Question 1

What are the three main steps in an ELISA assay?

Accepted Answer

ELISA involves three sequential steps: capture or immobilization of the target analyte on a microplate, detection of the analyte using target-specific detection proteins, and enzyme reaction where a conjugated enzyme converts its substrate to a colored product. This three-step process forms the foundation for all ELISA variants, enabling sensitive quantification of biomolecules in biological samples.

Question 2

How does indirect ELISA differ from sandwich ELISA?

Accepted Answer

Indirect ELISA coats the plate with a specific capture antigen to detect antibodies in a sample, measuring immune responses. Sandwich ELISA uses a capture antibody to coat the plate and detects antigens sandwiched between capture and detection antibodies. Sandwich ELISA is better suited for analyzing complex samples like tissue culture supernatants where the antigen is part of a mixed sample.

Question 3

When should competitive ELISA be used instead of other ELISA types?

Accepted Answer

Competitive ELISA is used when only one antibody is available for a target antigen or when detecting small antigens with a single epitope that cannot accommodate two different antibodies due to steric hindrance. The plate is coated with purified antigen, and higher signal indicates lower antigen concentration in the sample, creating an inverse relationship.

Question 4

What enzymes are commonly used in ELISA and what do they do?

Accepted Answer

Horseradish peroxidase (HRP) and alkaline phosphatase (AP) are the most common enzymes conjugated to antibodies in ELISA. These enzymes convert colorless substrates into soluble, colored products that can be measured and quantified using a plate reader, enabling detection and quantification of antigen-antibody complexes.

Question 5

How is a standard curve created and used in sandwich ELISA?

Accepted Answer

A standard curve is generated by adding known concentrations of purified recombinant protein to designated wells and processing them alongside test samples. Mean absorbance values for known concentrations are plotted on the y-axis against protein concentrations on the x-axis. Test sample absorbance values are then compared to this curve to determine the unknown antigen concentration in the sample.

Question 6

Why is blocking an important step in ELISA protocols?

Accepted Answer

Blocking prevents non-specific binding of antibodies and proteins to empty sites on the microplate. After coating the plate with antigen or antibody, blocking buffer fills remaining protein-binding sites, reducing background signal and improving assay specificity. This step ensures that only target-specific interactions are detected.

Question 7

What information does serial dilution provide in indirect ELISA analysis?

Accepted Answer

Serial dilution of serum samples reveals antibody concentration patterns by showing how absorbance decreases as samples become more diluted. When mean absorbance values from serially diluted samples are plotted against dilution factors, the resulting curve demonstrates that less antibody is present in more diluted samples, enabling semi-quantitative assessment of immune response.

ELISA Assays: Indirect, Sandwich, and Competitive - Assessment Quiz