ELISA Practical Flashcards
What are the different types of ELISA ?
Direct ELISA
Antigen is immobilized on a microplate surface.
An enzyme-linked antibody directly binds to the antigen.
Substrate is added → enzyme catalyzes a color change.
Advantages: Fast, simple, high specificity.
Disadvantages: High background noise, no signal amplification.
Indirect ELISA:
Antigen is immobilized on the plate.
Primary antibody binds to the antigen.
Secondary antibody (enzyme-linked) binds to the primary antibody.
Substrate is added → enzyme activity generates a signal.
Advantages: Increased sensitivity (signal amplification), versatile.
Disadvantages: Risk of cross-reactivity from secondary antibody.
Sandwich ELISA:
Capture antibody is immobilized on the plate.
Antigen binds to the capture antibody.
Detection antibody (enzyme-linked) binds to antigen.
Substrate is added → signal generated.
Advantages: High specificity (dual recognition), high sensitivity.
Disadvantages: Requires well-matched antibody pairs.
Competitive ELISA:
Known antigen competes with sample antigen for binding to an antibody.
Less signal → more antigen in sample.
Advantages: Useful for small molecules and low-concentration samples.
Disadvantages: More complex setup and analysis.
Describe the principal of detection in ELISA:
Signal intensity (color change) is proportional to the amount of antigen or antibody present in the sample.
The enzyme catalyses a reaction that generates a measurable signal
Common enzymes - Horseradish peroxidase (HRP), Alkaline phosphatase (AP)
Common substrates:
- HRP → TMB (3,3′,5,5′-Tetramethylbenzidine) → blue color
- AP → p-Nitrophenyl phosphate (pNPP) → yellow color
Signal is measured using a spectrophotometer or plate reader at a specific wavelength
How do you analyse ELISA data ?
Standard curve construction - absorbance vs concentration then fit the data using a linear or four-parameter logistic (4PL) curve
Typical signal range follows a sigmoidal curve
Linear regression for direct or indirect ELISA
4PL regression for sandwich or competitive ELISA (more accurate for wide concentration ranges)
LOD (Limit of Detection) – lowest detectable concentration
LOQ (Limit of Quantification) – lowest measurable concentration with precision
IC50 – concentration at which 50% inhibition occurs (competitive ELISA)
Describe some common troubleshooting errors during an ELISA:
High background:
- caused by incomplete washing and non specific binding
- solution is increase washing steps and optimise blocking
- also caused by contaminated reagents, solution is sue fresh reagents and ensure they’re stored properly
No signal:
- caused by incorrect antibody or inactive enzyme
- solution is to check antibody sensitivity
Low sensitivity:
- caused by poor antigen antibody binding
- solution is to use higher affinity antibodies or increase the incubation time
Edge effects
- caused by uneven coating or temperature variation
Describe advantages of ELISA:
High sensitivity and specificity
Can handle multiple samples in parallel
Adaptable to automated systems
Relatively low cost and accessible
Describe disadvantages of ELISA:
Requires well-characterized antibodies
Some antigens may not immobilize well
Signal intensity can be affected by substrate instability
Describe the difference between direct and indirect ELISA:
Direct - Uses a single, enzyme-labelled primary antibody that binds directly to the antigen
Uses an unlabelled primary antibody to bind the antigen, followed by a labelled secondary antibody that binds the primary antibody
Describe the advantages and disadvantages of indirect ELISA:
Advantages:
- increased sensitivity due to signal amplification from the secondary antibody
- more flexible as different primary antibodies can be used with the same labelled secondary antibody
Disadvantages:
- increased risk of cross reactivity leading to background noise
- longer protocol with more incubation steps
Describe the importance of using BSA:
Importance of blocking with BSA before adding the anti-lactoferrin antibody
blocks non-specific binding sites on the plate, preventing antibodies from binding non specifically to the well surface
Reduces background noise and increases assay specificity
What is the importance of running samples and standards in triplicate ?
Ensures accuracy and reproducibility by accounting for pipetting errors or slight variations in conditions
If you skip this step, results could be unreliable, and a single erroneous measurement could lead to incorrect conclusions
Why does OPD and hydrogen peroxide produce an orange colour ?
OPD (o-phenylenediamine) reacts with hydrogen peroxide in the presence of horseradish peroxidase (HRP), producing a coloured red product (usually orange or yellow)
The intensity of the colour corresponds to the amount of enzyme activity, which is proportional to the antigen concentration
What is the name of the equation and style of curve used to assess ?
A 4 parameter fit and a sigmoidal curve
