The Enzyme-Linked Immunosorbent Assay (ELISA) is a versatile and widely used plate-based technique for detecting and quantifying soluble substances, such as peptides, proteins, antibodies, and hormones. Its high sensitivity and specificity stem from the specific binding between an antigen and an antibody, with the detection achieved via an enzyme-linked conjugate that produces a measurable signal.

(ELISA Flowchart)

In a typical ELISA designed to detect an antigen within a complex protein mixture, the antigen is first immobilized onto the wells of a microplate, either through direct adsorption or via a capture antibody coated on the plate surface. After blocking to prevent nonspecific binding, the antigen is detected using a specific detection antibody. This detection antibody may be directly conjugated to an enzyme or fluorophore for signal generation. Alternatively, it can be indirectly detected using an enzyme- or fluorophore-labeled secondary antibody or through avidin-biotin chemistry (see below). For enzyme-based detection, a suitable substrate is added to produce a measurable signal. The intensity of this signal is proportional to the amount of antigen present in the sample. Washing steps between each stage are critical to remove unbound materials, ensuring that only specific high-affinity interactions contribute to the final signal.

(Diagram of ELISA formats)

1. Materials

1.1 Bicarbonate/carbonate coating buffer (100 mM) , antigen or antibody should be diluted in coating buffer to immobilize them to the wells: 3.03 g Na2CO3, 6.0 g NaHCO3, 1000 mL distilled water, pH 9.6.

1.2 PBS: 1.16 g Na2HPO4, 0.1 g KCl, 0.1 g K3PO4, 4.0 g NaCl (500 mL distilled water) pH 7.4.

1.3 Blocking solution: Commonly used blocking agents are 5% BSA , non-fat dry milk, gelatin in PBS.

1.4 Wash solution: Usually PBS or Tris-buffered (pH 7.4) with detergent such as 0.05% (v/v) Tween20 (TBST).

1.5 Antibody dilution buffer: Primary and secondary antibody should be diluted in 1x blocking solution to reduce Non specific binding.

2. Method

2.1 Dilute the antigen to a final concentration of 10 µg/mL in PBS or other carbonate buffer. Coat the wells

of a PVC microtiter plate with the antigen by pipeting 100µL of the antigen dilution in the top wells of the

plate. Dilute down the plate as required. Seal the plate and incubate overnight at 4℃ or 2 h at room

temperature.

2.2 Wash plate 3 times with PBS.

2.3 Block the remaining protein-binding sites in the coated wells by adding 200 µL blocking buffer, 5% non

fat dry milk/PBS, per well. Alternative blocking reagents include Block BSA.

2.4 Cover the plate with an adhesive plastic and incubate for at least 2 h at room temperature or, if more

convenient, overnight at 4℃.

2.5 Wash the plate twice with PBS

2.6 Add 100 µL of the antibody, diluted at the optimal concentration (according to the manufacturer’s

instructions) in blocking buffer immediately before use.

2.7 Cover the plate with an adhesive plastic and incubate for 2 h at room temperature.

2.8 Wash the plate 5 times with PBS.

2.9 Dispense 100 µL (or 50 µL) of the substrate solution per well with a multichannel pipet or a multipipet.

2.10 After sufficient color development (if it is necessary) add 50-100 µL of stop solution to the wells.

2.11 Record the absorbance at 450 nm on a plate reader within 30 minutes of stopping the reaction.

1. Materials

1. Bicarbonate/carbonate coating buffer (100 mM) , antigen or antibody should be diluted in coating buffer to immobilize them to the wells: 3.03 g Na2CO3, 6.0 g NaHCO3, 1000 mL distilled water, pH 9.6.

2. PBS: 1.16 g Na2HPO4, 0.1 g KCl, 0.1 g K3PO4, 4.0 g NaCl (500 mL distilled water) pH 7.4.

3. Blocking solution: Commonly used blocking agents are 5% BSA , non-fat dry milk, gelatin in PBS.

4. Wash solution: Usually PBS or Tris-buffered (pH 7.4) with detergent such as 0.05% (v/v) Tween20 (TBST).

5. Antibody dilution buffer: Primary and secondary antibody should be diluted in 1x blocking solution to reduce Non specific binding.

