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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). It will cover the following sections:

  1. Preparation
  2. Assay Protocol
  3. Data Analysis

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.