Chemiluminescence assays MCQs With Answer

Chemiluminescence assays MCQs With Answer

Introduction: Chemiluminescence assays are powerful immunodetection techniques widely used in M.Pharm research and diagnostics due to their high sensitivity, broad dynamic range, and compatibility with automation. This blog provides targeted multiple-choice questions that explore fundamental principles (flash vs glow kinetics), common luminophores (luminol, acridinium esters, CDP‑Star), label chemistries, assay formats, instrument components, and practical issues such as signal quenching, substrate handling, and calibration. Each question is designed to deepen understanding of assay design, optimization, and interpretation—skills essential for M.Pharm students planning work in bioassay development, clinical laboratories, or pharmaceutical analytics.

Q1. What is the fundamental principle underlying chemiluminescence immunoassays?

• Emission of light from a fluorophore after excitation by a laser
• Emission of light resulting from a chemical reaction producing an electronically excited product
• Measurement of heat released during an antigen–antibody interaction
• Generation of a color change via enzyme-substrate chromogenic reaction

Correct Answer: Emission of light resulting from a chemical reaction producing an electronically excited product

Q2. Which statement correctly differentiates “flash” from “glow” chemiluminescence kinetics?

• Flash is a low-intensity long-duration signal; glow is a high-intensity short burst
• Flash is rapid, intense and short-lived; glow is lower intensity but sustained over time
• Flash requires enzyme labels only; glow requires non-enzymatic labels only
• Flash signals are always wavelength-shifted compared with glow signals

Correct Answer: Flash is rapid, intense and short-lived; glow is lower intensity but sustained over time

Q3. Which substrate system is most commonly used with horseradish peroxidase (HRP) in chemiluminescent immunoassays?

• CDP‑Star with magnesium ions
• Luminol with hydrogen peroxide and an enhancer
• Acridinium ester in alkaline solution

Correct Answer: Luminol with hydrogen peroxide and an enhancer

Q4. What is a defining advantage of acridinium ester (AE) labels in CLIA?

• They produce a sustained glow signal lasting hours without trigger
• They require enzymatic turnover to generate light
• They generate a rapid flash-type signal upon addition of a trigger solution without enzyme
• They emit light only when bound to metallic nanoparticles

Correct Answer: They generate a rapid flash-type signal upon addition of a trigger solution without enzyme

Q5. CDP‑Star is a chemiluminescent substrate for which enzyme label commonly used in immunoassays?

• Horseradish peroxidase (HRP)
• Alkaline phosphatase (AP)
• Beta-galactosidase
• Glucose oxidase

Correct Answer: Alkaline phosphatase (AP)

Q6. Which is the principal analytical advantage of chemiluminescent immunoassays compared with traditional colorimetric ELISAs?

• Lower cost of reagents for routine assays
• Ability to detect non-protein analytes only
• Higher analytical sensitivity and wider dynamic range
• No requirement for calibration curves

Correct Answer: Higher analytical sensitivity and wider dynamic range

Q7. Which factor most directly determines the limit of detection (LOD) in a chemiluminescent assay?

• The color of the microtiter plate
• Signal-to-noise ratio (background noise and photon yield)
• The number of washing steps only
• The presence of fluorescent dyes in the sample

Correct Answer: Signal-to-noise ratio (background noise and photon yield)

Q8. In which scenario is a glow-type chemiluminescent reagent preferred?

• When an ultrafast read is required within milliseconds of addition
• When assays are automated on high-throughput platforms needing stable signal integration
• When no instrument is available and visual detection is needed
• When only qualitative yes/no results are required without quantitation

Correct Answer: When assays are automated on high-throughput platforms needing stable signal integration

Q9. What is the role of chemiluminescence enhancers (e.g., p‑iodophenol) in luminol systems?

• They quench background light to lower sensitivity
• They increase reaction pH and denature antibodies
• They increase photon yield and often prolong emission by stabilizing intermediates
• They convert glow kinetics into flash kinetics by removing peroxide

Correct Answer: They increase photon yield and often prolong emission by stabilizing intermediates

Q10. Which sample constituent is most likely to cause quenching of a chemiluminescent signal?

