🔬 ELISA (Enzyme-Linked Immunosorbent Assay)
Definition:
ELISA is a plate-based assay technique designed for detecting and quantifying soluble
substances such as peptides, proteins, antibodies, and hormones. It relies on specific antigen-
antibody binding and an enzyme-mediated color change to detect the presence and quantity of
target molecules.
🧪 Practical ELISA Procedure (Generic Format)
Materials:
96-well ELISA plate (high binding)
Antigen or antibody (depending on assay type)
Coating buffer (e.g., carbonate-bicarbonate buffer, pH 9.6)
Blocking buffer (e.g., 5% BSA or skim milk in PBS)
Washing buffer (e.g., PBS-Tween 20)
Primary antibody (if applicable)
Enzyme-linked secondary antibody (e.g., HRP-conjugated)
Substrate (e.g., TMB for HRP)
Stop solution (e.g., 2N sulfuric acid)
Plate reader (450 nm)
Standard Procedure (Sandwich ELISA Example):
1. Plate Coating
Add 100 µL of capture antibody (diluted in coating buffer) to each well.
Incubate overnight at 4°C or 1-2 hours at 37°C.
2. Blocking
Discard coating solution and add 200 µL of blocking buffer to block nonspecific
binding.
Incubate for 1 hour at room temperature.
3. Washing
Wash plate 3–5 times with PBS-T.
4. Sample Addition
Add 100 µL of sample or standard (diluted appropriately).
Incubate for 1–2 hours at 37°C.
5. Washing
Wash plate 3–5 times with PBS-T.
6. Detection Antibody
Add 100 µL of enzyme-linked detection antibody.
Incubate for 1 hour at room temperature.
7. Washing
Wash 3–5 times.
8. Substrate Addition
Add 100 µL of TMB substrate solution.
Incubate for 10–30 minutes (protected from light).
9. Stopping Reaction
Add 50 µL of stop solution (2N
H₂SO₄).
Blue color turns yellow.
10. Measurement
Read absorbance at 450 nm using an
ELISA plate reader.
🔁 Types of ELISA
Type Description Use Case
Antigen is immobilized, enzyme-
Direct ELISA Fast, fewer steps, but less sensitive.
conjugated antibody binds directly.
Primary antibody binds antigen;
Indirect Higher sensitivity; useful for antibody
enzyme-linked secondary antibody
ELISA detection.
binds primary.
Sandwich Capture antibody binds antigen, then Very specific and sensitive. Best for
ELISA detection antibody is added. complex samples.
Type Description Use Case
Competitive Sample antigen competes with labeled Ideal for small antigens or
ELISA antigen for binding to antibody. detecting low concentrations.
🧬 Key Components
Antigen: Target molecule (protein, peptide, etc.)
Primary Antibody: Binds specifically to antigen.
Secondary Antibody: Conjugated to an enzyme; binds to primary antibody.
Enzyme: Usually HRP (Horseradish Peroxidase) or AP (Alkaline Phosphatase).
Substrate: TMB (3,3',5,5'-Tetramethylbenzidine) for HRP gives blue/yellow color upon
reaction.
⚠️Important Notes
Precision: Consistent washing and incubation times are crucial.
Controls: Include positive and negative controls for validation.
Optimization: Antibody concentrations, incubation times, and buffer pH must be
optimized for each system.
Storage: Coated plates can be stored at 4°C if sealed and dried properly.
🧪 Common Enzymes Used in ELISA
In ELISA (Enzyme-Linked Immunosorbent Assay), enzymes are conjugated to antibodies or
antigens to produce a detectable signal upon reaction with a suitable substrate. The most
commonly used enzymes are:
1. Horseradish Peroxidase (HRP)
Advantages:
o Rapid kinetics leading to shorter assay times.
o High sensitivity and stability.
o Cost-effective and widely available.
Common Substrates:
o TMB (3,3',5,5'-Tetramethylbenzidine): Produces a blue color that turns yellow
upon acidification.
o ABTS (2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)): Produces a green
color.
o OPD (o-Phenylenediamine dihydrochloride): Produces an orange color.
Mabtechinfo.gbiosciences.com
2. Alkaline Phosphatase (AP)
Advantages:
o Provides a linear response over a wide range, allowing for continuous readings.
o Stable under various conditions.
Common Substrates:
o pNPP (p-Nitrophenyl Phosphate): Produces a yellow color upon
dephosphorylation. Mabtechinfo.gbiosciences.com+1NCBI+1
3. β-Galactosidase (β-Gal)
Note: Less commonly used due to limited substrate options and lower sensitivity
compared to HRP and AP. Cell Signaling Technology+2NCBI+2Thermo Fisher
Scientific - US+2
