ELISA Types & Procedure
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The document describes four main types of ELISA assays: direct, indirect, sandwich, and competitive. It provides details on the procedure for each type. Direct ELISA uses one antibody directly conjugated to an enzyme for detection. Indirect ELISA uses an unlabeled primary antibody detected by a labeled secondary antibody, allowing for signal amplification. Sandwich ELISA requires two antibodies that bind to different epitopes of the target antigen. Competitive ELISA measures competition between a labeled antigen and unlabeled antigen in a sample.
ELISA Types & Procedure
- 1. ELISA Types & Procedure By:https://www.bosterbio.com/
- 2. Types of ELISA Basically there are 4 different types of ELISA namely; 1. Direct ELISA 2. Indirect ELISA 3. Sandwich ELISA 4. Competitive ELISA
- 3. Direct ELISA For direct detection, an antigen coated to a multi-well plate is detected by an antibody that has been directly conjugated to an enzyme. This detection method is a good option if there is no commercially available ELISA kits for your target protein. 1. Advantages ● Quick because only one antibody and fewer steps are used. ● Cross-reactivity of secondary antibody is eliminated. 1. Disadvantages ● Cell Smear: Adhere non-adherent cells on coverslip with chemical bond ● Immunoreactivity of the primary antibody might be adversely affected by labeling with enzymes or tags. ● Labeling primary antibodies for each specific ELISA system is time-consuming and expensive. ● No flexibility in choice of primary antibody label from one experiment to another. ● Minimal signal amplification.
- 4. Direct Elisa Procedure This is a general protocol in which antigen coating and blocking may not be required if the wells from the manufacturer have been pre-adsorbed with the antigen. 1. Antigen Coating ● Dilute purified antigens to a final concentration of 1-10 μg/ml in bicarbonate/carbonate antigen- coating ● Buffer (100 mM NaHCO3 in deionized water; pH adjusted to 9.6). ● Pipette 100 μL of diluted antigen to each well of a microtiter plate. ● Cover the plate with adhesive plastic and incubate at 4°C overnight (or 37C for 30 min). ● Remove the coating solution and wash the plate 3X with 200 μL PBS (Phosphate Buffered Saline) buffer (10 mM Na2HPO4 and 1.8 mM NaH2PO4 in deionized water with 0.2% Tween 20; pH Adjusted to 7.4) with for 5 minutes each time. The coating/washing solutions can be removed by flicking the plate over a sink. The remaining drops can be removed by patting the plate on a paper towel or by aspiration. Do not allow the wells to dry out at any time.
- 5. Direct Elisa Procedure 2. Blocking ● Pipette 200 μL blocking buffer (5% w/v non-fat dry milk in PBS buffer) per well to block residual protein-binding sites. Alternatively, BSA or BlockACE can be used to replace non-fat dry milk. ● Cover the plate with adhesive plastic and incubate for 1-2 hour(s) at 37°C (or at 4°C overnight). ● Remove the blocking solution and wash the plate 2X with 200 μL PBS for 5 min each time. Flick the plate and pat the plate as described in the coating step. 3. Reagent Preparation ● Prepare for the diluted standard solutions.
- 6. Direct Elisa Procedure 4. Primary Antibody Incubation ● Serially dilute the conjugated primary antibody with blocking buffer immediately before use. The optimal dilution should be determined by a titration assay according to the antibody manufacturer. ● Pipette 100 μL of diluted secondary antibody solution to each well. ● Cover the plate with adhesive plastic and incubate for 2 hours at room temperature. ● Remove the content in the wells and wash them 3X with 200 μL PBS buffer for 5 min each time. Flick the plate and pat the plate as described in the coating step. 5. Substrate Preparation Prepare the substrate solution immediately before use or bring the pre-made substrate to room temperature. The two widely used enzymes for signal detection are horse radish peroxidase (HRP) and alkaline phosphatase (AP), and their corresponding substrates, stopping solutions, detection absorbance wavelengths and color developed are as follows:
- 7. Direct Elisa Procedure ● The TMB substrate must be kept at 37°C for 30 min before use. ● Hydrogen peroxide can also act as a substrate for HRP. ● Sodium azide is an inhibitor of HRP. Do not include the azide in buffers or wash solutions if HRP- labeled conjugate is used for detection.
