Immunoassay methods: heterogeneous and homogeneous systems MCQs With Answer

Introduction: Immunoassay techniques are indispensable in pharmacological and toxicological screening, offering sensitive and specific detection of drugs, biomarkers, and xenobiotics. This quiz set focuses on heterogeneous and homogeneous immunoassay systems, covering principles, formats (sandwich, competitive), common labels (enzymes, fluorescence, chemiluminescence, radioisotopes), and practical issues such as hook effect, cross-reactivity, interference (biotin, heterophile antibodies), assay validation parameters (LOD, LOQ, precision, dynamic range), and troubleshooting. Designed for M.Pharm students, these MCQs emphasize deeper understanding of assay selection, kinetics, solid-phase choices, and analytical performance to prepare you for designing, interpreting and validating immunoassays in drug screening and toxicology applications.

Q1. Which single feature fundamentally distinguishes heterogeneous immunoassays from homogeneous immunoassays?

• They always use radioactive labels
• They require a physical separation or wash step to discriminate bound from free label
• They never use antibodies
• They are always point-of-care lateral flow tests

Correct Answer: They require a physical separation or wash step to discriminate bound from free label

Q2. Which of the following is an example of a homogeneous immunoassay commonly used for therapeutic drug monitoring?

• Sandwich ELISA
• Radioimmunoassay (RIA)
• Enzyme Multiplied Immunoassay Technique (EMIT)
• Capture (direct) immunoblot

Correct Answer: Enzyme Multiplied Immunoassay Technique (EMIT)

Q3. A sandwich ELISA is particularly advantageous for which type of analyte and why?

• Small haptens, because they bind two antibodies simultaneously
• Low molecular weight drugs, because they require competitive binding
• Large proteins with two distinct epitopes, because two antibodies (capture and detection) can bind simultaneously
• Highly hydrophobic molecules, because they partition to the solid phase

Correct Answer: Large proteins with two distinct epitopes, because two antibodies (capture and detection) can bind simultaneously

Q4. Historically which label provided the highest analytical sensitivity in immunoassays?

• Enzymatic labels like horseradish peroxidase
• Chromogenic dyes
• Radioisotopic labels (RIA)
• Colloidal gold

Correct Answer: Radioisotopic labels (RIA)

Q5. The prozone or “hook” effect in sandwich immunoassays results in:

• Apparent plateau at high analyte concentrations that increases signal
• Falsely elevated readings due to reagent contamination
• Falsely low signal at very high analyte concentrations due to antibody saturation preventing sandwich formation
• Increased assay background from non-specific binding

Correct Answer: Falsely low signal at very high analyte concentrations due to antibody saturation preventing sandwich formation

Q6. Cross-reactivity of an antibody in an immunoassay is best described as:

• The degree to which the antibody binds structurally related compounds in addition to the target analyte
• The nonspecific adsorption of proteins to plastic surfaces
• The proportion of labeled antigen bound at equilibrium
• The percentage of antigen degraded during assay incubation

Correct Answer: The degree to which the antibody binds structurally related compounds in addition to the target analyte

Q7. Which analytical parameter defines the lowest concentration of analyte that can be reliably distinguished from blank?

• Upper limit of quantitation (ULOQ)
• Limit of detection (LOD)
• Limit of blank (LOB)
• Dynamic range

Correct Answer: Limit of detection (LOD)

Q8. Which homogeneous immunoassay technique measures changes in molecular rotation of a fluorescent tracer upon antibody binding?

• Enzyme-linked immunosorbent assay (ELISA)
• Fluorescence polarization immunoassay (FPIA)
• Radioimmunoassay (RIA)
• Chemiluminescent microparticle immunoassay (CMIA)

Correct Answer: Fluorescence polarization immunoassay (FPIA)

Q9. In EMIT homogeneous assays the measured signal is directly proportional to analyte concentration because:

• Antibody binding activates the enzyme label producing more signal
• Antibody binding irreversibly degrades the enzyme label
• Free enzyme-labeled analyte retains enzymatic activity while antibody-bound conjugate has reduced activity, so signal increases with analyte concentration
• The assay measures precipitated complexes after centrifugation

Correct Answer: Free enzyme-labeled analyte retains enzymatic activity while antibody-bound conjugate has reduced activity, so signal increases with analyte concentration

Q10. Which solid phase is most commonly used in heterogeneous microplate immunoassays?

