Standardized Protocol for ELISA Assay
1
Direct ELISA Protocol
Materials
- Bicarbonate/carbonate coating buffer (100mM): Antigen or antibody should be diluted in this coating buffer to facilitate their immobilization on the wells. Preparation: 3.03g Na₂CO₃, 6.0g NaHCO₃, dissolved in 1000ml distilled water, adjusted to pH 9.6.
- PBS: 1.16g Na₂HPO₄, 0.1g KCl, 0.1g K₃PO₄, 4.0g NaCl, dissolved in 500ml distilled water, pH 7.4.
- Blocking solution: Commonly used blocking agents include 1% BSA, serum, non-fat dry milk, casein, or gelatin, all prepared in PBS.
- Wash solution: Typically PBS or Tris-buffered saline (pH 7.4) supplemented with a detergent such as 0.05% (v/v) Tween20 (TBST).
- Antibody dilution buffer: Both primary and secondary antibodies should be diluted in 1x blocking solution to minimize non-specific binding.
Method
- Dilute the antigen to a final concentration of 10µg/ml in PBS or other carbonate buffer. Add 100µl of the antigen dilution to the top wells of a microtiter plate, then perform serial dilutions as needed. Seal the plate and incubate overnight at 4℃ or for 2 hours at room temperature.
- Wash the plate 3 times with PBS.
- Block the remaining protein-binding sites in the coated wells by adding 200µl of blocking buffer (5% non-fat dry milk in PBS) to each well. Alternative blocking reagents include BlockACE or BSA.
- Cover the plate with an adhesive plastic cover and incubate for at least 2 hours at room temperature, or overnight at 4℃ for greater convenience.
- Wash the plate twice with PBS.
- Immediately before use, dilute the antibody to the optimal concentration (following instructions) in blocking buffer, then add 100µl to each well.
- Cover the plate with an adhesive plastic cover and incubate for 2 hours at room temperature.
- Wash the plate 5 times with PBS.
- Use a multichannel pipette or multipipette to add 100µl (or 50µl) of substrate solution to each well.
- Once sufficient color development is achieved (if required), add 50-100µl of stop solution to each well.
- Measure the absorbance at 450 nm using a plate reader within 30 minutes of stopping the reaction.
2
Indirect ELISA Protocol
Materials
- Bicarbonate/carbonate coating buffer (100mM): Antigen or antibody should be diluted in this coating buffer to facilitate their immobilization on the wells. Preparation: 3.03g Na₂CO₃, 6.0g NaHCO₃, dissolved in 1000ml distilled water, adjusted to pH 9.6.
- PBS: 1.16g Na₂HPO₄, 0.1g KCl, 0.1g K₃PO₄, 4.0g NaCl, dissolved in 500ml distilled water, pH 7.4.
- Blocking solution: Commonly used blocking agents include 1% BSA, serum, non-fat dry milk, casein, or gelatin, all prepared in PBS.
- Wash solution: Typically PBS or Tris-buffered saline (pH 7.4) supplemented with a detergent such as 0.05% (v/v) Tween20 (TBST).
- Antibody dilution buffer: Both primary and secondary antibodies should be diluted in 1x blocking solution to minimize non-specific binding.
Method
- Dilute the antigen to a final concentration of 1-20μg/mL using PBS or bicarbonate/carbonate coating buffer. Add 50µl of the antigen dilution to the top wells of a microtiter plate, then perform serial dilutions as needed. Seal the plate and incubate overnight at 4℃ or for 2 hours at room temperature.
- Wash the plate 3 times with PBS.
- Block the remaining protein-binding sites in the coated wells by adding 200µl of blocking buffer (5% non-fat dry milk in PBS) to each well. Alternative blocking reagents include BlockACE or BSA.
- Cover the plate with an adhesive plastic cover and incubate for at least 2 hours at room temperature, or overnight at 4℃ for greater convenience.
- Wash the plate 3 times with PBS.
- Add 100µl of diluted primary antibody to each well.
- Cover the plate with an adhesive plastic cover and incubate for 2 hours at room temperature.
- Wash the plate 4 times with PBS.
- Immediately before use, dilute the conjugated secondary antibody to the optimal concentration (following instructions) in blocking buffer, then add 100µl to each well.
