8-oxoG Elisa figure

Question 1

What were the results of the ELISA

Question 2

What are the principles of the ELISA

Answer 2

ELISA is based on the specific binding of an antigen (target molecule) to its corresponding antibody, which is either immobilized on a plate or detected via a secondary antibody. The assay uses an enzyme-linked detection system, where an enzyme (such as HRP—horseradish peroxidase or AP—alkaline phosphatase) catalyzes a reaction that produces a colorimetric, fluorescent, or chemiluminescent signal, allowing quantification of the target molecule.

Unlike sandwich ELISA, where antigen binding is detected directly, competitive ELISA relies on the competition between two antigens:
1. Sample Antigen (Unknown concentration in biological samples)
2. Labeled/Standard Antigen (Known concentration, pre-coated or enzyme-linked)
The higher the sample antigen concentration, the fewer antibody binding sites are available for the labeled antigen, resulting in a lower signal intensity.

Key Rule: Signal is inversely proportional to the target antigen concentration
* More antigen in the sample → Less labeled antigen binds → Lower signal
* Less antigen in the sample → More labeled antigen binds → Higher signal

Question 3

What are the advantages and limitations to an ELISA?

Answer 3

Advantages
✔ High Sensitivity (pg/mL detection range).
✔ High Specificity (antigen-antibody interactions).
✔ Quantitative & Reproducible.
✔ High-Throughput (96-well or 384-well plates).

Limitations
✖ Antibody Dependency: Requires well-validated antibodies to avoid cross-reactivity.
✖ Time-Consuming: Typically takes 3–6 hours per run.
✖ Interference: Sample contaminants (e.g., hemoglobin, lipids) may affect results.

Question 4

What are alternatives to measuring 8-oxoG ?

Question 5

What is the workflow for ELISA?

Answer 5

ELISA Workflow
Step 1: Coating the Plate (For Sandwich ELISA or Direct ELISA)
* A 96-well plate (typically made of polystyrene) is coated with a capture antibody or antigen and incubated overnight.
Step 2: Blocking Non-Specific Binding
* Blocking buffer (BSA, casein, or Tween-20 in PBS) is added to prevent non-specific binding.
Step 3: Adding the Sample
* Biological samples (serum, plasma, cell lysates) are added to the wells and incubated to allow antigen-antibody binding.
Step 4: Detection with an Enzyme-Linked Antibody
* Either a directly labeled antibody (HRP/AP-conjugated) or a secondary detection system is used.
Step 5: Substrate Addition & Signal Detection
* HRP Substrates: TMB (3,3’,5,5’-Tetramethylbenzidine) produces a blue color (converted to yellow with acid stop solution).
* AP Substrates: pNPP (p-Nitrophenyl Phosphate) produces a yellow color.
Step 6: Quantification
* The color intensity is measured using a plate reader at 450 nm (HRP) or 405 nm (AP).
A standard curve is generated using known antigen concentrations to determine sample concentrations.