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ELISA Protocols

There are 4 types of ELISA with 3 common detection systems. On this page, you can find detailed protocols and side-to-side comparisons of all of them.

What Is ELISA?

The enzyme-linked immunosorbent assay (ELISA) is a gold standard technique for the quantification of biomolecules such as peptides, proteins, antibodies and hormones. The quantity of a biomolecule is calculated by measuring the intensity of a signal produced at the end of the reaction.

The ELISA (enzyme-linked immunosorbent assay) is recognized by scientists for its convenient, quick, and simple execution. Below we provide general protocols for our PicoKine™ ELISA kits and EZ-Set™ ELISA Kits (DIY Antibody Pairs). When using Boster's ELISA kits, please refer to the manuals provided with your ELISA kits for batch-specific information.

What is ELISA used for?

ELISA’s versatility to detect peptides, proteins, antibodies, and hormones, and its ability to generate quantitative and qualitative data make it one of the most popular and powerful immunoassays available. ELISA is widely used in clinical medicine, biotechnology, pharmacology, and food industry.

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Typical ELISA workflow Summary

ELISA is a highly versatile procedure and different formats with different detection strategies have been developed according to the needs, but they all are based on the following basic elements:

  • Coating of micro-titration wells:

    Immobilization of antigens to the surface of polystyrene microtitration wells, either directly or indirectly.

  • Formation of antigen-antibody complex:

    Incubation with antigen-specific antibodies leads to the formation of antigen-antibody complex. The production of this complex is necessary for signal generation.

  • Detection

    A signal generated (either a color change, fluorescence, or chemiluminescence) is measured by specific detectors. Usually, greater the signal generated, greater the amount of target analyte in the sample.

Comparison of 4 types of ELISA protocols

Protocol Detect Advantages Disadvantages
Direct ELISA Antibody
  • Time efficient protocol with no cross reactivity from secondary antibody.
  • Time consuming and low flexibility with high background signal.
Indirect ELISA Antibody
  • Highly versatile and sensitive due to use of labelled secondary antibodies.
  • Cost-effective as fewer labelled antibodies are used.
  • Different detection strategies can be used. (Colorimetry, chemiluminescence, etc.)
  • Chances of cross-reactivity with the secondary antibody.
  • Complex protocol with an additional step as compared to Direct ELISA.
Sandwich ELISA Antigen
  • Highly flexible, sensitive, and specific protocol as two antibodies bind to the same antigen, at different binding sites.
  • Different detection strategies can be used. (Colorimetry, chemiluminescence, etc.)
  • Antigen of interest should be large enough to enable attachment of antibodies at different binding sites.
Competitive ELISA Antigen
  • Ideal for samples having low concentration of target antigen.
  • Antigens having small size and limited number of antibody binding sites are evaluated.
  • Relatively complex protocol.
  • Requires the use of inhibitor antigen.

Comparison of 3 ELISA detection methods

Colorimetric detection Fluorescent detection Chemiluminescence detection
Equipment Required Microplate spectrophotometer Fluorometer Luminometer
Cost of hardware Starting from $13,000 Starting from $15,000 Starting from $54,000
Cost of assay High High Medium
Potential for multiplexing None Bead based multiplexing Planar based multiplexing
Turnaround time (approx.) 2-3 hours 1-2 hours 1 hour
Detection Colorimetric Fluorescence Luminescence
Sensitivity Low (Narrow linear range) High High (Wide linear range)
Reporter Enzymatic Fluorescent tag Chemiluminescence
Sample Volume High volume Low volume Low volume
Operator skill Basic with minimum 2 weeks training Basic with minimum 2 weeks training Basic with minimum 2 weeks training

Protocol Details

Detailed protocols for sandwich, competitive, direct and indirect ELISA

In this section: 1. Reagent Preparation, 2. Detailed Protocol

This is a sample protocol for the most common kit set up in which the standard recombinant protein is 10ng and the highest concentration of standard well is 5000pg/ml. These numbers might differ for each individual kit.

