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Troubleshooting Common Issues In Elisa Kits: Tips And Strategies

Sep 22nd 2023

Troubleshooting Common Issues In Elisa Kits: Tips And Strategies

Since its invention almost half a century ago, the Enzyme-Linked Immunosorbent Assay (ELISA) has established itself as the industry standard for diagnostic testing and research on therapeutic efficacy. With an ELISA, you can measure the amount of a target molecule (like a protein, peptide, or hormone) in a fluid sample by sticking it to a microplate with certain antibodies that attach to that molecule and spotting the specific binding with high accuracy.

Even though new techniques like flow cytometry have emerged, Elisa test kits remain common because it is specific and can be used in many different ways.

But similar to other laboratory techniques and solutions, it has some downsides and errors.

Learn more about the tips and strategies used to resolve issues related to this type of immunoassay.

Let's start by exploring different approaches to Elisa kit troubleshooting, like detecting false negatives in Elisa, and non-specific binding in Elisa.

Weak or Zero Signal

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Photo by National Cancer Institute on Unsplash

Let's explore some common issues in Elisa kits related to zero or low signal in Elisa and their respective measures.

  • Not starting with reagents at room temperature: Before beginning the assay, it is best if all the chemicals are at room temperature. Let the chemicals rest on the bench at room temperature for 15–20 minutes.
  • Poor storage and handling of components: The kit's sticker will tell you its storage procedure. Make sure you adhere to the instructions for storing the gear. The recommended temperature range for storing most kits is between 2 and 8 degrees Celsius.
  • Old reagents: Verify the expiration dates of each reagent. Reagents that have passed their expiry date should not be used.
  • Improper addition or preparation of reagents: Verify that the correct dilution was used and that the reagents were introduced in the appropriate sequence.
  • Mistakes in the diluting process: Verify the pipetting procedure (detailed further down this guide) and crosscheck your calculations.
  • Capture antibody failed to adhere to the plate: If you employ a prepared-to-use kit, the leaves with the premade kit will already have the capture antibody coated.Ensure you have an ELISA plate, not a tissue culture plate if you're coating your plate using an Antibody Pair Kit. Use PBS to dilute the antibody. Ensure that the coating and blocking processes are properly prepared and incubated.

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Photo by CDC on Unsplash

  • Use of insufficient detector antibody: The methods for making kits have greatly improved, so ensure you dilute the antibodies correctly. Optimization of the assay may be required while creating ELISA utilizing an Antibody Pair Kit.
  • Scratches made in the wells using pipettes or washing tips: Always exercise extreme caution whenever administering from or into wells. The automated plate washers may need to adjust their calibration so that the ends don't hit the bottom of the wells.
  • Plate scanned at the wrong wavelength: A crucial step for troubleshooting Elisa plate reading is to use the suggested filter or wavelength and check that the plate reader is correctly configured for the type of substrate you wish to use.

Excessive Signal