• High salt concentration alone without oxidizable species
• Substances that scavenge reactive intermediates or absorb emitted photons (e.g., thiols, hemoglobin)
• Buffer with optimal pH recommended by the manufacturer
• Surfactants that improve wetting of the plate surface

Correct Answer: Substances that scavenge reactive intermediates or absorb emitted photons (e.g., thiols, hemoglobin)

Q11. Which detector is most commonly used in dedicated chemiluminescence plate readers to convert photons into a measurable electrical signal?

• Photomultiplier tube (PMT)
• Thermocouple sensor
• Mass spectrometer
• Electrochemical amperometric cell

Correct Answer: Photomultiplier tube (PMT)

Q12. Which conjugation chemistry is commonly used to attach acridinium ester labels to antibodies?

• NHS‑ester coupling to lysine residues
• Biotin‑streptavidin non-covalent adsorption
• Thiolation using cyanogen bromide
• Direct ionic binding to the Fc region

Correct Answer: NHS‑ester coupling to lysine residues

Q13. Which immunoassay format typically employs a labeled antigen competing with analyte in the sample for antibody binding sites?

• Sandwich immunoassay
• Competitive immunoassay
• Indirect immunofluorescence
• Capture‑release mass spectrometry assay

Correct Answer: Competitive immunoassay

Q14. Which of the following strategies will most effectively expand the usable dynamic range of a chemiluminescent assay?

• Increasing substrate concentration without changing detection settings
• Optimizing integration time, minimizing background, and selecting reagents with linear response over concentration ranges
• Using colored plates to mask high signal wells
• Performing fewer replicates to reduce data spread

Correct Answer: Optimizing integration time, minimizing background, and selecting reagents with linear response over concentration ranges

Q15. What artifact can occur in flash-type assays when very high analyte concentrations are present?

• Substrate depletion leading to a distorted or truncated signal peak
• Conversion of the flash signal into a glow signal spontaneously
• Complete elimination of background noise
• Spontaneous enzyme inactivation that improves accuracy

Correct Answer: Substrate depletion leading to a distorted or truncated signal peak

Q16. What are the recommended storage conditions for most chemiluminescent substrates to preserve activity?

• Room temperature in direct sunlight for rapid activation
• Frozen at –80°C in unlabeled containers
• Refrigerated or frozen, protected from light and oxygen, as per manufacturer instructions
• Mixed with bleach to prevent microbial growth

Correct Answer: Refrigerated or frozen, protected from light and oxygen, as per manufacturer instructions

Q17. Why is spectral multiplexing more challenging with chemiluminescence than with fluorescence?

• Chemiluminescent emitters require laser excitation that interferes with multiplexing
• Chemiluminescent emissions are often broadband and labels lack distinct, narrow emission bands
• Because chemiluminescence is not sensitive enough for multiplex detection
• Because chemiluminescent labels cannot be conjugated to antibodies

Correct Answer: Chemiluminescent emissions are often broadband and labels lack distinct, narrow emission bands

Q18. What is the purpose of a trigger or initiator solution in acridinium ester-based assays?

• To stabilize the acridinium ester and prevent light emission
• To generate alkaline peroxide conditions that rapidly oxidize the ester and produce a flash of light
• To bind nonspecifically to the plate preventing carryover
• To reduce background by precipitating proteins

Correct Answer: To generate alkaline peroxide conditions that rapidly oxidize the ester and produce a flash of light

Q19. What is the typical shape of a calibration curve for immunoassays measured over a wide concentration range and how is it commonly fitted?

• Linear and fitted with a simple linear regression across all concentrations
• S-shaped (sigmoidal) and commonly fitted with a four‑parameter logistic (4PL) or five‑parameter logistic model
• Parabolic and fitted with a quadratic polynomial
• Random and cannot be fitted meaningfully

Correct Answer: S-shaped (sigmoidal) and commonly fitted with a four‑parameter logistic (4PL) or five‑parameter logistic model

Q20. Which safety hazard is especially relevant when working with chemiluminescent reagents in the laboratory?

• Extensive gamma radiation generation from luminophores
• Use of strong oxidizers (e.g., hydrogen peroxide, peracids) and organic solvents requiring PPE and proper waste handling
• Emission of toxic levels of carbon monoxide during light generation
• Spontaneous combustion of antibody conjugates at room temperature

Correct Answer: Use of strong oxidizers (e.g., hydrogen peroxide, peracids) and organic solvents requiring PPE and proper waste handling

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