1. Reagent preparation

Bring all reagents to 37°C prior to use. The assay can also be done at room temperature. However, we recommend doing it at 37°C for best consistency with our QC results.

  • Standard Solutions:

    It is recommended that the standards be prepared no more than 2 hours prior to performing the experiment. Use 10ng of lyophilized standard for each experiment. Gently spin the vial prior to use. Reconstitute the standard to a stock concentration of 10ng/ml using 1ml of sample diluent. Allow the standard to sit for a minimum of 10 minutes with gentle agitation prior to making dilutions.

    1. Number tubes 1-8. Final concentrations should be: Tube # 1 –5000pg/ml, #2 –2500pg/ml, #3 – 1250pg/ml, #4 – 625pg/ml, #5 – 312.5pg/ml, #6 –156.25pg/ml, #7 – 78.125pg/ml, #8 – Sample Diluent serves as the zero standard (0pg/ml).
    2. To generate standard #1–5000pg/ml, add 500µl of the reconstituted standard stock solution of 10ng/ml and 500µl of sample diluent to tube #1 for a final volume of 1000µl. Mix thoroughly.
    3. Add 300 µl of sample diluent to tubes # 2-7.
    4. To generate standard #2–2500pg/ml, add 300 µl of standard #1 from tube #1 to tube #2 for a final volume of 600 µl. Mix thoroughly.
    5. To generate standard #3–1250pg/ml, add 300 µl of standard #2 from tube #2 to tube #3 for a final volume of 600 µl. Mix thoroughly.
    6. Continue the serial dilution for tube #4-7.

  • 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), mixing thoroughly, and using within 2 hours of generation.

  • Avidin-Biotin-Peroxidase Complex (ABC):

    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), mixing thoroughly, and using within 2 hours of generation.

  • Samples:

    The user needs to estimate the concentration of the target protein in the sample and use an appropriate dilution factor so that the diluted target protein concentration falls in the middle range of O.D. values of the standard curve. Dilute the sample using the provided diluent buffer. Pilot tests using a dilution series of each sample type is necessary. The sample must be mixed thoroughly with Sample Diluent.

2. PicoKine™ Sandwich ELISA Protocol Details

All of the ELISA kits from Boster use the sandwich format and biotin-streptavidin chemistry. Our ELISA assays protocol requires the dilutions of standard solutions, biotinylated antibody (detection antibody), and avidin-biotin-peroxidase complex.

  • Reagent Preparation:

    Prepare for the diluted standard solutions, biotinylated antibody and ABC solutions as shown in the above Reagent Preparation section.

    Remove excess microplate strips from the plate frame. Seal and store them in the original packaging.

  • Sample (Standard) Incubation:

    Add 100 µl of the standard, samples, or control per well. Add 100 µl of the sample diluent buffer into the zero well. At least two replicates of each standard, sample, or control is recommended.

    Cover with the plate sealer provided and incubate for 120 minutes at RT (or 90 min. at 37 °C).

    Remove the cover and discard the liquid in the wells into an appropriate waste receptacle. Invert the plate on the benchtop onto a paper towel and tap the plate to gently blot any remaining liquid. It is recommended that the wells are not allowed to completely dry at any time.

  • Biotinylated Antibody Incubation:

    Add 100 µl of the prepared 1x Biotinylated Antibody to each well.

    Cover with plate sealer and incubate for 90 minutes at RT (or 60 minutes at 37°C).

    Wash the plate 3 times with the 1x wash buffer.

    1. Discard the liquid in the wells into an appropriate waste receptacle. Then, invert the plate on the benchtop onto a paper towel and tap the plate to gently blot any remaining liquid. It is recommended that the wells are not allowed to completely dry at any time.
    2. Add 300 µl of the 1x wash buffer to each assay well (For cleaner background, incubate for 60 seconds between each wash).
    3. Repeat steps a-b two additional times.

  • ABC Incubation:

    Add 100 µl of the prepared 1x Avidin-Biotin-Peroxidase Complex into each well. Cover with the plate sealer provided and incubate for 40 minutes at RT (or 30 minutes at 37°C).