• Glass capillaries
• Microtiter polystyrene wells coated with capture antibody
• Free-solution droplets
• Liposome vesicles in suspension

Correct Answer: Microtiter polystyrene wells coated with capture antibody

Q11. Competitive immunoassay formats are generally preferred for detection of which type of analyte?

• Large proteins with two epitopes
• Small haptens and drugs with a single epitope
• High molecular weight polysaccharides
• Whole cells or viruses

Correct Answer: Small haptens and drugs with a single epitope

Q12. Time-resolved fluorescence immunoassays (TRFIA) improve signal-to-noise primarily because:

• They use enzymes that amplify light emission
• Lanthanide chelate labels have long-lived emission which is measured after short-lived background fluorescence decays
• They do not use antibodies, eliminating background
• The assays run at very low temperatures to reduce noise

Correct Answer: Lanthanide chelate labels have long-lived emission which is measured after short-lived background fluorescence decays

Q13. Exogenous biotin supplementation in patients can interfere with immunoassays that use streptavidin-biotin capture by causing:

• No measurable effect on any immunoassay
• Saturation of streptavidin binding sites leading to falsely low or falsely high results depending on assay format
• Complete loss of antibody specificity
• Increased endogenous peroxidase activity

Correct Answer: Saturation of streptavidin binding sites leading to falsely low or falsely high results depending on assay format

Q14. Which type of antibody generally provides the highest specificity for a single epitope in immunoassays?

• Polyclonal antibodies raised in rabbits
• Monoclonal antibodies directed to a single epitope
• Serum from immunized animals without purification
• Secondary antibodies only

Correct Answer: Monoclonal antibodies directed to a single epitope

Q15. When designing an assay for a large therapeutic protein, which immunoassay format is typically preferred and why?

• Competitive assay, because large proteins compete better with labeled antigen
• Sandwich assay, because two non-overlapping antibodies can capture and detect the protein increasing specificity and sensitivity
• Homogeneous assay like FPIA, because it requires no wash steps
• RIA, because radioactivity is required for large proteins

Correct Answer: Sandwich assay, because two non-overlapping antibodies can capture and detect the protein increasing specificity and sensitivity

Q16. Which metric is used to evaluate precision of an assay across different runs or days?

• Intra-assay accuracy
• Inter-assay coefficient of variation (CV)
• Limit of detection (LOD)
• Recovery percentage

Correct Answer: Inter-assay coefficient of variation (CV)

Q17. Immunoassay calibration curves that are sigmoidal are most appropriately fitted using which regression model?

• Linear least squares regression
• Four-parameter logistic (4PL) or five-parameter logistic (5PL) models
• Polynomial of degree 10
• Simple moving average

Correct Answer: Four-parameter logistic (4PL) or five-parameter logistic (5PL) models

Q18. Lateral flow immunoassays (LFIA) are primarily characterized by which of the following statements?

• They require complex wash and incubation steps like plate ELISAs
• They are rapid, point-of-care tests that provide qualitative or semi-quantitative results often readable visually
• They always provide highly quantitative results with low limits of detection
• They cannot be used for protein detection

Correct Answer: They are rapid, point-of-care tests that provide qualitative or semi-quantitative results often readable visually

Q19. Heterophile antibodies in a patient sample can cause which common immunoassay problem?

• False-negative results by degrading the analyte enzymatically
• False-positive results by cross-linking capture and detection antibodies
• Complete inhibition of substrate conversion in enzymatic labels
• Precipitation of assay buffer salts

Correct Answer: False-positive results by cross-linking capture and detection antibodies

Q20. In assay validation, the term “dynamic range” most accurately refers to:

• The number of wash steps required for an assay
• The concentration interval over which the assay provides acceptable accuracy and precision
• The specific activity of the label used
• The shelf-life of the antibody reagent

Correct Answer: The concentration interval over which the assay provides acceptable accuracy and precision

Download