ELISA Protocol

5. Sample Preparation

The procedure below provides a general guidance for the preparation of commonly tested samples for use in ELISA assays.
Generally:
- Protein extract concentration should be at 1-2 mg/mL.
- Cell and tissue extracts are diluted 1:1 with binding buffer.
- Samples are centrifuged at 10,000 rpm for 5 min at 4°C before use, to remove any precipitate.

• Cell Culture Supernatants
Centrifuge cell culture media at 1,500 rpm for 10 min at 4°C, aliquot and assay supernatant immediately. Hold at -80°C and avoiding freeze thawing.

• Cell Extracts
Place tissue culture plates on ice. Remove the media and gently wash cells once with chilled (10 mM Na2HPO4 and 1.8 mM NaH2PO4 in deionized water with 0.2% Tween 20; pH Adjusted to 7.4). Remove the PBS and add 0.5 ml extraction buffer per 100 mm plate. Tilt the plate and scrape the cells into a pre-chilled tube. Vortex briefly and incubate on ice for 15-30 min. Centrifuge at 13,000 rpm for 10 min at 4°C to pellet the insoluble content. Aliquot the supernatant in chilled tubes on ice, store at -80°C and avoid freeze thawing.

• Conditioned Media
Plate the cells in complete growth medium (with serum) until the desired level of confluence is achieved. Remove the growth media and gently wash cells twice with 2- 3 ml of warm PBS. Remove the PBS, gently add serum-free growth medium and incubate for 1-2 days. Transfer the medium into a centrifuge tube and centrifuge at 1,500 rpm for 10 min at 4°C. Aliquot the supernatant, store samples at -80°C and avoid freeze thawing.

• Tissue Extract
Homogenize tissue on ice in ice-cold buffer, containing protease inhibitors. Place the tissue in micro-centrifuge tubes and snap-freeze in liquid nitrogen. Store samples at -80°C or keep on ice for immediate homogenization. Add 300 μL of extraction buffer per 5 mg of tissue and homogenize.

• Extraction buffer
00 mM Tris, pH 7.4
150 mM NaCL
1 mM EGTA
1 mM EDTA
1% Triton X-100 0.5%
0.5% sodium deoxycholate (SDS)

Extraction buffer can be prepared in advance and stored at 4°C. Add phosphatase inhibitor (according to manufacturers guidelines), protease inhibitor (according to manufacturers guidelines) and PMSF to 1 mM before use.

Rinse the blade of the homogenizer twice with 300 μL extraction buffer. Place the sample on a shaker at 4°C for 2 hours. Centrifuge the sample for 20 min at 13,000 rpm at 4°C. Aliquot the supernatant into pre-chilled tubes on ice. Keep the samples at -80°C and avoid freeze-thawing
Note: Lysis buffer volume must be determined according to the amount of tissue present. Typical concentration of final protein extract is at least 1 mg/mL.

Biotinylated Antibody
Calculate the total volume needed for the assay by multiplying 0.1 mL/well and the number of wells required. Add 2-3 extra wells to the calculated number of wells to account for possible pipetting errors. Generate the required volume of diluted antibody by performing a 1:100 dilution (For each 1 μL concentrated antibody, add 99 μL antibody dilution buffer) and mixing thoroughly.

Avidin-Biotin-Peroxidase (ABC)
Diluted ABC solution has to be prepared freshly. Calculate the total volume needed for the assay by multiplying 0.1 mL/well and the number of wells required. Add 2-3 extra wells to the calculated number of wells to account for possible pipetting errors. Generate the required volume of diluted ABC solution by performing a 1:100 dilution (For each 1 μL concentrated ABC solution, add 99 μL ABC dilution buffer) and mixing thoroughly.

• Direct ELISA protocol
This is a general protocol in which antigen coating and blocking may not be required if the wells have been pre-adsorbed with the antigen by the manufacturer.

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 minutes.
- Remove the coating solution and wash the plate 3 times with 200 μL PBS buffer 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.

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 twice with 200 μL PBS for 5 min each time. Flick the plate and pat the plate as described in the coating step.

Reagent Preparation
Prepare for the diluted standard solutions as shown on above.

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.
- Remove the content in the wells and wash them 3 times with 200 μL PBS buffer for 5 min each time. Flick and pat the plate as described in the coating step.

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 colour developed are as follows:

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.