- 8. Direct Elisa Procedure 6. Signal Detection ● Pipette 90 μL of substrate solution to the wells with the control and standard solutions. ● Incubate the plate at 37°C in the dark. If TMB is used, shades of blue will be observed in the wells with the most concentrated solutions. Other wells may show no obvious color. ● Color should be developed in positive wells after 15 min. After sufficient color development, pipette 100 μL of stopping solution to the wells (if necessary). ● Read the absorbance (OD: Optical Density) of each well with a plate reader. 7. Data Analysis ● Prepare a standard curve using the data produced from the diluted standard solutions. Use absorbance on the Y-axis (linear) and concentration on the X-axis (log scale). ● Interpret the sample concentration from the standard curve.
- 9. Indirect ELISA For indirect detection, the antigen coated to a multi-well plate is detected in two stages or layers. First an unlabeled primary antibody, which is specific for the antigen, is applied. Next, an enzyme-labeled secondary antibody is bound to the first antibody. The secondary antibody is usually an anti-species antibody and is often polyclonal. The indirect assay, the most popular format for ELISA, has the advantages and disadvantages: 1. Advantages ● A wide variety of labeled secondary antibodies are available commercially. ● Versatile because many primary antibodies can be made in one species and the same labeled secondary antibody can be used for detection. ● Maximum immunoreactivity of the primary antibody is retained because it is not labeled. ● Sensitivity is increased because each primary antibody contains several epitopes that can be bound by the labeled secondary antibody, allowing for signal amplification. 1. Disadvantages ● Cell Smear: Adhere non-adherent cells on coverslip with chemical bond ● Cross-reactivity might occur with the secondary antibody, resulting in nonspecific signal. ● An extra incubation step is required in the procedure.
- 10. Indirect ELISA Procedure This is a general protocol in which antigen coating and blocking may not be required if the wells from the manufacturer have been pre-adsorbed with the antigen. 1. Antigen Coating ● Dilute purified antigens to a final concentration of 1-10 μg/mL in bicarbonate/carbonate antigen- coating ● Buffer (100 mM NaHCO3 in deionized water; pH adjusted to 9.6). ● Pipette 100 μL of diluted antigen to each well of a microtiter plate. ● Cover the plate with adhesive plastic and incubate at 4°C overnight (or 37°C for 30 min). ● Remove the coating solution and wash the plate 3X with 200 μL PBS (Phosphate Buffered Saline) buffer (10 mM Na2HPO4 and 1.8 mM NaH2PO4 in deionized water with 0.2% Tween 20; pH Adjusted to 7.4) with for 5 minutes each time. The coating/washing solutions can be removed by flicking the plate over a sink. The remaining drops can be removed by patting the plate on a paper towel or by aspiration. Do not allow the wells to dry out at any time.
- 11. Indirect ELISA Procedure 2. Blocking ● Pipette 200 μL blocking buffer (5% w/v non-fat dry milk in PBS buffer) per well to block residual protein-binding sites. Alternatively, BSA or BlockACE can be used to replace non-fat dry milk. ● Cover the plate with adhesive plastic and incubate for 1-2 hour(s) at 37°C (or at 4°C overnight). ● Remove the blocking solution and wash the plate 2X with 200 μL PBS for 5 minutes each time. Flick the plate and pat the plate as described in the coating step. 3. Reagent Preparation ● Prepare for the diluted standard solutions
- 12. Indirect ELISA Procedure 4. Primary Antibody Incubation ● Serially dilute the primary antibody of choice with blocking buffer. The optimal dilution should be determined by a titration assay according to the antibody manufacturer. ● Pipette 100 μL of each diluted antibody per well. Repeat in duplicate or triplicate for accuracy. The negative control should be species- and isotype-matched as well as non-specific immunoglobulin diluted in PBS buffer. ● Cover the plate with adhesive plastic and incubate for 1 hour at 37°C (or 2 hours at room temperature). These incubation times should be sufficient to receive a strong signal. However, if a weak signal is observed, perform incubation overnight at 4°C for a stronger signal. ● Remove the diluted antibody solution and wash the wells 3X with 200 μL PBS for 5 min each time. Flick the plate and pat the plate as described in the coating step. ● Serially dilute the primary antibody of choice with blocking buffer. The optimal dilution should be determined by a titration assay according to the antibody manufacturer.
