ELISA

What is ELISA used for?

To detect and/or quantify a specific protein (antigen or antibody) in a sample. Used in disease diagnosis, hormone testing, allergen detection, and research.

Step 1: Coating the Plate

Antigen/antibody immobilized on wells. Importance: Provides a fixed target. If wrong: Weak or inconsistent signal.

Step 2: Blocking

Blocking solution fills empty spaces. Importance: Prevents nonspecific binding. If wrong: High background or hindered binding.

Step 3: Sample Addition

Sample fluid added (contains antigen/antibody). Importance: Introduces unknown. If wrong: Contamination, wrong dilution, or no detection.

Step 4: Washing

Wells rinsed with buffer. Importance: Removes unbound material. If wrong: Insufficient = background; Excessive = loss of target.

Step 5: Detection Antibody

Enzyme-linked antibody binds antigen. Importance: Provides specificity + enzyme. If wrong: Wrong antibody or too much causes errors.

Step 6: Washing Again

Removes unbound detection antibodies. Importance: Prevents false positives. If wrong: Insufficient = false positives; Excessive = loss.

Step 7: Substrate Addition

Enzyme substrate produces color. Importance: Converts binding into measurable signal. If wrong: No color, or overdevelopment.

Step 8: Signal Detection

Color measured with spectrophotometer. Importance: Provides quantitative results. If wrong: Timing/measurement errors cause inaccuracy

Why is washing repeated?

To ensure unbound antibodies are removed, preventing false positives.

What is the role of the enzyme?

It reacts with substrate to produce a color signal proportional to the amount of target present.