Immunoassay evaluation and protocol preparation (digoxin, insulin) MCQs With Answer

Introduction

This MCQ set focuses on immunoassay evaluation and protocol preparation for digoxin and insulin—key topics in Pharmacological and Toxicological Screening Methods-I for M.Pharm students. Questions are designed to deepen understanding of assay validation parameters (sensitivity, specificity, precision, accuracy, LOD/LLOQ), matrix effects, cross-reactivity, calibration and QC strategies, and practical protocol steps (sample collection, incubation, wash, conjugates, and detection). Emphasis is placed on digoxin and insulin assay nuances: therapeutic ranges, cross-reacting compounds, dilutional linearity, and interference such as biotin and heterophile antibodies. Answers are provided to aid preparation for exams and lab protocol design.

Q1. What is the most appropriate definition of the lower limit of quantification (LLOQ) in immunoassay validation?

• The lowest analyte concentration that gives a signal distinguishable from blank with CV ≤20% and accuracy within ±20%
• The lowest concentration that can be detected but not necessarily quantified with acceptable precision
• The concentration at which the calibration curve deviates from linearity
• The highest concentration above which hook effect occurs

Correct Answer: The lowest analyte concentration that gives a signal distinguishable from blank with CV ≤20% and accuracy within ±20%

Q2. Which regression model is most commonly recommended for calibration curve fitting in sandwich immunoassays for hormones like insulin?

• Simple linear regression
• Log-log linear regression
• 4-parameter logistic (4PL) or 5-parameter logistic (5PL) non-linear regression
• Polynomial of degree 3

Correct Answer: 4-parameter logistic (4PL) or 5-parameter logistic (5PL) non-linear regression

Q3. During validation, which experiment best detects a high-dose (hook) effect in a sandwich immunoassay for digoxin?

• Spike-recovery at a single low concentration
• Serial dilution of very high-concentration samples to assess non-linearity
• Comparing two different anticoagulants
• Measuring blank samples multiple times

Correct Answer: Serial dilution of very high-concentration samples to assess non-linearity

Q4. Which anticoagulant is generally preferred for routine insulin immunoassays to minimize proteolytic degradation and preserve peptide analytes?

• EDTA plasma
• Heparin plasma
• Serum without anticoagulant
• Citrate plasma

Correct Answer: EDTA plasma

Q5. What is the main purpose of including low, medium, and high quality control (QC) samples in every immunoassay run?

• To increase the sample throughput
• To evaluate assay stability across the calibration range and detect run-to-run variability
• To replace the need for a standard curve
• To reduce the incubation time required for the assay

Correct Answer: To evaluate assay stability across the calibration range and detect run-to-run variability

Q6. Which of the following acceptance criteria is commonly applied to within-run (intra-assay) precision for bioanalytical immunoassays (except at LLOQ)?

• CV ≤5%
• CV ≤15%
• CV ≤30%
• CV ≤50%

Correct Answer: CV ≤15%

Q7. In digoxin immunoassays, which substance is known to cause clinically significant cross-reactivity and potentially false-positive results?

• Digoxin-specific Fab fragments
• Digitoxin and some plant-derived cardiac glycosides
• Exogenous insulin
• Human serum albumin

Correct Answer: Digitoxin and some plant-derived cardiac glycosides

Q8. What procedural control helps identify matrix effects and recovery issues during insulin assay validation?

• Using only buffer calibrators without matrix
• Performing spike-and-recovery experiments using the actual sample matrix
• Omitting wash steps to improve signal
• Measuring only pooled control materials

Correct Answer: Performing spike-and-recovery experiments using the actual sample matrix

Q9. Which label/enzyme is commonly used in ELISA detection systems for immunoassays and allows sensitive colorimetric readout?

• Horseradish peroxidase (HRP)
• Green fluorescent protein (GFP)
• Luciferase without substrate
• DNA polymerase

Correct Answer: Horseradish peroxidase (HRP)

Q10. Which interference is particularly important to consider when using biotin-streptavidin based immunoassays for hormones like insulin?