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 colour.
- Colour should be developed in positive wells after 15 min. After sufficient colour 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.

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.

• Indirect ELISA protocol
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.

Antigen Coating
- Dilute the 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 the microtiter plate.

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 the 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 twice with 200 μL PBS for 5 minutes each time. Flick and pat the plate as described in the coating step.

Reagent Preparation
- Prepare for the diluted standard solutions as shown on above.

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 achieve a strong signal. However, if a weak signal is observed, perform incubation overnight at 4°C to obtain a stronger signal.
- Remove the diluted antibody solution and wash the wells 3 times with 200 μL PBS for 5 minutes each time. Flick the plate and pat the plate as described in the coating step.

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.
- Cover the plate with adhesive plastic and incubate for 2 hours at room temperature.
- Remove the content in the wells and wash them 3 times with 200 μL PBS buffer for 5 minutes each time. Flick and pat the plate as described in the coating step.

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 colour developed are as follows:

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.

Sandwich ELISA protocol
Capture Antibody Coating
- 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 3 times with 200 μL PBS 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.

Blocking
- Pipette 200 μL blocking buffer (5% w/v non-fat dry milk in PBS buffer; Alternatively, BSA or BlockACE can be used to replace non-fat dry milk.) per well to block residual protein-binding sites.
- 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 twice with 200 μL PBS for 5 minutes each time. Flick and tap the plate as described in the coating step.

Reagent Preparation
Prepare biotinylated antibody and ABC solutions as described previously for the diluted standard solutions.

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 3 times with 200 μL PBS buffer for 5 minutes each. Flick the plate and pat the plate as described in the coating step.

Biotinylated Antibody Incubation
- Pipette 100 μL of diluted antibody to the 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 3 times with 200 μL PBS for 5 min each time. Flick the plate and pat the plate as described in the coating step.

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 30 minutes at 37°C.
- Remove the content in the wells and wash them 3 times with 200 μL PBS buffer for 5 minutes each. Flick the plate and pat the plate as described in the coating step.

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 colour developed are as follows:

Note:
• The TMB substrate must be kept at 37°C for 30 minutes 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.

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 colour.
- A colourimetric signal should be developed in positive wells after 15 minutes. Stop the reaction by adding 100 μL of stop solution.
- Read the absorbance (OD: Optical Density) of each well with a plate reader.

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). Competitive ELISA yields an inverse curve: Higher values of antigen in the samples yield a smaller amount of colour change.
- Interpret the sample concentration from the standard curve.

• Competitive ELISA protocol
This is a general protocol in which antigen coating and blocking may not be required if the wells have been pre-adsorbed with the antigen by the manufacturer.

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 37°C for 30 minutes.
- Remove the coating solution and wash the plate 3X with 200 μL PBS buffer 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.

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 twice with 200 μL PBS for 5 minutes each time. Flick and pat the plate as described in the coating step.

Reagent Preparation - Prepare for the diluted standard solutions as shown on p.9.

Sample 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 diluted sample 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 three times with 200 μL PBS buffer for 5 minutes each time. Flick and pat the plate as described in the coating step.

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 3 times with 200 μL PBS for 5 minutes each time. Flick the plate and pat the plate as described in the coating step.

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.
- Cover the plate with adhesive plastic and incubate for 2 hours at room temperature.
- Remove the content in the wells and wash them 3 times with 200 μL PBS buffer for 5 minutes each time. Flick the plate and pat the plate as described in the coating step.

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 colour developed are as follows:

Note:
• The TMB substrate must be kept at 37°C for 30 minutes 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.

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 colour.
- Colour should be developed in positive wells after 15 minutes. After sufficient colour 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.

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).
- Competitive ELISA yields an inverse curve: Higher values of antigen in the samples yield a smaller amount of colour change.
- Interpret the sample concentration from the standard curve

7. Troubleshooting Guide

The following guide serves as a checklist for the possible causes and solutions with respect to some of the most commonly encountered problems from the ELISA assays.

• Weak / no signal

• Saturated signal

• High Background

• Low sensitivity

• Poor standard curve

• Poor replicate data

• Inconsistent Assay-to-Assay results

• Slow colour development

• Plate imaging problems

Gain Data

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Researchers just simply copy their ELISA data from excel and paste to our GainDataTM  then  follow the tool, less than a minute the result will be calculated automatically.