    Wash the plate 5 times with the 1x wash buffer.

    1. Discard the liquid in the wells into an appropriate waste receptacle. Then, invert the plate on the benchtop onto a paper towel and tap the plate to gently blot any remaining liquid. It is recommended that the wells are not allowed to completely dry at any time.
    2. Add 300 µl of the 1x wash buffer to each assay well (For cleaner background, incubate for 60 seconds between each wash).
    3. Repeat steps a-b two additional times.

  • Signal Detection:

    Add 90 µl of Color Developing Reagent to each well. Cover with the plate sealer provided and incubate in the dark for 30 minutes at RT (or 15-25 minutes at 37°C). The optimal incubation time must be empirically determined. A guideline to look for is blue shading for the top four standard wells while the remaining standards remain clear.

    Add 100 µl of Stop Solution to each well. The color should immediately change to yellow.

    Within 30 minutes of stopping the reaction, the O.D. absorbance should be read with a microplate reader at 450nm.

Browse PicoKine™ ELISA kits

In this section: 1. Reagent Preparation, 2. Detailed Protocol

This protocol is to serve as a guide for researchers when using Boster’s EZ-Set™ ELISA Kits (DIY Antibody Pairs).

1. Reagent Preparation

Bring all reagents to room temperature before use. Allow all components to sit for a minimum of 15 minutes with gentle agitation after initial reconstitution. Working dilutions should be prepared and used immediately.

  • Plate Preparation:

    • Dilute the Capture Antibody to the working concentration in 1:100 with Capture Antibody Diluent (i.e. Add 1μL Capture Antibody into 99μL Capture Antibody Diluent.) Immediately coat a 96-well microplate with 100 μL per well of the diluted Capture Antibody. Seal the plate and incubate overnight at 4°C.
    • Block plates by adding 200 μL of Reagent Diluent to each well. Incubate at room temperature for 2 hours.
    • Aspirate each well and wash with PBS, repeating the process two times for a total of three washes. Wash by filling each well with PBS (300-350 μL) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining PBS by aspirating or by inverting the plate and blotting it against clean paper towels. (Plate Washing Method)

  • Reconstitution of Standard:

    It is recommended that the standards be prepared no more than 2 hours prior to performing the experiment. Use one 10 ng of lyophilized standard for each experiment. Gently spin the vial prior to use. Reconstitute the standard to a stock concentration of 10ng/ml using 1ml of Reagent Diluent. Allow the standard to sit for a minimum of 10 minutes with gentle agitation prior to making dilutions.

    Dilution of Standard

    • Number tubes 1-8. Final Concentrations to be Tube #1 – 1000pg/ml, #2 – 500pg/ml, #3 – 250pg/ml, #4 – 125pg/ml, #5 – 62.5pg/ml, #6 – 31.2pg/ml, #7 – 15.6pg/ml, #8 – 0.0 (Blank).
    • To generate standard #1, add 100µl of the reconstituted standard stock solution of 10ng/ml and 900µl of sample diluent to tube #1 for a final volume of 1000µl. Mix thoroughly.
    • Add 300 µl of Reagent Diluent to tubes # 2-7.
    • To generate standard #2, add 300 µl of standard #1 from tube #1 to tube #2 for a final volume of 600 µl. Mix thoroughly.
    • To generate standard #3, add 300 µl of standard #2 from tube #2 to tube #3 for a final volume of 600 µl. Mix thoroughly.
    • Continue the serial dilution for tube #4-7.
    • Tube #8 is a blank standard to be used with every experiment.

  • Preparation of the polyclonal antibody working solution:

    • Each vial contains 500 μL of polyclonal antibody.
    • The polyclonal antibody should be diluted in 1:100 with Capture Antibody Diluent and mixed thoroughly (i.e. Add 1 μL polyclonal antibody to 99 μL Capture Antibody Diluent).

  • Preparation of biotinylated polyclonal antibody working solution:

    • Each vial contains 500 μL of biotinylated polyclonal antibody.
    • Biotinylated polyclonal antibody should be diluted in 1:100 with Reagent Diluent and mixed thoroughly (i.e. Add 1 μL biotinylated detection antibody to 99 μL Reagent Diluent).

