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how to reduce CV in ELISA

Guest susan

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Hi Susan, and welcome to the forum!

I’m sorry to hear that you get high CV%. Before being able to give you a specific answer, we need to know a few more details:

1. Is it the CV% of inter-assay (when comparing different experiments) or intra-assay (within one plate) you are referring to?

2. Is it the standard curve or the samples (or both) that worry you?

3. How high CV% do you get?

4. Are the CV's for OD values or to back-calculated concentrations? Please note that for very low OD values, such as the buffer background, CV values will always be high.

Regardless of your answers above, here are some general things to consider in order to minimize high CV%:

  • Use calibrated pipettes, and preferably the same pipettes in all experiments.
  • If your samples need to be diluted with a high dilution factor, make one or several pre-dilutions, and do not take too low sample volumes (lower than 5 ul).
  • Adjust all reagents, except Mabtech’s TMB substrate, to room temperature before use and preferably incubate plates at the same temperature.
  • Also, all incubation times need to be the same between experiments.
  • Include several replicates of the samples and at least duplicates of the standard (as recommended).

Please also be advised that for human IFN-g (3420-1H-6) and Latent TGF-b (3550-1H-6) we offer validated ELISA Pro kits (3420-1HP-1 and 3550-1-HP-1, respectively) with pre-coated plates, buffers and substrate that ensure reproducibility and thus minimize risk of high CV.

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