Principles and applications of ELISA MCQs With Answer

Introduction:
This quiz collection on Principles and Applications of ELISA is designed for M.Pharm students aiming to deepen their practical and theoretical understanding of enzyme-linked immunosorbent assays. The questions focus on core principles — antigen–antibody interactions, enzyme labels, substrates, assay formats (direct, indirect, sandwich, competitive), and critical assay variables such as blocking, washing, sensitivity, and matrix effects. Applications and troubleshooting topics like the hook effect, amplification strategies, plate coating conditions, and validation concepts are included to bridge laboratory practice with method development and quality control. Use these MCQs to assess comprehension, prepare for exams, and sharpen problem-solving for real-world immunoassay challenges.

Q1. What is the fundamental principle behind an ELISA?

• Physical separation of analytes on a gel matrix followed by staining
• Size-based filtration of proteins and colorimetric staining
• Specific antigen–antibody binding detected by an enzyme-linked conjugate producing a measurable signal
• Direct measurement of analyte mass by spectrometry

Correct Answer: Specific antigen–antibody binding detected by an enzyme-linked conjugate producing a measurable signal

Q2. Which statement best describes a sandwich ELISA?

• The antigen and labeled antibody compete for binding to a limited number of plate-bound antibodies
• Antigen is directly labeled and captured on the plate for detection
• An antigen is captured between a plate-bound capture antibody and a detection antibody, forming an antibody–antigen–antibody complex
• A secondary antibody binds nonspecifically to all proteins and is measured

Correct Answer: An antigen is captured between a plate-bound capture antibody and a detection antibody, forming an antibody–antigen–antibody complex

Q3. How does indirect ELISA differ from direct ELISA?

• Indirect ELISA uses a labeled primary antibody which increases specificity
• Indirect ELISA uses an unlabeled primary antibody and a labeled secondary antibody, amplifying signal
• Indirect ELISA measures enzyme activity without any antibodies
• Indirect ELISA requires no blocking step

Correct Answer: Indirect ELISA uses an unlabeled primary antibody and a labeled secondary antibody, amplifying signal

Q4. In a competitive ELISA assay, how does increasing analyte concentration affect the measured signal?

• Signal increases linearly with analyte concentration
• Signal remains constant regardless of analyte concentration
• Signal decreases as analyte concentration increases
• Signal increases exponentially at high analyte concentrations

Correct Answer: Signal decreases as analyte concentration increases

Q5. What is the primary purpose of adding a blocking agent to ELISA wells after coating?

• To denature the coated antigen for better binding
• To increase enzymatic turnover of the substrate
• To prevent nonspecific adsorption of proteins to the plate surface and reduce background
• To enhance color development by providing extra chromogenic substrate

Correct Answer: To prevent nonspecific adsorption of proteins to the plate surface and reduce background

Q6. Which enzyme label is most commonly used in ELISA due to its high turnover and stability?

• β-galactosidase
• Horseradish peroxidase (HRP)
• Glucose oxidase
• Catalase

Correct Answer: Horseradish peroxidase (HRP)

Q7. Which chromogenic substrate is typically used with HRP in a colorimetric ELISA and yields a blue product that turns yellow after acid stop?

• p-Nitrophenyl phosphate (pNPP)
• 3,3′,5,5′-Tetramethylbenzidine (TMB)
• o-Phenylenediamine dihydrochloride (OPD) only
• Luciferin

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

Q8. After stopping a TMB reaction with sulfuric acid, at which wavelength is the absorbance typically read?

• 340 nm
• 405 nm
• 450 nm
• 620 nm

Correct Answer: 450 nm

Q9. What is the “hook effect” (prozone) in sandwich ELISA and why is it problematic?

• High background due to blocked wells; it increases apparent signal
• Very high analyte concentrations saturate both capture and detection antibodies causing a falsely low signal
• Low analyte concentrations produce signal noise that mimics positive results
• It is caused by improper washing leading to carryover

Correct Answer: Very high analyte concentrations saturate both capture and detection antibodies causing a falsely low signal

Q10. How does the streptavidin–biotin system enhance ELISA sensitivity?

