Biological Tests: Assays and potency testing of vaccines (tetanus, diphtheria, rabies, heparin, oxytocin, antivenom) MCQs With Answer

Introduction: Biological tests and potency assays are central to ensuring the safety, efficacy, and consistency of vaccines and biologics. For M.Pharm students specializing in advanced pharmaceutical analysis, mastering the principles and methodologies used to evaluate tetanus, diphtheria, rabies vaccines, heparin, oxytocin, and antivenoms is essential. This blog provides focused multiple-choice questions that probe in vivo neutralization assays, in vitro serological and chromogenic methods, reference standards, units of potency (IU, Lf, neutralizing units), and statistical approaches to LD50/ED50 estimation. These MCQs are designed to deepen conceptual understanding and prepare students for practical and regulatory challenges in biological testing and potency determination.

Q1. What is the primary objective of a biological potency assay for a vaccine?

• To quantify the total protein content of the vaccine
• To determine the vaccine’s sterility under defined conditions
• To measure the biological activity compared with an international or reference standard
• To assess the chemical purity of adjuvants present in the formulation

Correct Answer: To measure the biological activity compared with an international or reference standard

Q2. Which in vivo method is classically used to determine potency of tetanus toxoid vaccines?

• Haemagglutination inhibition assay
• Mouse toxin neutralization assay (NIH test)
• Chromogenic anti-Xa assay
• Enzyme-linked immunosorbent assay (ELISA) for total IgG

Correct Answer: Mouse toxin neutralization assay (NIH test)

Q3. The “Lf” unit commonly associated with diphtheria and tetanus toxoids refers to which of the following?

• The international potency in IU per dose
• The limit of flocculation indicating antigenic mass
• The lethal fraction determined in animal challenge
• The light fluorescence intensity in an ELISA

Correct Answer: The limit of flocculation indicating antigenic mass

Q4. For potency testing of inactivated rabies vaccines, which test is used to evaluate protective efficacy in vivo?

• Rapid fluorescent focus inhibition test (RFFIT) alone
• Mouse challenge/neutralization potency test (NIH-like mouse test)
• Chromogenic anti-Xa assay
• Broth microdilution bacterial assay

Correct Answer: Mouse challenge/neutralization potency test (NIH-like mouse test)

Q5. Which assay is considered the gold-standard for determining heparin potency in pharmacopeial testing?

• Activated partial thromboplastin time (aPTT) clotting assay
• Chromogenic anti-factor Xa assay calibrated against a heparin reference standard
• Direct ELISA for heparin mass
• In vivo bleeding time measurement in rabbits

Correct Answer: Chromogenic anti-factor Xa assay calibrated against a heparin reference standard

Q6. The standard biological assay used to determine oxytocin potency is based on which physiological response?

• Antibody production measured by ELISA
• Increase in blood pressure in anesthetized rats
• Uterine contraction (isolated uterine strip bioassay)
• Renal sodium excretion rate

Correct Answer: Uterine contraction (isolated uterine strip bioassay)

Q7. Antivenom potency is typically expressed as which of the following?

• Milligrams of protein per mL
• Neutralizing units per mL based on ED50 or the amount of venom neutralized
• International Units of enzyme inhibition per dose
• Optical density units in an ELISA

Correct Answer: Neutralizing units per mL based on ED50 or the amount of venom neutralized

Q8. Which statistical method is commonly used to calculate LD50 or ED50 values in biological assays?

• Kaplan-Meier estimator
• Spearman-Karber method
• Student’s t-test
• ANOVA followed by Dunnett’s test

Correct Answer: Spearman-Karber method

Q9. Why is a calibrated reference standard required in potency assays for vaccines and biologics?

