ELISA Technical Resources

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In this article you can find the following information:

  1. Reagent Preparation
  2. Sandwich ELISA Protocol
  1. Reagent Preparation

    1. Standard Solutions
      • 10,000 pg/mL: Add 1 mL of sample diluent buffer into one tube of standard (10 ng per tube) and mix thoroughly. Note: Store this solution at 4°C for up to 12 hours (or -20°C for 48 hours) and avoid freeze-thaw cycles.
      • 5,000 pg/mL: Mix 0.3 mL of 10,000 pg/mL with 0.3 mL of sample diluent buffer and mix thoroughly.
      • 2,500 pg/mL: Mix 0.3 mL of 5,000 pg/mL with 0.3 mL of sample diluent buffer and mix thoroughly.
      • Perform similar dilutions until the standard solutions with these concentrations (pg/mL) are made:
      • 1,250, 625, 312, 156 and 78.
      • Add 100 µL of each of the diluted standard solutions to the appropriate empty wells. Repeat in duplicate or triplicate for accuracy.

      Note: The standard solutions are best used within 2 hours.

    2. 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.
    3. 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) and mixing thoroughly.

      Note: The diluted ABC solution should not be prepared more than 1 hour prior to the experiment.

  2. Sandwich ELISA Protocol

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

    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.
    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 shown in the above Reagent Preparation section.

      Note: The diluted ABC solution should not be prepared more than 1 hour prior to the experiment.

    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.
    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. Flick the plate and pat the plate as described in the coating step.
    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:

      Enzyme Substrate* Stop Solution Absorbance (nm) Color Developed
      HRP TMB 2M H2SO4 450 Yellow
      AP pNPP 0.75M NaOH 405 Yellow

      * TMB: 3,3’,5,5’-tetramethylbenzidine; pNPP: p-nitrophenyl-phosphate

      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.
    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.