- 13. Indirect ELISA Procedure ● Pipette 100 μL of each diluted antibody per well. Repeat in duplicate or triplicate for accuracy. The negative control should be species- and isotype-matched as well as non-specific immunoglobulin diluted in PBS buffer. ● Cover the plate with adhesive plastic and incubate for 1 hour at 37°C (or 2 hours at room temperature). These incubation times should be sufficient to receive a strong signal. However, if a weak signal is observed, perform incubation overnight at 4°C for a stronger signal. ● Remove the diluted antibody solution and wash the wells 3X with 200 μL PBS for 5 min each time. Flick the plate and pat the plate as described in the coating step. 5. Secondary Antibody Incubation ● Serially dilute the conjugated secondary antibody with blocking buffer immediately before use. The optimal dilution should be determined by a titration assay according to the antibody manufacturer. ● Pipette 100 μL of diluted secondary antibody solution to each well.
- 14. Indirect ELISA Procedure ● Cover the plate with adhesive plastic and incubate for 2 hours at room temperature. ● Remove the content in the wells and wash them 3X with 200 μL PBS buffer for 5 min each time. Flick the plate and pat the plate as described in the coating step. 6. Substrate Preparation Prepare the substrate solution immediately before use or bring the pre-made substrate to room temperature. The two widely used enzymes for signal detection are horse radish peroxidase (HRP) and alkaline phosphatase (AP), and their corresponding substrates, stopping solutions, detection absorbance wavelengths and color developed are as follows:
- 15. Indirect ELISA Procedure Note: ● The TMB substrate must be kept at 37°C for 30 min before use. ● Hydrogen peroxide can also act as a substrate for HRP. ● Sodium azide is an inhibitor of HRP. Do not include the azide in buffers or wash solutions if HRP- labeled conjugate is used for detection. 7. Signal Detection ● Pipette 90 μL of substrate solution to the wells with the control and standard solutions. ● Incubate the plate at 37°C in the dark. If TMB is used, shades of blue will be observed in the wells with the most concentrated solutions. Other wells may show no obvious color. ● Color should be developed in positive wells after 15 min. After sufficient color development, pipette 100 μL of stop solution to the wells (if necessary). ● Read the absorbance (OD: Optical Density) of each well with a plate reader.
- 16. Indirect ELISA Procedure 8. Data Analysis ● Prepare a standard curve using the data produced from the diluted standard solutions. Use absorbance on the Y-axis (linear) and concentration on the X-axis (log scale). ● Interpret the sample concentration from the standard curve.
- 17. Sandwich ELISA Sandwich ELISAs typically require the use of matched antibody pairs, where each antibody is specific for a different, non- overlapping part (epitope) of the antigen molecule. A first antibody (known as capture antibody) is coated to the wells. The sample solution is then added to the well. A second antibody (known as detection antibody) follows this step in order to measure the concentration of the sample. This type of ELISA has the following advantages: ● High specificity: the antigen/analyte is specifically captured and detected ● Suitable for complex (or crude/impure) samples: the antigen does not require purification prior to measurement ● Flexibility and sensitivity: both direct or indirect detection methods can be used
- 18. Sandwich ELISA Procedure All of the ELISA kits from Boster use the sandwich format and avidin-biotin chemistry. Our ELISA assays require the dilutions of standard solutions, biotinylated antibody (detection antibody) and avidin-biotin- peroxidase. 1. Capture Antibody Coating ● (These steps are not required if the pre-adsorbed Picokine ELISA kits from Boster are used) ● Dilute the capture antibody to a final concentration of 1-10 μg/mL in bicarbonate/carbonate antigen-coating buffer (100 mM NaHCO3 in deionized water; pH adjusted to 9.6). ● Pipette 100 μL of diluted antibody to each well of a microtiter plate. ● Cover the plate with adhesive plastic and incubate at 4°C overnight (or 37°C for 30 min). ● Remove the coating solution and wash the plate 3X with 200 μL PBS (Phosphate Buffered Saline) buffer (10 mM Na2HPO4 and 1.8 mM NaH2PO4 in deionized water with 0.2% Tween 20; pH Adjusted to 7.4) with for 5 minutes each time. The coating/washing solutions can be removed by flicking the plate over a sink. The remaining drops can be removed by patting the plate on a paper towel or by aspiration. Do not allow the wells to dry out at any time.
