Methods, limitations, and applications of Radio-Immunoassay MCQs With Answer

Introduction

Radioimmunoassay (RIA) is a highly sensitive immunoanalytical technique used in B.Pharm to quantify hormones, drugs and biomarkers by competitive binding between a radiolabelled tracer and unlabeled analyte for a limited antibody. Key methods include competitive and solid‑phase separation, using radiolabels such as iodine‑125 and gamma counting to measure counts per minute (CPM). Important concepts are sensitivity, specificity, cross‑reactivity, standard curves, limit of detection and assay validation. Limitations include radiation safety, tracer half‑life, disposal and potential matrix effects. Applications span clinical diagnostics, therapeutic drug monitoring and pharmacokinetics. Now let’s test your knowledge with 30 MCQs on this topic.

Q1. What is the fundamental principle of radioimmunoassay (RIA)?

• Formation of an immune complex between a labelled antigen and antibody in a competitive binding format
• Direct measurement of radioactivity in a sample without antibodies
• Covalent labelling of antibodies to increase antigen affinity
• Use of enzymes to produce a color change proportional to antigen

Correct Answer: Formation of an immune complex between a labelled antigen and antibody in a competitive binding format

Q2. Which radiolabel is most commonly used in classical RIA laboratories?

• Carbon‑14
• Technetium‑99m
• Iodine‑125
• Fluorine‑18

Correct Answer: Iodine‑125

Q3. In RIA, what is the main role of the tracer?

• To precipitate unbound antibody
• To act as a non‑specific blocker
• To compete with unlabeled analyte for antibody binding and provide a measurable signal
• To alter the antigen’s immunogenicity

Correct Answer: To compete with unlabeled analyte for antibody binding and provide a measurable signal

Q4. Which method is commonly used to separate bound from free fraction in solid‑phase RIA?

• Gel electrophoresis
• Solid phase precipitation by antibody immobilized on tubes or beads
• Thin layer chromatography
• Liquid‑liquid extraction

Correct Answer: Solid phase precipitation by antibody immobilized on tubes or beads

Q5. What does the term B/B0 represent in RIA data analysis?

• Binding at equilibrium divided by baseline binding of a heterologous assay
• Bound radioactivity in sample divided by bound radioactivity in zero standard (percent binding)
• Background counts divided by baseline counts
• Booster effect in antibody affinity maturation

Correct Answer: Bound radioactivity in sample divided by bound radioactivity in zero standard (percent binding)

Q6. The hook (prozone) effect in RIA results in which erroneous outcome when analyte concentration is extremely high?

• Falsely high measured concentrations due to signal saturation
• Falsely low measured concentrations due to antibody saturation and reduced complex formation
• Improved assay precision
• No effect; RIA is immune to prozone phenomena

Correct Answer: Falsely low measured concentrations due to antibody saturation and reduced complex formation

Q7. Which factor is a primary limitation of RIA compared with non‑radioactive immunoassays?

• Lower sensitivity than colorimetric assays
• Requirement for radioactive handling, regulatory controls and waste disposal
• Inability to measure small molecules
• Complete lack of specificity

Correct Answer: Requirement for radioactive handling, regulatory controls and waste disposal

Q8. Which clinical analyte is classically measured by RIA?

• Serum creatinine
• Thyroxine (T4) and other hormones
• Complete blood count
• Blood glucose by enzymatic assay

Correct Answer: Thyroxine (T4) and other hormones

Q9. In constructing an RIA standard curve, what is typically plotted on the x‑axis and y‑axis?

• X: percent bound; Y: linear concentration
• X: log concentration; Y: percent bound or B/B0
• X: time; Y: radioactivity decay
• X: antibody concentration; Y: tracer half‑life

Correct Answer: X: log concentration; Y: percent bound or B/B0

Q10. Cross‑reactivity in RIA refers to:

• The tendency of the tracer to degrade in storage
• The antibody binding to structurally related compounds causing false readings
• Non‑specific adsorption of radioactivity to labware
• The variation of CPM with instrument temperature

Correct Answer: The antibody binding to structurally related compounds causing false readings

Q11. Which detector is used to measure I‑125 radioactivity in RIA?

• Beta counter
• Gamma counter
• Mass spectrometer
• Fluorimeter

Correct Answer: Gamma counter

Q12. Radiochemical purity of the tracer is important because:

• Impure tracer increases assay sensitivity
• Impurities can produce non‑specific signals and degrade accuracy
• It affects only the color of the reagent
• It determines antibody affinity directly

Correct Answer: Impurities can produce non‑specific signals and degrade accuracy

Q13. Which step helps reduce non‑specific binding in RIA?

• Omitting washing steps
• Adding blocking proteins (e.g., BSA) and thorough washing
• Increasing incubation temperature to boiling
• Using unpurified serum as diluent

Correct Answer: Adding blocking proteins (e.g., BSA) and thorough washing

Q14. Which of the following best describes the competitive format typical of RIA for small molecules?

