Complement fixation test MCQs With Answer

Introduction

The complement fixation test (CFT) is a classical serological assay used to detect specific antibodies by measuring their ability to fix complement in the presence of antigen. For M.Pharm students, understanding CFT is important for grasping foundations of immunodiagnostics, assay design, and interpretation of immune responses. This blog covers practical and theoretical aspects of the test including principle, components, controls, indicator systems, sensitivity, limitations, and modern alternatives. The MCQs that follow are designed to deepen conceptual understanding, reinforce laboratory considerations such as complement source and heat inactivation, and prepare students for applied questions in immunotechnology and pharmaceutical immunodiagnostics.

Q1. What is the fundamental principle of the complement fixation test (CFT)?

• Detection of antigens by their ability to lyse indicator cells directly
• Measurement of complement activation by antigen–antibody complexes leading to consumption of complement
• Quantification of free complement components in serum using ELISA
• Detection of cell-mediated immunity by complement-dependent cytotoxicity

Correct Answer: Measurement of complement activation by antigen–antibody complexes leading to consumption of complement

Q2. Which pathway of the complement system is primarily involved in the classical CFT?

• Alternative pathway
• Lectin pathway
• Classical pathway
• Terminal pathway only (C5–C9)

Correct Answer: Classical pathway

Q3. Why must patient serum be heat-inactivated before performing a CFT?

• To remove antibodies that would otherwise interfere
• To inactivate endogenous complement present in the serum
• To enhance antigen binding to antibodies
• To sterilize the serum and remove pathogens

Correct Answer: To inactivate endogenous complement present in the serum

Q4. Which antibody is most efficient at fixing complement and therefore most readily detected by CFT in a primary infection?

• IgA
• IgG1 only
• IgM
• IgE

Correct Answer: IgM

Q5. In a standard hemolytic indicator system for CFT, what serves as the indicator of unfixed complement?

• Rabbit erythrocytes sensitized with human complement
• Sheep red blood cells (SRBC) coated with anti-sheep RBC antibody
• Human neutrophils labeled with fluorescent dye
• Latex particles agglutinated by complement

Correct Answer: Sheep red blood cells (SRBC) coated with anti-sheep RBC antibody

Q6. What does a positive CFT (i.e., no hemolysis in indicator step) indicate about the patient serum?

• Patient serum lacks specific antibody against the test antigen
• Complement in the system was dysfunctional
• Specific antibody is present and has consumed complement
• Antigen alone caused complement fixation without antibody

Correct Answer: Specific antibody is present and has consumed complement

Q7. Which control is essential to confirm that complement in the assay was active and able to lyse the indicator system?

• Antigen control (antigen + complement without serum)
• Complement control (complement + indicator alone)
• Serum control (patient serum + indicator without antigen)
• Negative dilution control (buffer only)

Correct Answer: Complement control (complement + indicator alone)

Q8. Which of the following is a major limitation of the complement fixation test compared to modern immunoassays?

• It detects only IgA antibodies
• It requires large volumes of antigen and complement and is less sensitive and more labor-intensive
• It provides quantitative molar concentrations of antibodies directly
• It cannot distinguish between different antibody isotypes

Correct Answer: It requires large volumes of antigen and complement and is less sensitive and more labor-intensive

Q9. How is an antibody titer determined using the CFT?

• By measuring optical density of hemolysis
• By finding the highest serum dilution that still prevents hemolysis (complement fixation)
• By counting the number of lysed erythrocytes microscopically
• By quantifying residual complement proteins with mass spectrometry

Correct Answer: By finding the highest serum dilution that still prevents hemolysis (complement fixation)

Q10. Which component of complement is specifically consumed first in classical pathway activation during CFT?

• C3 convertase only
• C1 complex (C1q, C1r, C1s)
• C9 polymerization
• Factor B

Correct Answer: C1 complex (C1q, C1r, C1s)

Q11. What is the role of the “antigen control” in a CFT?