- 19. Sandwich ELISA Procedure 2. Blocking ● (These steps are not required if the pre-adsorbed Picokine ELISA kits from Boster are used) ● Pipette 200 μL blocking buffer (5% w/v non-fat dry milk in PBS buffer) per well to block residual protein-binding sites. Alternatively, BSA or BlockACE can be used to replace non-fat dry milk. ● Cover the plate with adhesive plastic and incubate for 1-2 hour(s) at 37°C (or at 4°C overnight). ● Remove the blocking solution and wash the plate 2X with 200 μL PBS for 5 minutes each time. Flick the plate and pat the plate as described in the coating step. 3. Reagent Preparation Prepare for the diluted standard solutions, biotinylated antibody and ABC solutions as
- 20. Sandwich ELISA Procedure 4. Sample (Antigen) Incubation ● Serially dilute the sample with blocking buffer immediately before use. The optimal dilution should be determined by a titration assay according to the antibody manufacturer. ● Pipette 100 μL of each of the diluted sample solutions and control to each empty well. Repeat in duplicate or triplicate for accuracy. The negative control should be species- and isotype-matched as well as non- specific immunoglobulin diluted in PBS buffer. ● Cover the plate with adhesive plastic and incubate for 2 hours at room temperature. ● Remove the content in the wells and wash them 3X with 200 μL PBS buffer for 5 minutes each time. Flick the plate and pat the plate as described in the coating step.
- 21. Sandwich ELISA Procedure 5. Biotinylated Antibody Incubation ● Pipette 100 μL of diluted antibody to the wells with control, standard solutions and diluted samples. ● Cover the plate with adhesive plastic and incubate for 1 hour at 37°C (or 2 hours at room temperature). These incubation times should be sufficient to receive a strong signal. However, if a weak signal is observed, perform incubation overnight at 4°C for a stronger signal. ● Remove the content in the wells and wash them 3X with 200 μL PBS for 5 min each time. Flick the plate and pat the plate as described in the coating step. 6. ABC Incubation ● Pipette 100 μL of diluted ABC solution to the wells with control, standard solutions and diluted samples. ● Cover the plate with adhesive plastic and incubate for 0.5 hour at 37°C. ● Remove the content in the wells and wash them 3X with 200 μL PBS buffer for 5 min each time.
- 22. Sandwich ELISA Procedure 7. Substrate Preparation Prepare the substrate solution immediately before use or bring the pre-made substrate to room temperature. The two widely used enzymes for signal detection are horse radish peroxidase (HRP) and alkaline phosphatase (AP), and their corresponding substrates, stopping solutions, detection absorbance wavelengths and color developed are as follows: ● The TMB substrate must be kept at 37°C for 30 min before use. ● Hydrogen peroxide can also act as a substrate for HRP. ● Sodium azide is an inhibitor of HRP. Do not include the azide in buffers or wash solutions if HRP- labeled conjugate is used for detection.
- 23. Sandwich ELISA Procedure 8. Signal Detection ● Pipette 90 μL of substrate solution to the wells with the control, standard solutions and diluted samples. ● Incubate the plate at 37°C in the dark. If TMB is used, shades of blue will be observed in the wells with the most concentrated solutions. Other wells may show no obvious color. ● Color should be developed in positive wells after 15 min. After sufficient color development, pipette 100 μL of stop solution to the appropriate wells (if necessary). ● Read the absorbance (OD: Optical Density) of each well with a plate reader. 9. Data Analysis ● Prepare a standard curve using the data produced from the diluted standard solutions. Use absorbance on the Y-axis (linear) and concentration on the X-axis (log scale). ● Interpret the sample concentration from the standard curve.
- 24. Competitive ELISA Procedure This ELISA kit is of competitive format. Competitive ELISA, also known as inhibition ELISA, is a surface/plate based assay, where the plate is coated with capture antibodies reactive to the molecule of interest. The sample (containing native molecule of interest) and enzyme conjugated recombinant protein (the competing molecule) are added to the coated wells. Since the amount of enzyme conjugated molecule in each well is constant, the level of native molecule in the sample will determine the binding ratio of enzyme conjugated molecule vs. native molecule. After an incubation period, any unbound antibody is washed off. Enzyme substrate (for example, TMB for HRP) is added to each well and will be transformed into a blue precipitate, the amount of which is linearly proportional to the amount of enzyme in the well. The precipitate is then turned into yellow by adding the acid stop solution and the concentration of yellow precipitate is read at 450nm for light absorbance (O.D. value). The O.D. is then used to calculate the amount of molecule of interest in each well, by comparing each sample well against the standard curve. The standard curve is generated using the same principle but instead of adding samples, a series of recombinant molecules with known concentrations are added to 6-8 wells.