  • Preparation of Avidin-Biotin-Peroxidase Complex (ABC) working solution:

    • Each vial contains 500 μL of Avidin-Biotin-Peroxidase Complex (ABC).
    • Avidin-Biotin-Peroxidase Complex (ABC) should be diluted in 1:100 with Reagent Diluent and mixed thoroughly (i.e. Add 1 μL ABC to 99 μL Reagent Diluent).

2. EZ-Set™ ELISA Protocol Details

  • Prepare all reagents and working standards as directed previously.
  • Remove excess microplate strips from the plate frame and seal and store them in the original packaging.
  • Add 100 µl of the standard, samples, or control per well. At least two replicates of each standard, sample, or control is recommended.
  • Cover with the plate sealer provided and incubate for 120 minutes at RT (or 90 minutes at 37 °C).
  • Remove the cover and discard the liquid in the wells into an appropriate waste receptacle. Invert the plate on the benchtop onto a paper towel and tap the plate to gently blot any remaining liquid. It is recommended that the wells are not allowed to completely dry at any time.
  • Add 100 µl of the prepared 1x biotinylated polyclonal antibody to each well.
  • Cover with plate sealer and incubate for 90 minutes at RT (or 60 minutes at 37°C).
  • Wash the plate 3 times with PBS. Discard the liquid in the wells into an appropriate waste receptacle. Then, invert the plate on the benchtop onto a paper towel and tap the plate to gently blot any remaining liquid. It is recommended that the wells are not allowed to completely dry at any time.
    • Add 300 µl of PBS to each assay well (For cleaner background, incubate for 60 seconds between each wash).
    • Repeat steps a and b for 2 additional times.
  • Add 100 µl of the prepared 1x Avidin-Biotin-Peroxidase Complex into each well and incubate for 40 minutes at RT (or 30 minutes at 37°C).
  • Wash the plate 5 times with PBS-T.
    • Discard the liquid in the wells into an appropriate waste receptacle. Then, invert the plate on the benchtop onto a paper towel and tap the plate to gently blot any remaining liquid. It is recommended that the wells are not allowed to completely dry at any time.
    • Add 300 µl of PBS-T to each assay well (For cleaner background, incubate for 60 seconds between each wash).
    • Repeat steps a and b for 4 additional times.
  • Add 90 µl of Color Developing Reagent to each well and incubate in the dark for 30 minutes at RT (or 25-30 minutes at 37°C). (The optimal incubation time must be empirically determined. A guideline to look for is blue shading the top four standard wells while the remaining standards remain clear.)
  • Add 100 µl of Stop Solution to each well. The color should immediately change to yellow.
  • Within 30 minutes of stopping the reaction, the O.D. absorbance should be read with a microplate reader at 450nm.
Browse EZ-Set™ ELISA kits

In this section: 1. Materials Required, 2. Checklist/Workflow, 3. Detailed Protocol

Sandwich ELISA is based on the detection and quantification of target protein (antigen), which is sandwiched between primary and secondary antibodies, each binding to a different epitope of the target antigen. This is a general protocol for sandwich ELISA.

1. Materials Required

  • Bicarbonate/carbonate antigen coating buffer (100 mM NaHCO3 in deionized water; pH adjusted to 9.6)
  • 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)
  • Blocking buffer (5% w/v non-fat dry milk in PBS buffer)
  • Washing solution, usually PBS or Tris-buffered saline (pH 7.4)
  • Diluted Biotinylated Antibody
  • Diluted Avidin-Biotin-Peroxidase (ABC)
  • Substrate solution
  • Stop solution
  • ELISA plate reader

2. Checklist/Workflow

Pre-Assay Preparation:

  • Capture Antibody Coating
  • Blocking
  • Reagent Preparation

Assay Procedure:

  • Sample (antigen) incubation
  • Addition of Biotinylated antibody
  • Addition of Avidin-Biotin-Peroxidase enzyme
  • Substrate addition (signal production)
  • Addition of stop solution
  • Signal detection
  • Data analysis

3. Detailed Protocol

  • Capture Antibody Coating:

    (This step is not required when 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 antigencoating buffer.
    • 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. 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:

    (This step is not required when 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.
    • 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.

