Radioimmunoassay principles MCQs With Answer

Radioimmunoassay (RIA) is a classical and highly sensitive analytical technique that uses radiolabeled antigens or antibodies to measure very low concentrations of hormones, drugs, and other biomolecules. This set of MCQs is designed for M.Pharm students to deepen understanding of RIA principles, including tracer selection, antibody-antigen binding kinetics, competitive versus noncompetitive formats, separation techniques, and data interpretation such as %bound, B/B0, and calibration curves. Practical considerations like assay sensitivity, specificity, cross-reactivity, the hook effect, and safety in handling radioisotopes are also covered. These questions and answers will aid exam preparation and reinforce laboratory application of immunotechnology concepts.

Q1. Which radioactive isotope is most commonly used as a tracer in routine radioimmunoassays due to its suitable half-life and gamma emission?

• Iodine-125
• Carbon-14
• Phosphorus-32

Correct Answer: Iodine-125

Q2. Radioimmunoassay primarily relies on which principle to quantify an unlabeled analyte?

• Competition between labeled and unlabeled antigen for a limited number of antibody binding sites
• Direct measurement of antibody enzymatic activity
• Electrochemical detection of antigen-antibody complexes
• Separation of free antigen by chromatography

Correct Answer: Competition between labeled and unlabeled antigen for a limited number of antibody binding sites

Q3. In a standard competitive RIA, what happens to the bound radioactivity as the concentration of unlabeled antigen in the sample increases?

• Bound radioactivity decreases
• Bound radioactivity increases
• Bound radioactivity remains constant
• Bound radioactivity fluctuates randomly

Correct Answer: Bound radioactivity decreases

Q4. Which separation method is commonly used in RIA to separate bound from free tracer by precipitating the antibody complex?

• PEG (polyethylene glycol) precipitation
• High-performance liquid chromatography
• Dialysis
• Size-exclusion filtration without antibody

Correct Answer: PEG (polyethylene glycol) precipitation

Q5. The parameter B0 in the context of RIA refers to which of the following?

• Bound radioactivity in the absence of unlabeled competitor (maximum binding)
• Background radioactivity from the buffer alone
• Baseline drift in the gamma counter
• Binding at zero incubation time

Correct Answer: Bound radioactivity in the absence of unlabeled competitor (maximum binding)

Q6. Sensitivity of an RIA is best described as:

• The lowest concentration of analyte that can be distinguished from zero with defined confidence
• The maximum concentration measurable without dilution
• The degree of cross-reactivity with similar compounds
• The time required to reach equilibrium

Correct Answer: The lowest concentration of analyte that can be distinguished from zero with defined confidence

Q7. Which factor most directly affects the specificity of a radioimmunoassay?

• The antibody’s cross-reactivity profile
• The gamma counter model used
• The scintillation fluid brand
• The room temperature during counting

Correct Answer: The antibody’s cross-reactivity profile

Q8. The “hook effect” in immunoassays can lead to which of the following artifacts?

• False low results at very high analyte concentrations
• False high results at low analyte concentrations
• Increased assay precision
• Complete loss of signal due to tracer decay

Correct Answer: False low results at very high analyte concentrations

Q9. In RIA kinetics, equilibrium between labeled antigen, unlabeled antigen, and antibody is important because:

• Only at equilibrium does the bound/free ratio accurately reflect analyte concentration
• Equilibrium eliminates the need for separation
• Equilibrium prevents tracer decay
• Equilibrium increases antibody cross-reactivity

Correct Answer: Only at equilibrium does the bound/free ratio accurately reflect analyte concentration

Q10. Which of the following calculations is commonly used to transform raw counts to a normalized value for calibration curves in RIA?

• Percent bound (%B) or B/B0
• Signal-to-noise ratio (S/N)
• Specific activity per mole
• Half-life correction factor only

Correct Answer: Percent bound (%B) or B/B0

Q11. A high-affinity antibody in an RIA will result in which of the following assay characteristics?

• Increased sensitivity and lower detection limits
• Complete nonspecific binding
• Faster tracer decay
• Broader standard curve requiring more dilutions

Correct Answer: Increased sensitivity and lower detection limits

Q12. Which of the following is an advantage of using I-125 as a tracer over tritium (H-3) in RIA?

• Higher energy gamma emission allows easier and more precise counting
• Shorter half-life reducing waste concerns
• No shielding required during handling
• It does not require purification after labeling

Correct Answer: Higher energy gamma emission allows easier and more precise counting

Q13. Cross-reactivity in RIA is best defined as:

• The degree to which structurally similar compounds compete with the analyte for antibody binding
• The affinity of the tracer for the gamma counter
• The amount of background radiation from lab equipment
• The percentage of labeled antigen that is bound

Correct Answer: The degree to which structurally similar compounds compete with the analyte for antibody binding

Q14. During assay validation, which parameter assesses the closeness of measured values to the true value?

• Accuracy
• Precision
• Linearity
• Robustness

Correct Answer: Accuracy

Q15. In a competitive RIA, what does a Scatchard plot primarily provide?

• Estimates of antibody affinity and binding site concentration
• Direct measure of tracer specific activity
• Gamma counter calibration factors
• Stability profile of the radiolabeled antigen

Correct Answer: Estimates of antibody affinity and binding site concentration

Q16. Which sample matrix effect can interfere with RIA and often requires matrix-matched standards or extraction?

• Presence of binding proteins or endogenous antibodies that alter free analyte availability
• Ambient light exposure during incubation
• Using a different gamma counter brand
• Tracer half-life variability

Correct Answer: Presence of binding proteins or endogenous antibodies that alter free analyte availability

Q17. What is the primary safety consideration when performing RIA in the laboratory?

• Minimizing radiation exposure by time, distance, and shielding and proper radioactive waste disposal
• Ensuring pH of all buffers is exactly 7.00
• Always using fresh antibodies for every sample
• Using only disposable plasticware to avoid contamination

Correct Answer: Minimizing radiation exposure by time, distance, and shielding and proper radioactive waste disposal

Q18. Which analytical step reduces nonspecific binding in an RIA?

• Inclusion of blocking proteins or detergents in the assay buffer
• Increasing tracer activity indefinitely
• Reducing antibody concentration to zero
• Avoiding incubation to prevent equilibrium

Correct Answer: Inclusion of blocking proteins or detergents in the assay buffer

Q19. The specific activity of a radiolabeled antigen refers to:

• The radioactivity per unit mass or mole of labeled compound
• The affinity constant of the antibody
• The proportion of bound to free antigen at equilibrium
• The incubation time required for equilibrium

Correct Answer: The radioactivity per unit mass or mole of labeled compound

Q20. When converting RIA raw counts to concentration using a standard curve, which interpolation method is commonly used for better accuracy over a wide range?

• Four-parameter logistic (4PL) or log-logit transformations of the calibration curve
• Simple linear regression through origin only
• Arithmetic mean of standards without fitting
• Using the median standard value for all samples

Correct Answer: Four-parameter logistic (4PL) or log-logit transformations of the calibration curve

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