• Tube containing the highest concentration standard
• Tube used to measure free tracer only

Correct Answer: Tube with maximum bound tracer in absence of competing unlabeled analyte

Q28. What is a common method to prepare a radioactive standard curve in RIA?

• Prepare serial dilutions of a known unlabeled analyte and measure B/B0 for each
• Measure only one standard and extrapolate the rest
• Use increasing amounts of antibody rather than analyte to generate the curve
• Count standards without any antibody to derive curve

Correct Answer: Prepare serial dilutions of a known unlabeled analyte and measure B/B0 for each

Q29. Which regulatory/documentation practice is essential when working with RIA in a B.Pharm lab?

• Keeping informal verbal records of counts
• Documented SOPs, radioactive material logs, waste records, and personnel dosimetry
• Disposing all radioactive waste in regular trash after dilution
• Sharing tracer between labs without inventory control

Correct Answer: Documented SOPs, radioactive material logs, waste records, and personnel dosimetry

Q30. Comparing RIA to non-radioactive immunoassays like ELISA, which statement is accurate?

• RIA always has better specificity but drastically lower sensitivity than ELISA
• RIA generally offers superior sensitivity for low-abundance analytes but involves radioactivity handling and disposal; ELISA avoids radioactivity and is safer for routine use
• ELISA cannot quantify small molecules; only RIA can
• RIA requires no calibration while ELISA does

Correct Answer: RIA generally offers superior sensitivity for low-abundance analytes but involves radioactivity handling and disposal; ELISA avoids radioactivity and is safer for routine use

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