  • Reagent Preparation:

    • Prepare the diluted solutions of standard, biotinylated antibody and Avidin-Biotin-Peroxidase (ABC).

  • Sample (antigen) Incubation:

    • Pipette 100 μL of each of the diluted sample solutions and control to each empty well. Repeat in duplicate or triplicate for accuracy.
    • 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 to ensure complete removal of PBS buffer.

  • 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).
    • Remove the content in the wells and wash them 3X with 200 μL PBS for 5 min each time.

  • Avidin-Biotin-Peroxidase 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.

  • 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).

  • Signal Production and Detection:

    • Pipette 90 μL of substrate solution to the wells with the control, standard and sample 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.
    • Color should be developed in positive wells after 15 min. After sufficient color development, pipette 100 μL of stop solution to the wells.
    • 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.

In this section: 1. Materials Required, 2. Checklist/Workflow, 3. Detailed Protocol

Competitive ELISA, also referred to as inhibition ELISA, is based on the immobilization of an antibody specific to the target protein, which competes with a standard for binding to a limited amount of antibodies immobilized on the plate. This is a general protocol for competitive ELISA.

1. Materials Required

  • Bicarbonate/carbonate antigen coating buffer (100 mM NaHCO3 in deionized water; pH adjusted to 9.6)
  • 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)
  • Blocking buffer (5% w/v non-fat dry milk in PBS buffer)
  • Washing solution, usually PBS or Tris-buffered saline (pH 7.4)
  • Diluted primary and secondary antibodies
  • Substrate solution
  • Stop solution
  • ELISA plate reader

2. Checklist/Workflow

Pre-Assay Preparation:

  • Antigen Coating
  • Blocking
  • Reagent Preparation

Assay Procedure:

  • Sample (target antigen) incubation
  • Incubation with primary antibody
  • Incubation with secondary antibody
  • Substrate addition (signal production)
  • Addition of stop solution
  • Signal detection
  • Data analysis

3. Detailed Protocol

  • Antigen Coating:

    (This step is not required when pre-adsorbed PicoKine ELISA kits from Boster are used.)

    • Dilute purified antigens to a final concentration of 1-10 μg/mL in bicarbonate/carbonate antigen coating buffer.
    • 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. 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:

    (This step is not required when 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.
    • 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.

  • Reagent Preparation:

    • Prepare the diluted solutions of standard, primary and secondary antibodies, along with substrate solution.

  • Sample (Antigen) Incubation:

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

  • Primary Antibody Incubation:

    • Pipette 100 μL of diluted primary antibody in each well. Repeat in duplicate or triplicate for accuracy.
    • Cover the plate with adhesive plastic and incubate for 1 hour at 37°C (or 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 to ensure complete removal of PBS buffer.

  • Secondary Antibody Incubation:

    • Pipette 100 μL of diluted secondary antibody in each well. Cover the plate with adhesive plastic and incubate for 1 hour at 37°C (or 2 hours at room temperature)
    • Remove the content in the wells and wash them 3X with 200 μL PBS for 5 min each time.

  • 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).

  • Signal Production and Detection:

    • Pipette 90 μL of substrate solution to the wells with the control, standard and sample 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.
    • Color should be developed in positive wells after 15 min. After sufficient color development, pipette 100 μL of stop solution to the wells.
    • 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.

In this section: 1. Materials Required, 2. Checklist/Workflow, 3. Detailed Protocol

In Direct ELISA, the target antigen is adhered to the surface of microtitration wells and incubated with an enzyme-labeled antibody. The enzyme attached with the antigen-antibody complex, acts on the substrate, which is added in the next step and converts it into a colored product. The intensity of the color change produced is directly proportional to the target analyte present in the sample. Below is a general protocol for direct ELISA.

