ELISA technique MCQs With Answer

ELISA technique MCQs With Answer

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ELISA technique MCQs With Answer for M.Pharm Students

Enzyme-Linked Immunosorbent Assay (ELISA) is a cornerstone technique in modern pharmaceutical analytical methods, enabling sensitive and specific quantification of biomolecules such as proteins, hormones, cytokines, and antibodies. For M.Pharm students, mastering ELISA encompasses understanding assay formats (direct, indirect, sandwich, competitive), surface chemistry, enzyme-substrate systems, optimization variables, and data analysis using logistic models. This set of MCQs is designed to reinforce theoretical knowledge and practical decision-making—from plate selection and blocking to minimizing interference, managing hook effects, and ensuring precision. Each question includes a detailed answer option to help you self-evaluate and prepare for laboratory work, research, and advanced analytical roles in the pharmaceutical sciences.

Q1. Which statement best describes the fundamental principle of ELISA?

  • Enzyme-linked detection of antigen–antibody complexes measured by a substrate-driven signal
  • Color development due to protein migration during electrophoresis
  • Quantification of analytes by radioisotope-labeled antibodies
  • Direct mass spectrometric quantitation of antigen

Correct Answer: Enzyme-linked detection of antigen–antibody complexes measured by a substrate-driven signal

Q2. Which ELISA format offers the highest specificity and sensitivity for detecting low-abundance protein antigens?

  • Direct ELISA
  • Indirect ELISA
  • Sandwich ELISA
  • Dot blot assay

Correct Answer: Sandwich ELISA

Q3. In an indirect ELISA, how is the primary antibody typically configured?

  • Enzyme-conjugated primary antibody targeting the antigen
  • Biotinylated primary antibody detected by labeled streptavidin only
  • Unlabeled and detected with an enzyme-conjugated secondary antibody
  • Radiolabeled primary antibody detected by scintillation

Correct Answer: Unlabeled and detected with an enzyme-conjugated secondary antibody

Q4. Which is the most commonly used chromogenic substrate for HRP-based ELISA?

  • 3,3′,5,5′-Tetramethylbenzidine (TMB)
  • p-Nitrophenyl phosphate (pNPP)
  • 4-Methylumbelliferyl phosphate (4-MUP)
  • 3,3′-Diaminobenzidine (DAB)

Correct Answer: 3,3′,5,5′-Tetramethylbenzidine (TMB)

Q5. The most suitable coating buffer for passive adsorption of proteins onto high-binding polystyrene ELISA plates is:

  • Carbonate–bicarbonate buffer, pH ~9.6
  • Acetate buffer, pH 4.0
  • PBS with 0.1% Tween-20
  • Tris-glycine with SDS

Correct Answer: Carbonate–bicarbonate buffer, pH ~9.6

Q6. The primary purpose of the blocking step in ELISA is to:

  • Increase enzyme activity on the plate
  • Reduce nonspecific binding by occupying uncoated plastic sites
  • Enhance antigen–antibody affinity by changing pH
  • Promote faster color development

Correct Answer: Reduce nonspecific binding by occupying uncoated plastic sites

Q7. A typical washing buffer composition that minimizes nonspecific interactions is:

  • Deionized water alone
  • PBS or TBS containing 0.05% Tween-20
  • Carbonate–bicarbonate buffer with 1% SDS
  • Citrate buffer with 0.5% Triton X-100

Correct Answer: PBS or TBS containing 0.05% Tween-20

Q8. The “hook” (prozone) effect in sandwich ELISA is caused by:

  • Low antibody affinity at high pH
  • Incomplete blocking of the plate
  • Very high antigen concentration that prevents proper sandwich formation
  • Excessive wash cycles removing the capture antibody

Correct Answer: Very high antigen concentration that prevents proper sandwich formation

Q9. For a symmetric sigmoidal ELISA standard curve, the most commonly used data-fitting model is:

  • Linear regression
  • Four-parameter logistic (4PL) regression
  • Polynomial (third-order) regression
  • Exponential regression

Correct Answer: Four-parameter logistic (4PL) regression

Q10. In ELISA, the limit of detection (LOD) is generally defined as the signal equivalent to:

  • Mean of low standard minus 1 SD
  • Mean of blank plus 3 SD
  • Mean of blank plus 10 SD
  • Mean of highest standard minus 3 SD

Correct Answer: Mean of blank plus 3 SD

Q11. What is a commonly used stop solution for the HRP–TMB reaction in colorimetric ELISA?

  • 0.1 N hydrochloric acid
  • 2 N sulfuric acid
  • 0.5 N sodium hydroxide
  • 1 M acetic acid

Correct Answer: 2 N sulfuric acid

Q12. After stopping an HRP–TMB reaction with acid, the absorbance is typically read at:

  • 405 nm
  • 450 nm
  • 560 nm
  • 620 nm

Correct Answer: 450 nm

Q13. Which ELISA format is most appropriate for quantifying small molecules (haptens) that cannot bind two antibodies simultaneously?

  • Direct ELISA
  • Indirect ELISA
  • Sandwich ELISA
  • Competitive ELISA

Correct Answer: Competitive ELISA

Q14. An effective strategy to reduce heterophilic antibody interference in human serum ELISAs is to:

  • Increase substrate concentration twofold
  • Use F(ab′)2 or species-specific heterophilic blockers during incubation
  • Decrease the number of wash steps
  • Switch to a nonbinding plate

Correct Answer: Use F(ab′)2 or species-specific heterophilic blockers during incubation

Q15. For high-efficiency antigen adsorption, the best plate type is:

  • Low-binding polypropylene plate
  • Untreated polystyrene plate
  • High-binding polystyrene ELISA plate (e.g., MaxiSorp)
  • Glass microplate

Correct Answer: High-binding polystyrene ELISA plate (e.g., MaxiSorp)

Q16. Using a biotin–streptavidin system in ELISA primarily serves to:

  • Reduce incubation time by half
  • Increase sensitivity via high-affinity, multivalent binding
  • Prevent hook effect at high antigen levels
  • Change the detection from colorimetric to fluorometric

Correct Answer: Increase sensitivity via high-affinity, multivalent binding

Q17. Inter-assay precision in ELISA is appropriately evaluated by calculating:

  • Coefficient of determination (R²) of the standard curve
  • Coefficient of variation (CV%) of controls across different runs
  • The slope of the calibration curve
  • The absorbance of the blank

Correct Answer: Coefficient of variation (CV%) of controls across different runs

Q18. Running samples and standards in duplicate or triplicate primarily helps to:

  • Increase the dynamic range
  • Estimate technical variability and identify outliers
  • Reduce nonspecific binding
  • Accelerate incubation kinetics

Correct Answer: Estimate technical variability and identify outliers

Q19. Which practice best minimizes edge effects caused by evaporation during incubations?

  • Increase incubation temperature to 40°C
  • Reduce wash volume to conserve reagents
  • Use plate sealers and prefill perimeter wells with buffer to stabilize humidity
  • Switch to a black plate

Correct Answer: Use plate sealers and prefill perimeter wells with buffer to stabilize humidity

Q20. Compared with colorimetric ELISA, chemiluminescent ELISA generally provides:

  • Lower sensitivity but greater robustness
  • Higher sensitivity and a broader dynamic range
  • No need for a calibration curve
  • Increased resistance to matrix effects

Correct Answer: Higher sensitivity and a broader dynamic range

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