ELISA for Fungal Toxin Detection

Antibody Based Technologies
1

I am wondering if anyone can share their experience with using ELISA assays for detecting aflatoxins in food and feed. I have seen pre-made plates, but also thinking about making my own. Would be interested in the community’s thoughts.

2

@Marcu

Thank you for joining the Discussion Boards. Making your own plates will cut down on cost drastically while using the pre made plates, you can rely on the reproducibility and avoid batch variations. What’s your ultimate goal? Are you looking for the expertise to prepare your own ELISA for detection or any other purpose. Provide us with more information and we can help you better

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3

Thank you for the response. This is for a commercial testing lab, and want to offer it as a screening service. My goal is to develop a protocol for creating an ELISA Plate for Mycotoxin Screening (and of course be able to assess threshold levels or quantitate in a variety of matrices but that is a sample prep issue I believe).

Generative AI suggest this method: Coating the ELISA Plate with Capture Antibody

  1. Dilute the Capture Antibody
  • Prepare antibody solution at 1-10 µg/mL in coating buffer.
  • Example: Add 100 µL antibody solution per well.
  1. Incubate Overnight at 4°C
  • Cover the plate with a plastic seal or parafilm.
  1. Wash the Plate (3x)
  • Remove coating buffer and wash wells 3 times with 300 µL PBST per well.
  • Remove excess liquid by tapping the plate on a paper towel.
  1. Block the Plate (to Prevent Non-Specific Binding)
  • Add 200 µL blocking buffer (1% BSA in PBS) per well.
  • Incubate for 1 hour at 37°C.
  • Wash 3x with PBST.
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4

Marcu

Generative AI response is highly generalized and with the Antibody recommendation as per it, your ELISA cost will be way too high. Do you have an in house Antibody preparation or are planning to buy one (buy monoclonal - if you intend to). Once you have the antibody, i recommend checking the antibody titer first to check its binding affinity and then we can determine the concentration required for coating/ ELISA.

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5

Marcu

In addition to @ARGERINE’s suggestions, PBST based systems can also interfere with your detection signal if you intend to use an HRP based detection system for your ELISA. I recommend using TBST and not PBST even for coating of Antibody.

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