• Hemolysis only
• High biotin intake causing competitive displacement and signal distortion
• Elevated sodium levels in plasma
• Temperature fluctuations during incubation

Correct Answer: High biotin intake causing competitive displacement and signal distortion

Q11. During validation, what does dilutional linearity (parallelism) test demonstrate for endogenous insulin measurement?

• That measured concentration increases when diluting sample
• That sample matrix effect disappears after dilution and the assay responds proportionally to analyte concentration
• That only calibrators are linear and endogenous samples need no testing
• That random errors are reduced by dilution

Correct Answer: That sample matrix effect disappears after dilution and the assay responds proportionally to analyte concentration

Q12. For digoxin therapeutic drug monitoring, what is the commonly cited therapeutic plasma concentration range in ng/mL used in clinical interpretation?

• 0.01–0.05 ng/mL
• 0.5–2.0 ng/mL
• 5–10 ng/mL
• 10–20 ng/mL

Correct Answer: 0.5–2.0 ng/mL

Q13. Which validation parameter quantifies the closeness of measured values to the true value in immunoassays?

• Precision
• Accuracy (bias)
• Linearity range
• Specificity

Correct Answer: Accuracy (bias)

Q14. If a digoxin immunoassay shows significant positive bias due to heterophile antibodies, which corrective step is most appropriate?

• Increase incubation temperature
• Use heterophile blocking reagents or perform assay using an alternate method less affected by heterophiles
• Reduce sample volume
• Change substrate for enzyme reaction

Correct Answer: Use heterophile blocking reagents or perform assay using an alternate method less affected by heterophiles

Q15. What is the recommended back-calculated accuracy acceptance criterion for calibration standards in immunoassay validation (except at LLOQ)?

• Within ±5% of nominal
• Within ±15% of nominal
• Within ±50% of nominal
• Within ±100% of nominal

Correct Answer: Within ±15% of nominal

Q16. Which sample handling practice is important to preserve insulin integrity before immunoassay analysis?

• Repeated freeze-thaw cycles without limits
• Immediate processing, aliquoting, and storage at −80°C to avoid proteolysis and degradation
• Leaving samples at room temperature overnight to equilibrate
• Adding sodium chloride to samples to stabilize insulin

Correct Answer: Immediate processing, aliquoting, and storage at −80°C to avoid proteolysis and degradation

Q17. In method comparison between a new immunoassay and reference method, which statistical approach best evaluates agreement across the measurement range?

• Pearson correlation coefficient alone
• Bland-Altman plot and deming regression to assess bias and agreement
• Comparison of means only
• ANOVA without pairwise analysis

Correct Answer: Bland-Altman plot and deming regression to assess bias and agreement

Q18. What is the primary reason to include wash steps with optimized buffer in ELISA-based immunoassay protocols?

• To deliberately lower assay sensitivity
• To remove unbound reagents and reduce background, improving specificity and precision
• To dilute calibrators during incubation
• To denature capture antibodies

Correct Answer: To remove unbound reagents and reduce background, improving specificity and precision

Q19. Which action is appropriate if recovery during spike-and-recovery experiments for digoxin is 60% across multiple concentrations?

• Accept results as typical for immunoassays
• Investigate matrix effects, adjust sample preparation, or use matrix-matched calibrators or alternative extraction procedures
• Increase incubation time only
• Ignore recovery and report measured values

Correct Answer: Investigate matrix effects, adjust sample preparation, or use matrix-matched calibrators or alternative extraction procedures

Q20. Which statement best describes cross-reactivity in an immunoassay validation for digoxin?

• Cross-reactivity refers to assay precision measured across runs
• Cross-reactivity is the ability of structurally related compounds to bind the antibody and produce signal, expressed as percentage relative to analyte
• Cross-reactivity defines the limit of detection only
• Cross-reactivity is irrelevant for small molecule assays like digoxin

Correct Answer: Cross-reactivity is the ability of structurally related compounds to bind the antibody and produce signal, expressed as percentage relative to analyte

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