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Question about the standard curve of ELISA Flex

  • GL
    Guest Liu2022-12-15T09:21:56Z

    Dear Mabtech support team,

    I bought the ELISA Flex used for detecting human IL-2 (Cat:3445-1H-20) and I have a question about the repeatability of the standard curve.

    Refer to the instruction of the ELISA Flex, I have tried to repeat the standard curve several times, but the actual numerical value of OD450 was much lower than the standard curve showed in the instruction. For example, the standard, with the concentration of 1000ng/ml, was actually detected with the result of 1.5 in OD450. I wonder what I should do to repeat the standard curve.

    Looking forward to your suggestions!

    Thanks,

    Liu

  • C
    Christian@mabtech.com2022-12-15T10:41:30Z
    Admin

    Dear Liu,

    The low OD values could be caused by different factors. For the ELISA to work optimal it is important to use high protein binding plates. It is also important that the ELISA standard is reconstituted according to our instructions, and that the standard stock is thereafter aliquoted (at that same high concentration of 1 ug/ml) and kept at -20°C. Make sure not to freeze-thaw the standard. At 1000 pg/ml (not ng/ml) maximal OD should be obtained. Could you please get back to us with information of the kit batch number? The TMB substrate used can also influence the OD-values. We recommend our ELISA substrate: TMB for HRP product code 3652-F10.

  • GL
    Guest Liu2022-12-16T09:28:20Z

    Dear Christian,

    Thanks a lot for your reply! I am sure I operated as your advice as well as the instruction and used high binding type of plates in the past several experiments. And I actually started from 1000 pg/ml (not 1000 ng/ml, I am sorry for my previous typing error about the concentration) and set the serial concentration according to instruction. As for the ELISA substrate, I will use another new one to try again. By the way, the kit batch number is 39.

    Best,

    Liu

  • C
    Christian@mabtech.com2022-12-16T11:21:53Z
    Admin

    The ELISA substrate may very well be the culprit. Which substrate have you used for these earlier experiments?

    Please report back when you have re-run the ELISA with another substrate manufacturer, preferentially 3652-F10 from Mabtech.