• To check for non-specific hemolysis caused by antigen preparation
• To ensure serum contains live cells
• To measure complement concentration in patient serum
• To neutralize antibodies present in complement source

Correct Answer: To check for non-specific hemolysis caused by antigen preparation

Q12. Why is guinea pig serum commonly used as a source of complement in CFT?

• Because guinea pig complement is heat-stable and not inactivated at 56°C
• Because it contains high classical pathway activity and low natural inhibitors against many species’ antibodies
• Because it naturally contains sheep RBCs that act as indicators
• Because it lacks C1q and prevents background fixation

Correct Answer: Because it contains high classical pathway activity and low natural inhibitors against many species’ antibodies

Q13. Which statement best explains why IgG subclasses differ in complement fixation efficiency?

• All IgG subclasses bind complement equally but differ in half-life
• Structural differences in Fc regions among IgG subclasses affect their ability to bind C1q
• Only glycosylated IgG molecules can fix complement
• Complement fixation is determined solely by antigen valency, not IgG subclass

Correct Answer: Structural differences in Fc regions among IgG subclasses affect their ability to bind C1q

Q14. Which modification can improve the specificity of a CFT when antigen preparations contain contaminating proteins?

• Increasing complement concentration tenfold
• Absorption of serum with irrelevant antigens to remove cross-reacting antibodies
• Heat-treating antigen at 95°C for 10 minutes
• Adding EDTA to chelate calcium and magnesium

Correct Answer: Absorption of serum with irrelevant antigens to remove cross-reacting antibodies

Q15. In a typical two-step CFT procedure, what occurs during the first incubation step?

• Complement is tested for hemolytic activity with indicator cells
• Serum antibodies react with specific antigen and, if present, fix complement in that mixture
• Indicator erythrocytes are sensitized with antibody
• Residual complement is quantified using colorimetric readout

Correct Answer: Serum antibodies react with specific antigen and, if present, fix complement in that mixture

Q16. Which experimental factor can produce false-negative CFT results?

• Use of fresh, active complement source
• Too low antigen concentration leading to incomplete immune complex formation
• Appropriate heat inactivation of patient serum
• Using an indicator system with high sensitivity to complement

Correct Answer: Too low antigen concentration leading to incomplete immune complex formation

Q17. How can prozone effect influence interpretation of CFT and how is it resolved?

• Prozone causes exaggerated hemolysis; resolved by increasing serum concentration
• Prozone yields false-negative due to antibody excess; resolved by testing serial dilutions of serum
• Prozone leads to complement degradation; resolved by adding more complement
• Prozone results in nonspecific agglutination; resolved by heating antigen

Correct Answer: Prozone yields false-negative due to antibody excess; resolved by testing serial dilutions of serum

Q18. Which of the following modern assays is commonly cited as a more sensitive replacement for CFT in many diagnostic applications?

• Latex fixation test
• Western blotting only
• ELISA (enzyme-linked immunosorbent assay)
• Ouchterlony double immunodiffusion

Correct Answer: ELISA (enzyme-linked immunosorbent assay)

Q19. When interpreting rising antibody titers by CFT in paired sera, what fold-rise is generally considered diagnostic of recent infection?

• No rise; a single elevated titer is sufficient
• Two-fold rise
• Four-fold or greater rise in titer between acute and convalescent samples
• Ten-fold rise is required

Correct Answer: Four-fold or greater rise in titer between acute and convalescent samples

Q20. Which safety or quality control practice is important when performing CFT in a teaching or research laboratory?

• Omitting controls to save reagents if results are obvious
• Using non-standardized complement sources interchangeably without validation
• Including positive and negative control sera, standardizing complement activity, and validating antigen preparations
• Performing the assay at ambient temperatures without monitoring

Correct Answer: Including positive and negative control sera, standardizing complement activity, and validating antigen preparations

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