Introduction

PeproTech ELISA Development kits contain key components required for the quantitative measurement of native and/or recombinant proteins in a sandwich ELISA format. These ready-to-use kits are available for a variety of targets available in both ABTS and TMB formats for flexibility. Each kit is validated for quality and reproducibility and meets rigorous specifications for sensitivity, dynamic range, precision, specificity, recovery, and lot-to-lot consistency.

Note: The ELISA assay protocol provided is representative of most ready-to-use ELISA development kits. Protocols for individual kits may differ. Depending on the protein of interest, this general protocol may need to be optimized.

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PeproTech ABTS Sandwich ELISA Protocol

ABTS ELISA Protocol

Materials

Typical ABTS ELISA Development Kit components

  • Capture antibody (PeproTech antigen-affinity-purified polyclonal or monoclonal antibody)
  • Detection antibody (PeproTech biotinylated antigen-affinity-purified polyclonal antibody)
  • Standard (PeproTech Recombinant Protein)
  • HRP conjugate (Avidin, HRP conjugate)

Note: PeproTech standards and antibodies should be reconstituted according to the data sheet included with each product.

ABTS ELISA Buffer Kit components

* Light sensitive

Note: The ABTS ELISA Buffer Kit is sold separately and is recommended to be used in conjunction with ABTS ELISA Development Kit for optimal performance. This buffer kit contains all the necessary components to assay ten 96-well ELISA plates (included) and contains detailed handling instructions. When preparing your own solutions containing BSA (blocking buffer, diluent etc.), filter-sterilize using a 0.2 µm filter to reduce the potential for any bacterial contamination. Store at 4°C for up to 1 week.

Additional materials recommended


Protocol

Note: Steps 1-5 involve plate preparation. Dilute all concentrated solutions (PBS, Wash buffer and Diluent) to 1X using sterile distilled water. It is recommended to bring all solutions, including blocking buffer and ABTS liquid substrate, to ambient temperature before use. Pour off the desired volume of blocking buffer and ABTS liquid substrate prior to use.

  1. Dilute Capture antibody (monoclonal or polyclonal) with 1X PBS to a concentration of 1 μg/mL (polyclonal) or at least 2 μg/mL (monoclonal). Immediately, add 100 μL of diluted capture antibody to each ELISA plate well. Seal the plate with Sealing film and incubate overnight at room temperature.
  1. Aspirate the wells to remove liquid and wash the plate four times with >300 μL of 1X Wash buffer per well, followed by aspiration. Following wash, invert and tap the plate on absorbent paper to remove residual buffer.
  1. Add 300 μL of Blocking buffer to each well. Incubate for 1 hour at room temperature (RT).
  1. Aspirate blocking buffer then invert, and tap the plate on absorbent paper.
  1. Wash the plate four times with >300 μL of 1X Wash buffer per well (same as step 2).
  1. Perform serial dilutions of the standard from 0.01 μg/mL to zero in the 1X Diluent. Add 100 μL of standard or sample to each well. It is recommended to run standard or sample in triplicates. Incubate at room temperature for at least 2 hours.
  1. Aspirate and wash the plate four times (same as step 2).
  1. Dilute the Detection antibody (biotinylated) in 1X Diluent to a concentration of 0.5 μg/mL (500 ng/mL). Immediately, add 100 μL of diluted detection antibody per well. Incubate at room temperature for 2 hours.
  1. Aspirate the liquid and wash the plate four times (same as step 2).
  1. Prepare a working solution of Avidin-HRP conjugate in 1X Diluent at 1:2000 dilution. Add 100 μL of Avidin-HRP per well. Incubate at room temperature for 30 minutes.
  1. Aspirate the solution and wash the plate four times (same as step 2).
  1. Add 100 μL of ABTS liquid substrate solution to each well. Incubate the plate at room temperature for color development.
  1. Monitor color development with an ELISA plate reader at 405 nm with wavelength correction set at 650 nm.

Note: Reliable standard curves are obtained when O.D. readings do not exceed 0.2 units for the zero standard concentrations, or 1.2 units for the highest standard concentration. The plate should be monitored at 5-minute intervals until desired O.D. readings are obtained. The typical range is 5–40 minutes. O.D. readings may vary.

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PeproTech TMB Sandwich ELISA Protocol

TMB ELISA Protocol

Materials

Typical TMB ELISA Development Kit components

Note: PeproTech standards and antibodies should be reconstituted according to the data sheet included with each product.

TMB ELISA Buffer Kit components

* Light sensitive

Note: The TMB ELISA Buffer Kit is sold separately and is recommended to be used in conjunction with TMB ELISA Development Kit for optimal performance. This buffer kit contains all the necessary components to assay ten 96-well ELISA plates (included) and contains detailed handling instructions. When preparing your own solutions containing BSA (blocking buffer, diluent etc.), filter-sterilize using a 0.2 μm filter to reduce the potential for any bacterial contamination. Store at 4°C for up to 1 week.

Additional materials recommended


Protocol

Note: Steps 1-5 involve plate preparation. Dilute all concentrated solutions (PBS, Wash buffer and Diluent) to 1X using sterile distilled water. It is recommended to bring all solutions, including blocking buffer and TMB liquid substrate, to ambient temperature before use. Pour off the desired volume of blocking buffer and TMB liquid substrate prior to use.

  1. Dilute Capture antibody (monoclonal or polyclonal) with 1X PBS (pH 7.2) to achieve a concentration of 1 μg/mL for polyclonal antibodies or at least 2 μg/mL for monoclonal antibodies. Immediately, add 100 μL of diluted capture antibody into each well. Seal the plate and incubate overnight at room temperature.
  1. Aspirate the wells to remove liquid and wash the plate four times with >300 μL of 1X Wash buffer per well, followed by aspiration. Following wash, invert and tap the plate on absorbent paper to remove residual buffer.
  1. Add 300 μL of Blocking buffer to each well and incubate for 1 hour at room temperature (RT).
  1. Aspirate blocking buffer then invert, and tap the plate on absorbent paper.
  1. Wash the plate four times with >300 μL of 1X Wash buffer per well (same as step 2).
  1. Perform serial dilutions of the standard from 0.01 μg/mL to zero in the 1X Diluent. Add 100 μL of standard or sample to each well. Run standard or sample in triplicates. Incubate at room temperature for at least 2 hours.
  1. Aspirate and wash the plate four times (follow step 2).
  1. Dilute the biotinylated Detection antibody in 1X Diluent to a concentration of 0.5 μg/mL (500 ng/mL). Immediately, add 100 μL of diluted detection antibody to each well. Incubate at room temperature for 2 hours.
  1. Aspirate the liquid from the wells and wash the plate four times (follow step 2).
  1. Prepare a working solution of Streptavidin-HRP conjugate in Diluent to a concentration of 0.10 µg/mL. Add 100 μL of Streptavidin-HRP conjugate per well. Incubate at room temperature for 30 minutes.
  1. Aspirate the liquid from the wells and wash the plate four times (follow step 2).
  1. Add 100 μL of TMB liquid substrate solution to each well. Incubate the plate at room temperature for 20 minutes.
  1. Add 100 µL of Stop solution (1M HCl) to each well.
  1. Monitor color development with an ELISA plate reader at 450 nm with wavelength correction set at 650 nm.

Note:Reliable standard curves are obtained when O.D. readings do not exceed 0.15 units for the zero standard concentrations. Development time and O.D. readings may vary.

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Stylesheet for Classic Wide Template adjustments

For Research Use Only. Not for use in diagnostic procedures.