• Analyte is sandwiched between two antibodies
• Labeled analyte and unlabeled analyte compete for a limited number of antibody binding sites
• Antibody is labeled and directly quantified
• Antigen is immobilized and detected by enzyme color change

Correct Answer: Labeled analyte and unlabeled analyte compete for a limited number of antibody binding sites

Q15. Limit of detection (LOD) in RIA is influenced mainly by:

• Only the antibody concentration
• Background counts, tracer specific activity, and assay precision at low signal
• The color change intensity
• The molecular weight of analyte exclusively

Correct Answer: Background counts, tracer specific activity, and assay precision at low signal

Q16. Why is tracer half‑life important when planning RIA experiments?

• Long half‑life always reduces sensitivity
• Half‑life determines usable shelf life, timing of assays and decay correction for CPM
• Short half‑life eliminates need for calibration
• Tracer half‑life affects only the antibody specificity

Correct Answer: Half‑life determines usable shelf life, timing of assays and decay correction for CPM

Q17. Which factor is commonly used to express assay precision in RIA quality control?

• Limit of detection
• Coefficient of variation (CV %) of replicates
• Tracer molecular weight
• Number of antibodies used

Correct Answer: Coefficient of variation (CV %) of replicates

Q18. An advantage of RIA over bioassays is:

• Lower regulatory requirements
• Higher specificity and quantitative sensitivity for low‑abundance analytes
• No need for trained personnel
• Unlimited shelf‑life of reagents

Correct Answer: Higher specificity and quantitative sensitivity for low‑abundance analytes

Q19. Which sample matrix effect can alter RIA results if not controlled?

• Hemolysis or lipemia causing interference with binding or counts
• Ambient room color affecting radioactivity
• Seasonal variation of tracer isotope
• Patient hair color

Correct Answer: Hemolysis or lipemia causing interference with binding or counts

Q20. For measurement of therapeutic drug levels (e.g., digoxin) RIA is useful because:

• It measures only metabolites not parent drug
• It cannot detect concentrations in therapeutic range
• It can sensitively quantify low levels in biological fluids for monitoring
• It requires no calibration standards

Correct Answer: It can sensitively quantify low levels in biological fluids for monitoring

Q21. How is a sample concentration determined from RIA results?

• By direct reading of CPM without reference to standards
• By interpolating the sample percent bound (B/B0) on the standard curve
• By measuring sample viscosity
• By counting only background counts

Correct Answer: By interpolating the sample percent bound (B/B0) on the standard curve

Q22. Which validation parameter ensures the assay yields accurate values near clinical decision limits?

• Linearity or reportable range assessment
• Tracer half‑life determination
• Sterility testing
• Colorimetric calibration

Correct Answer: Linearity or reportable range assessment

Q23. Why have many labs replaced RIA with ELISA or non‑radioactive methods?

• RIA has superior safety and lower cost than ELISA
• ELISA avoids radioactivity, has easier waste handling and similar sensitivity for many analytes
• ELISA cannot measure hormones
• RIA reagents are universally available without regulation

Correct Answer: ELISA avoids radioactivity, has easier waste handling and similar sensitivity for many analytes

Q24. Specific activity of a radiolabelled tracer refers to:

• The radioactivity per unit mass of tracer (e.g., Ci/mmol)
• The temperature at which tracer melts
• The concentration of antibody in the assay
• The number of binding sites on the antigen

Correct Answer: The radioactivity per unit mass of tracer (e.g., Ci/mmol)

Q25. Which practice is essential for radioactive safety in an RIA laboratory?

• Using open benches for dispersion of isotopes
• Proper shielding, monitoring, using PPE and designated waste containers
• Incinerating radioactivity with general waste
• Storing isotopes at room temperature without labeling

Correct Answer: Proper shielding, monitoring, using PPE and designated waste containers

Q26. Which assay design is generally NOT suitable for small analytes like steroids?

• Competitive RIA
• Sandwich immunoassay requiring two antibodies binding distinct epitopes
• Heterologous competitive assay
• Solid phase competitive assay

Correct Answer: Sandwich immunoassay requiring two antibodies binding distinct epitopes

Q27. Counts per minute (CPM) measured in RIA reflect:

• The number of antibody molecules present
• The detected radioevents from the radiolabel in the measured fraction
• The volume of sample added
• The pH of the reaction mixture

Correct Answer: The detected radioevents from the radiolabel in the measured fraction

Q28. A practical way to check for non‑specific binding in an RIA run is to include:

• Only high concentration standards
• A zero‑antigen blank and non‑immune serum control
• Excess tracer without antibody
• A calibration curve with no standards

Correct Answer: A zero‑antigen blank and non‑immune serum control

Q29. During RIA QC, an unexpectedly high coefficient of variation (CV) between replicates suggests:

• Excellent assay performance
• Instrument or pipetting error, inconsistent reagent addition or poor mixing
• That the analyte concentration is optimal
• That tracer purity has improved

Correct Answer: Instrument or pipetting error, inconsistent reagent addition or poor mixing

Q30. Regulatory and documentation requirements for RIA include which of the following?

• No record keeping is needed for radioactive reagents
• Detailed records of isotope inventory, personnel monitoring, waste disposal and assay validation
• Only verbal authorization for isotope purchase
• Disposal of records after a single day

Correct Answer: Detailed records of isotope inventory, personnel monitoring, waste disposal and assay validation

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