• To increase the yield of the manufacturing process
• To allow calculation of relative potency and express results in standard units
• To replace the need for animal testing entirely
• To provide sterility assurance for each batch

Correct Answer: To allow calculation of relative potency and express results in standard units

Q10. Compared with clotting-time assays, chromogenic anti-Xa/IIa assays for heparin are preferred because they are:

• Less specific but faster
• More specific, more reproducible and less influenced by plasma factors
• Cheaper and do not require reference standards
• Dependent on animal-derived reagents only

Correct Answer: More specific, more reproducible and less influenced by plasma factors

Q11. An International Unit (IU) in biological assays is best described as:

• A unit equal to 1 mg of biologic material
• The biologic activity contained in a reference preparation assigned by an international authority
• A measure of optical density at 450 nm in ELISA
• The mass equivalent determined by gravimetric analysis

Correct Answer: The biologic activity contained in a reference preparation assigned by an international authority

Q12. The parallel line assay (PLA) is used in vaccine potency testing to:

• Compare two unrelated antigens for cross-reactivity
• Estimate relative potency by comparing dose–response lines of test and reference preparations
• Measure absolute protein concentration by Bradford reagent
• Determine sterility of final product

Correct Answer: Estimate relative potency by comparing dose–response lines of test and reference preparations

Q13. The principle of an antivenom neutralization test involves which of the following?

• Quantifying the amount of free venom peptides by HPLC
• Demonstrating that antibodies in the antivenom bind and prevent the toxic effects of venom in vivo
• Measuring complement activation in vitro
• Assessing endotoxin levels in the antivenom formulation

Correct Answer: Demonstrating that antibodies in the antivenom bind and prevent the toxic effects of venom in vivo

Q14. Heparin potency is commonly stated as IU per mg. Typical pharmacopeial potencies for unfractionated heparin are:

• 1–5 IU/mg
• 10–50 IU/mg
• 180–220 IU/mg (approximate range depending on source)
• 1000–2000 IU/mg

Correct Answer: 180–220 IU/mg (approximate range depending on source)

Q15. In a toxin neutralization potency assay (e.g., tetanus), the principal endpoint used to define potency is:

• The optical density change in a colorimetric substrate
• Survival or protection of animals after challenge with toxin
• The concentration of endotoxin measured by LAL
• The viscosity change in a gel diffusion test

Correct Answer: Survival or protection of animals after challenge with toxin

Q16. Which in vitro serological method is frequently used as an alternative or correlate to in vivo rabies potency tests by measuring virus-neutralizing antibodies?

• RFFIT (rapid fluorescent focus inhibition test)
• Chromogenic anti-Xa assay
• Flocculation (Lf) assay
• Broth culture sterility assay

Correct Answer: RFFIT (rapid fluorescent focus inhibition test)

Q17. The flocculation test for diphtheria toxoid measures which interaction?

• Precipitation between toxoid antigen and a specific antitoxin forming visible floccules
• Enzymatic cleavage of a chromogenic substrate by toxoid
• Binding of toxoid to heparin in solution
• Neutralization of rabies virus by toxoid

Correct Answer: Precipitation between toxoid antigen and a specific antitoxin forming visible floccules

Q18. How do aluminum-containing adjuvants typically impact vaccine potency assays?

• They reduce antigenicity and lower measured potency
• They have no measurable effect on immunogenicity
• They enhance immunogenicity, often increasing antibody responses and apparent potency
• They enzymatically degrade toxoids during storage

Correct Answer: They enhance immunogenicity, often increasing antibody responses and apparent potency

Q19. Stability studies for vaccine potency evaluation commonly include which approaches?

• Only microbial limit tests at release
• Real-time and accelerated stability testing with periodic potency assays
• Single time-point potency testing after final fill
• Mass spectrometry quantitation of excipients only

Correct Answer: Real-time and accelerated stability testing with periodic potency assays

Q20. In antivenom neutralization studies, the ED50 is defined as which of the following?

• The dose of venom that causes 50% hemolysis in vitro
• The effective dose of antivenom that protects 50% of animals against a defined challenge of venom
• The endotoxin level equal to 50 EU/mL
• The electron density at 50% binding in ELISA

Correct Answer: The effective dose of antivenom that protects 50% of animals against a defined challenge of venom

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