2. Method

2.1 Dilute antigen to a final concentration of 1-20 μg/mL using PBS or Bicarbonate/carbonate coating

buffer. Coat the wells of a PVC microtiter plate with the antigen by pipeting 50 µL of the antigen dilution in

the top wells of the plate. Dilute down the plate as required. Seal the plate and incubate overnight at 4℃

or 2 h at room temperature.

2.2 Wash plate 3 times with PBS.

2.3 Block the remaining protein-binding sites in the coated wells by adding 200 µL blocking buffer, 5% non

fat dry milk/PBS, per well. Alternative blocking reagents include Block BSA.

2.4 Cover the plate with an adhesive plastic and incubate for at least 2 h at room temperature or, if more

convenient, overnight at 4℃.

2.5 Wash the plate 3 times with PBS.

2.6 Add 100 µL of diluted primary antibody to each well.

2.7 Cover the plate with an adhesive plastic and incubate for 2 h at room temperature.

2.8 Wash the plate 4 times with PBS.

2.9 Add 100 µL of conjugated secondary antibody, diluted at the optimal concentration (according to the

manufacturer) in blocking buffer immediately before use.

2.10 Cover the plate with an adhesive plastic and incubate for 1-2 h at room temperature.

2.11 Wash the plate 5 times with PBS.

2.12 Dispense 100 µL (or 5 0µL) of the substrate solution per well with a multichannel pipet or a multipipet.

2.13 After sufficient color development (if it is necessary) add 50-100 µL of stop solution to the wells.

2.14 Record the absorbance at 450 nm on a plate reader within 30 min of stopping the reaction.

1. Materials

1. Bicarbonate/carbonate coating buffer (100 mM) , antigen or antibody should be diluted in coating buffer to immobilize them to the wells: 3.03 g Na2CO3, 6.0 g NaHCO3, 1000 mL distilled water, pH 9.6.

2. PBS: 1.16 g Na2HPO4, 0.1 g KCl, 0.1 g K3PO4, 4.0 g NaCl (500 mL distilled water) pH 7.4.

3. Blocking solution: Commonly used blocking agents are 5% BSA , non-fat dry milk, gelatin in PBS.

4. Wash solution: Usually PBS or Tris-buffered (pH 7.4) with detergent such as 0.05% (v/v) Tween20 (TBST).

5. Antibody dilution buffer: Primary and secondary antibody should be diluted in 1x blocking solution to reduce Non specific binding.

2. Method

2.1 Coat the wells of a PVC microtiter plate with the capture antibody at a concentration of 1-10 µg/mL in

carbonate/bicarbonate buffer (pH7.4). Seal the plate and incubate overnight at 4℃or 2h at room

temperature.

2.2 Wash plate 3 times with PBS.

2.3 Block the remaining protein-binding sites in the coated wells by adding 200µL blocking buffer, 5% non

fat dry milk/PBS, per well.

2.4 Cover the plate with an adhesive plastic and incubate for at least 1-2 h at room temperature or, if more

convenient, overnight at 4℃.

2.5 Add 100µL of appropriately diluted samples to each well. For accurate quantitative results, always

compare signal of unknown samples against those of a standard curve. Standards (duplicates or

triplicates) and blank must be run with each plate to ensure accuracy. Incubate for 90 min at 37℃.

2.6 Wash the plate twice with PBS.

2.7 Add 100 µL of diluted detection antibody to each well.

2.8 Cover the plate with an adhesive plastic and incubate for 2 h at room temperature.

2.9 Wash the plate 4 times with PBS.

2.10 Add 100 µL of secondary antibody conjugated, diluted at the optimal concentration (according to the

manufacturer) in blocking buffer immediately before use.

2.11 Cover the plate with an adhesive plastic and incubate for 1-2 h at room temperature.

2.12 Wash the plate 5 times with PBS.

2.13 Dispense 100 µL (or 50 µL) of the substrate solution per well with a multichannel pipet or a multipipet.

2.14 After sufficient color development (if it is necessary) add 50-100 µL of stop solution to the wells.

2.15 Record the absorbance at 450 nm on a plate reader within 30 min of stopping the reaction.

Controls: Always include blank wells (no antigen) and negative controls.

Optimization: Antibody concentrations and incubation times should be optimized for each new assay.

Linearity: Ensure sample readings fall within the linear range of the standard curve.

Reproducibility: Use technical replicates for both standards and samples.