ELISA Support

If you are experiencing difficulties with a Bethyl product, please first consult the Frequently Asked Questions below. If your question isn't answered, please complete the form as accurately as possible. The information is helpful to troubleshoot the differences between the observed results and the expected results. Once submitted, a Technical Support representative will contact you within 1 business day. Please note that all comments and suggestions are taken seriously by Bethyl Laboratories.

ELISA Support FAQs

  • Yes, our ELISA kits contain antibodies and a reference standard to establish a curve. We test and qualify our kits using the reagents that are listed in the product specification sheet.

  • Polyclonal.

  • Samples can be of biological (i.e. serum, plasma, urine, feces) or cell culture origin.

  • We recommend that you store your samples at 4 °C. However, biological or cell culture samples can be initially frozen until use but should not be repeatedly freeze-thawed. Once the sample is thawed, it should be stored at 4 °C.

  • Since all experimental designs are different, it is difficult to determine an overall dilution for all samples. If you have a general idea of the concentration of your samples, you will want to make dilutions so that your samples will fall in the middle range (or linear range) of the standard curve. Otherwise, you may want to take a few of your samples and make several dilutions (ex. 1:100, 1:1000, and 1:10,000) to determine a dilution range for your samples. Each investigator must determine the best dilution for their samples.

  • If the raw OD values appear too low, allow the reaction to continue by incubating the TMB development step longer than the 30 minutes outlined in our ELISA kit protocol. If the raw OD values appear to be developing too high, apply stop solution to the wells sooner than the 30 minute TMB incubation time.

  • Usually background is caused by cross contamination of samples or reagents. If using an automated plate washer, make sure it is properly maintained and routinely decontaminated. If using a multi-channel pipette or squirt bottle method, be consistent with each wash application being careful to wash all wells equally.

    You may also add another wash to each step. Make new reagents if background problems continue and make sure that you are not using a BSA product with an ELISA kit that may cross react (Bovine Albumin ELISA, Bovine Transferrin ELISA, Bovine IgG ELISA, Goat IgG ELISA, and others).

  • Make sure that you are quantitating your samples in the linear region of the curve. Samples that quantitate too close to the extreme ends of the curve may be inaccurate and difficult to reproduce.

  • For your convenience, Bethyl Laboratories sells ready-to-use Stop Solution, catalog number E115.

  • Some of the software choices are SoftMax Pro by Molecular Devices, KC Jr., SigmaPlot, or others. You cannot directly plot a 4-parameter curve with Microsoft EXCEL.

  • You can use a linear regression curve in Microsoft EXCEL. If you use this type of curve, only use a maximum of 5 points on the curve. We recommend that you discard the upper and lower points and plot your standard curve with the 5 middle points.




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