- Cover the plate with an adhesive plastic cover and incubate for 1-2 hours at room temperature.
- Wash the plate 5 times with PBS.
- Use a multichannel pipette or multipipette to add 100µl (or 50µl) of substrate solution to each well.
- Once sufficient color development is achieved (if required), add 50-100µl of stop solution to each well.
- Measure the absorbance at 450 nm using a plate reader within 30 minutes of stopping the reaction.
3
Sandwich ELISA Protocol
Materials
- Bicarbonate/carbonate coating buffer (100mM): Antigen or antibody should be diluted in this coating buffer to facilitate their immobilization on the wells. Preparation: 3.03g Na₂CO₃, 6.0g NaHCO₃, dissolved in 1000ml distilled water, adjusted to pH 9.6.
- PBS: 1.16g Na₂HPO₄, 0.1g KCl, 0.1g K₃PO₄, 4.0g NaCl, dissolved in 500ml distilled water, pH 7.4.
- Blocking solution: Commonly used blocking agents include 1% BSA, serum, non-fat dry milk, casein, or gelatin, all prepared in PBS.
- Wash solution: Typically PBS or Tris-buffered saline (pH 7.4) supplemented with a detergent such as 0.05% (v/v) Tween20 (TBST).
- Antibody dilution buffer: Both primary and secondary antibodies should be diluted in 1x blocking solution to minimize non-specific binding.
Method
- Coat the wells of a microtiter plate with the capture antibody at a concentration of 1-10µg/ml in carbonate/bicarbonate buffer (pH7.4). Seal the plate and incubate overnight at 4℃ or for 2 hours at room temperature.
- Wash the plate 3 times with PBS.
- Block the remaining protein-binding sites in the coated wells by adding 200µl of blocking buffer (5% non-fat dry milk in PBS) to each well.
- Cover the plate with an adhesive plastic cover and incubate for at least 1-2 hours at room temperature, or overnight at 4℃ for greater convenience.
- Add 100µl of appropriately diluted samples to each well. For accurate quantitative results, always compare the signal of unknown samples against a standard curve. Standards (run in duplicates or triplicates) and a blank control must be included with each plate to ensure accuracy. Incubate for 90 minutes at 37℃.
- Wash the plate twice with PBS.
- Add 100µl of diluted detection antibody to each well.
- Cover the plate with an adhesive plastic cover and incubate for 2 hours at room temperature.
- Wash the plate 4 times with PBS.
- Immediately before use, dilute the conjugated secondary antibody to the optimal concentration (following instructions) in blocking buffer, then add 100µl to each well.
- Cover the plate with an adhesive plastic cover and incubate for 1-2 hours at room temperature.
- Wash the plate 5 times with PBS.
- Use a multichannel pipette or multipipette to add 100µl (or 50µl) of substrate solution to each well.
- Once sufficient color development is achieved (if required), add 50-100µl of stop solution to each well.
- Measure the absorbance at 450 nm using a plate reader within 30 minutes of stopping the reaction.
4
Three ELISA Methods Comparison Table
| Comparison Index | Direct ELISA | Indirect ELISA | Sandwich ELISA |
|---|---|---|---|
| Antigen Coating Volume | 100µl per well | 50µl per well | No antigen coating (coated with capture antibody) |
| Antigen Concentration | 10µg/ml | 1-20μg/mL | Not specified (samples diluted appropriately) |
| Antibody Usage | Only primary antibody (no secondary antibody) | Primary antibody + conjugated secondary antibody | Capture antibody + detection antibody + conjugated secondary antibody |
| Incubation Time for Samples/Antibodies | Primary antibody: 2h (room temperature) | Primary antibody: 2h; secondary antibody: 1-2h (room temperature) | Samples: 90min (37℃); detection antibody: 2h; secondary antibody: 1-2h (room temperature) |
| Washing Times | 3→2→5 times (PBS) | 3→3→4→5 times (PBS) | 3→2→4→5 times (PBS) |
| Key Feature | Simple operation, short time, low non-specific binding | High sensitivity, wide applicability, signal amplification | High specificity and sensitivity, suitable for quantitative detection |