- 25. Competitive ELISA Procedure This is a general protocol in which antigen coating and blocking may not be required if the wells from the manufacturer have been pre-adsorbed with the antigen. 1. Antigen Coating ● Dilute purified antigens to a final concentration of 20 μg/ml in bicarbonate/carbonate antigen- coating ● Buffer (100 mM NaHCO3 in deionized water; pH adjusted to 9.6). ● Pipette 100 μL of diluted antigen to each well of a microtiter plate. ● Cover the plate with adhesive plastic and incubate at 4°C overnight (or 37C for 30 min). ● Remove the coating solution and wash the plate 3X with 200 μL PBS (Phosphate Buffered Saline) buffer (10 mM Na2HPO4 and 1.8 mM NaH2PO4 in deionized water with 0.2% Tween 20; pH Adjusted to 7.4) with for 5 minutes each time. The coating/washing solutions can be removed by flicking the plate over a sink. The remaining drops can be removed by patting the plate on a paper towel or by aspiration. Do not allow the wells to dry out at any time.
- 26. Competitive ELISA Procedure 2. Blocking Pipette 200 μL blocking buffer (5% w/v non-fat dry milk in PBS buffer) per well to block residual protein- binding sites. Alternatively, BSA or BlockACE can be used to replace non-fat dry milk. Cover the plate with adhesive plastic and incubate for 1-2 hour(s) at 37°C (or at 4°C overnight). Remove the blocking solution and wash the plate 2X with 200 μL PBS for 5 min each time. Flick the plate and pat the plate as described in the coating step. 3. Reagent Preparation a. Prepare for the diluted standard solutions
- 27. Competitive ELISA Procedure 4. Sample (Antigen) Incubation a. Serially dilute the sample with blocking buffer immediately before use. The optimal dilution should be determined by a titration assay according to the antibody manufacturer. b. Pipette 100 μL of diluted sample to each well. c. Cover the plate with adhesive plastic and incubate for 2 hours at room temperature. d. Remove the content in the wells and wash them 3X with 200 μL PBS buffer for 5 minutes each time. Flick the plate and pat the plate as described in the coating step.
- 28. Competitive ELISA Procedure 5. Primary Antibody Incubation a. Serially dilute the primary antibody of choice with blocking buffer. The optimal dilution should be determined by a titration assay according to the antibody manufacturer. b. Pipette 100 μL of each diluted antibody per well. Repeat in duplicate or triplicate for accuracy. The negative control should be species- and isotype-matched as well as non-specific immunoglobulin diluted in PBS buffer. c. Cover the plate with adhesive plastic and incubate for 1 hour at 37°C (or 2 hours at room temperature). These incubation times should be sufficient to receive a strong signal. However, if a weak signal is observed, perform incubation overnight at 4°C for a stronger signal. d. Remove the diluted antibody solution and wash the wells 3X with 200 μL PBS for 5 min each time.
- 29. Competitive ELISA Procedure 6. Secondary Antibody Incubation a. Serially dilute the conjugated secondary antibody with blocking buffer immediately before use. The optimal dilution should be determined by a titration assay according to the antibody manufacturer. b. Pipette 100 μL of diluted secondary antibody solution to each well. c. Cover the plate with adhesive plastic and incubate for 2 hours at room temperature. d. Remove the content in the wells and wash them 3X with 200 μL PBS buffer for 5 min each time. Flick the plate and pat the plate as described in the coating step.
- 30. Competitive ELISA Procedure 7. Substrate Preparation Prepare the substrate solution immediately before use or bring the pre-made substrate to room temperature. The two widely used enzymes for signal detection are horse radish peroxidase (HRP) and alkaline phosphatase (AP), and their corresponding substrates, stopping solutions, detection absorbance wavelengths and color developed are as follows: a. The TMB substrate must be kept at 37°C for 30 min before use. b. Hydrogen peroxide can also act as a substrate for HRP. c. Sodium azide is an inhibitor of HRP. Do not include the azide in buffers or wash solutions if HRP- labeled conjugate is used for detection.
- 31. Competitive ELISA Procedure 8. Signal Detection a. Pipette 90 μL of substrate solution to the wells with the control, standard solutions and diluted samples. b. Incubate the plate at 37C in the dark. If TMB is used, shades of blue will be observed in the wells with the most concentrated solutions. Other wells may show no obvious color. c. Color should be developed in positive wells after 15 minutes. After sufficient color development, pipette 100 μL of stopping solution to the wells (if necessary). d. Read the absorbance (OD: Optical Density) of each well with a plate reader. 9. Data Analysis a. Prepare a standard curve using the data produced from the diluted standard solutions. Use absorbance on the Y-axis (linear) and concentration on the X-axis (log scale). b. Competitive ELISA yields an inverse curve: Higher values of antigen in the samples yield a smaller amount of color change. c. Interpret the sample concentration from the standard curve.
- 32. THE END