1. Materials Required

  • Bicarbonate/carbonate antigen coating buffer (100 mM NaHCO3 in deionized water; pH adjusted to 9.6)
  • 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)
  • Blocking buffer (5% w/v non-fat dry milk in PBS buffer)
  • Washing solution, usually PBS or Tris-buffered saline (pH 7.4)
  • Diluted primary antibody
  • Substrate solution
  • Stop solution
  • ELISA plate reader

2. Checklist/Workflow

Pre-Assay Preparation:

  • Capture Antibody Coating
  • Blocking
  • Reagent Preparation

Assay Procedure:

  • Sample (antigen) incubation
  • Incubation with conjugated primary antibody
  • Substrate addition (signal production)
  • Addition of stop solution
  • Signal detection
  • Data analysis

3. Detailed Protocol

  • Antigen Coating:

    (This step is not required when pre-adsorbed PicoKine ELISA kits from Boster are used.)

    • Dilute purified antigens to a final concentration of 1-10 μg/mL in bicarbonate/carbonate antigen coating buffer.
    • 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. 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:

    (This step is not required when 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.
    • 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.

  • Reagent Preparation:

    • Prepare the diluted solutions of standard, primary and secondary antibodies, along with substrate solution.

  • Primary Antibody Incubation:

    • Pipette 100 μL of diluted conjugated primary antibody in each well. Repeat in duplicate or triplicate for accuracy.
    • 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 to ensure complete removal of PBS buffer.

  • 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).

  • Signal Production and Detection:

    • Pipette 90 μL of substrate solution to the wells with the control, standard and sample 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.
    • Color should be developed in positive wells after 15 min. After sufficient color development, pipette 100 μL of stop solution to the wells.
    • 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.

In this section: 1. Materials Required, 2. Checklist/Workflow, 3. Detailed Protocol

The Indirect ELISA method utilizes a target antigen complementary to the primary antibody present in the test solution. After incubation, the antigen-antibody complex is formed. Enzyme labeled secondary antibody having specificity for the primary antibody is added in the next step, followed by the addition of substrate solution which leads to signal generation. Below is a general protocol for indirect ELISA.

1. Materials Required

  • Bicarbonate/carbonate antigen coating buffer (100 mM NaHCO3 in deionized water; pH adjusted to 9.6)
  • 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)
  • Blocking buffer (5% w/v non-fat dry milk in PBS buffer)
  • Washing solution, usually PBS or Tris-buffered saline (pH 7.4)
  • Diluted primary and secondary antibodies
  • Substrate solution
  • Stop solution
  • ELISA plate reader

2. Checklist/Workflow

Pre-Assay Preparation:

  • Capture Antibody Coating
  • Blocking
  • Reagent Preparation

Assay Procedure:

  • Sample (antigen) incubation
  • Incubation with primary antibody
  • Incubation with secondary antibody
  • Substrate addition (signal production)
  • Addition of stop solution
  • Signal detection
  • Data analysis

3. Detailed Protocol

  • Antigen Coating:

    (This step is not required when pre-adsorbed PicoKine ELISA kits from Boster are used.)

    • Dilute purified antigens to a final concentration of 1-10 μg/mL in bicarbonate/carbonate antigen coating buffer.
    • 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. 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:

    (This step is not required when 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.
    • 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.

  • Reagent Preparation:

    • Prepare the diluted solutions of standard, primary and secondary antibodies, along with substrate solution.

  • Primary Antibody Incubation:

    • Pipette 100 μL of diluted primary antibody in each well. Repeat in duplicate or triplicate for accuracy.
    • 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 to ensure complete removal of PBS buffer.

  • Secondary Antibody Incubation:

    • Pipette 100 μL of diluted secondary antibody in each well. Cover the plate with adhesive plastic and incubate for 1 hour at 37°C (or 2 hours at room temperature).
    • Remove the content in the wells and wash them 3X with 200 μL PBS for 5 min each time.

  • 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).

  • Signal Production and Detection:

    • Pipette 90 μL of substrate solution to the wells with the control, standard and sample 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.
    • Color should be developed in positive wells after 15 min. After sufficient color development, pipette 100 μL of stop solution to the wells.
    • 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.

