ELISA Kit Protocols

ELISA procedure can accurately assess soluble proteins in their native state, so they are ideal for samples such as urine or saliva. Check out our ELISA protocols to learn how to get the best results.

The ELISA (enzyme-linked immunosorbent assay) is recognized by scientists for its convenient, quick, and simple execution. 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. 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.

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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 one 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.

Sample Preparation

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.

PicoKine™ Sandwich ELISA Protocol

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 four 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.

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-logisticcurve.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.

EZ-Set™ ELISA Kit Protocol

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

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.

  1. 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)
  2. 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.
  3. 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).
  4. 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).
  5. 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).

Assay Procedure

  1. Prepare all reagents and working standards as directed previously.
  2. Remove excess microplate strips from the plate frame and seal and store them in the original packaging.
  3. Add 100 µl of the standard, samples, or control per well. At least two replicates of each standard, sample, or control is recommended.
  4. Cover with the plate sealer provided and incubate for 120 minutes at RT (or 90 minutes at 37 °C).
  5. 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.
  6. Add 100 µl of the prepared 1x biotinylated polyclonal antibody to each well.
  7. Cover with plate sealer and incubate for 90 minutes at RT (or 60 minutes at 37°C).
  8. 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.
  9. 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).
  10. 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.
  11. 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.)
  12. Add 100 µl of Stop Solution to each well. The color should immediately change to yellow.
  13. Within 30 minutes of stopping the reaction, the O.D. absorbance should be read with a microplate reader at 450nm.

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.

FAQs

Q. How Many Samples Can I Run On A Plate?

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.