• By reducing antibody affinity to decrease nonspecific binding
• By covalently cross-linking antigens to the plate surface
• By using high-affinity biotinylated antibodies and enzyme-conjugated streptavidin to amplify signal through multiple enzyme molecules per binding event
• By changing substrate specificity to chemiluminescent reactions only

Correct Answer: By using high-affinity biotinylated antibodies and enzyme-conjugated streptavidin to amplify signal through multiple enzyme molecules per binding event

Q11. Which coating buffer pH is commonly used to promote passive adsorption of proteins to polystyrene microtiter plates?

• pH 4.0 acetate buffer
• pH 7.4 phosphate-buffered saline (PBS)
• pH 9.6 carbonate–bicarbonate buffer
• pH 2.0 glycine buffer

Correct Answer: pH 9.6 carbonate–bicarbonate buffer

Q12. In assay validation terminology, what does LOD (limit of detection) refer to?

• The highest concentration that can be quantified with acceptable accuracy
• The lowest concentration that can be reliably distinguished from a blank but not necessarily quantified precisely
• The concentration at which the assay fully saturates the detector
• The minimum volume of sample required for the assay

Correct Answer: The lowest concentration that can be reliably distinguished from a blank but not necessarily quantified precisely

Q13. Which additive is commonly included in wash buffers to reduce nonspecific protein interactions in ELISA?

• EDTA
• Tween-20 detergent
• Sodium azide
• Glycerol

Correct Answer: Tween-20 detergent

Q14. Which antibody type is more likely to show cross-reactivity with structurally similar antigens?

• Monoclonal antibody
• Recombinant single-chain variable fragment only
• Polyclonal antibody
• Fab fragment after affinity purification

Correct Answer: Polyclonal antibody

Q15. What is the primary purpose of generating a standard curve in a quantitative ELISA?

• To verify plate coating uniformity only
• To determine the concentration of unknown samples by interpolation within the assay’s linear range
• To provide a qualitative positive/negative cutoff without quantification
• To calibrate the spectrophotometer wavelength

Correct Answer: To determine the concentration of unknown samples by interpolation within the assay’s linear range

Q16. Which control is essential to confirm that the assay reagents and enzyme–substrate system are functioning correctly?

• Negative control containing no enzyme
• Positive control containing a known concentration of target antigen or analyte
• Blank with unrelated protein only
• Blank containing only blocking buffer without substrate

Correct Answer: Positive control containing a known concentration of target antigen or analyte

Q17. What distinguishes a kinetic ELISA readout from an end-point readout?

• Kinetic ELISA measures the initial reaction rate over time, providing higher precision and dynamic range
• Kinetic ELISA measures color only after prolonged incubation without timing
• End-point readout continuously measures absorbance every second
• Kinetic ELISA does not require a substrate

Correct Answer: Kinetic ELISA measures the initial reaction rate over time, providing higher precision and dynamic range

Q18. Why might skim milk be a poor blocking agent when using alkaline phosphatase (AP) as the detection enzyme?

• Milk contains high concentrations of HRP which competes with AP
• Milk proteins denature the capture antibody
• Skim milk often contains endogenous phosphatases that can interfere with AP-based signals
• Milk blocks are too expensive for routine use

Correct Answer: Skim milk often contains endogenous phosphatases that can interfere with AP-based signals

Q19. What is a primary advantage of multiplexed ELISA platforms over single-analyte ELISAs?

• They eliminate the need for capture antibodies entirely
• They allow simultaneous measurement of multiple analytes in a single small-volume sample, conserving sample and time
• They always produce higher sensitivity for every analyte compared to single-plex assays
• Multiplexed platforms do not require validation

Correct Answer: They allow simultaneous measurement of multiple analytes in a single small-volume sample, conserving sample and time

Q20. Which sample-related factor is a common source of matrix effect that can bias ELISA results?

• Hemolysis releasing intracellular components
• Using ultrapure reagent-grade water
• Proper centrifugation of samples
• Standard curve dilution in assay buffer

Correct Answer: Hemolysis releasing intracellular components

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