Data Analysis

Average the duplicate readings for each standard, sample, and control. Subtract the average zero standard O.D. reading.

It is recommended that a standard curve be created using computer software to generate a four parameter logistic (4-PL) curve-fit. A free program capable of generating a four parameter logistic (4-PL) curve-fit can be found online at www.myassays.com/four-parameter-logistic-curve.assay.

Alternatively, plot the mean absorbance for each standard against the concentration. The measured concentration in the sample can be interpolated by using linear regression of each average relative OD against the standard curve generated using curve fitting software. This will generate an adequate but less precise fit of the data.

For diluted samples, the concentration reading from the standard curve must be multiplied by the dilution factor.

We have an entire page dedicated to ELISA data analysis that provides step-by-step guidance. Check out the ELISA Data Analysis page for more information.

Go to ELISA Data Analysis

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FAQs

The number of samples that can be tested on each plate will depend on the number of standard points, controls, and replicates you choose to use. For a 96-well plate, it is common to run an 8-point standard curve with duplicate wells. The remaining 80 wells can be used for controls and/or samples. If controls are not included your assay, 80 samples can be run in singlicate. However, we strongly recommend testing ELISA samples in duplicate or triplicate because this will generate enough data for statistical validation of the results. If samples are tested in duplicates without controls, 40 samples can be run for one ELISA plate.

For a 96T double antibody ELISA kit, the sample volume needed for testing one sample is 100μL. As the samples should be run in duplicate, or triplicate, you should have about 250 uL of sample separated.

If washing is done manually, please ensure the exact preparation of wash solution as recommended in your ELISA kit. During manual washing, please consider using multi-channel pipette. ELISA automatic washers can also be used, just make sure that no contamination occurs. Rinse the plate gently, and dry it before proceeding to the next step.

A number of reasons could lead to this outcome. Either the reagents were wrongly used, or the substrate solution got contaminated. Pipetting error, or dispensing reagents in the wrong well can also lead to no color development in the positive control well. Repeat the procedure with strict vigilance.

Mix-up of positive and negative control and contamination during the initial steps might lead to erroneous results. Ensure full concentration and follow GLPs while performing ELISA to minimize such errors.

Ensure the filter used is of wavelength as recommended in the kit insert. Make sure that the plate is placed correctly inside the plate reader and there is no blockage of any kind. Ideally, you should measure the absorbance within 30 minutes after addition of stop solution.

TMB substrate is a clear, colorless solution used for development of color change in the microtitration well. You should always have a glance at the tip before dispensing it, or check its color by dispensing it in a clean container.

High concentrations of the target analyte might result in this. Dilute the sample and repeat the procedure. Also ensure that the substrate solution is not contaminated and incubation time is strictly monitored.

Calculate the mean OD for each standard concentration. Next, calculate the mean OD for the blank and subtract this value from the mean OD of each standard concentration. The standard curve can now be plotted as the standard concentration (x-axis) versus the corresponding OD (y-axis).

Inconsistent assay-to-assay results can be due to fluctuations in temperature, washing, incubation time, and pipetting error. Adherence to the same protocol each time, and calibration of the plate reader is recommended.

References

  • Elshal MF, McCoy JP. Multiplex bead array assays: performance evaluation and comparison of sensitivity to ELISA. Methods. 2006;38(4):317-323.
  • Bolton, J.S., Chaudhury, S., Dutta, S. et al. Comparison of ELISA with electro-chemiluminescence technology for the qualitative and quantitative assessment of serological responses to vaccination. Malar J 19, 159 (2020). https://doi.org/10.1186/s12936-020-03225-5
  • Coutlee F, Viscidi RP, Yolken RH. Comparison of colorimetric, fluorescent, and enzymatic amplification substrate systems in an enzyme immunoassay for detection of DNA-RNA hybrids. J Clin Microbiol. 1989 May;27(5) 1002-1007. doi:10.1128/jcm.27.5.1002-1007.1989. PMID: 2473088; PMCID: PMC267471.

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