Hi, I would like to ask on what to do when an ELISA kit protocol requires higher volumes of serum compared to what you have. In my case I have serum volumes of 10 microliters while the experiment needs 50 microliters. I have tried reducing the volumes of the reagents in the experiment to suit my 10 microliters serum but the problem arises when I try to validate the positive and negative controls of the kits. they fail the validation test when I use reduced volumes. Thank you!
Is there any reason why you could not dilute the 10 µl of serum to 50 µl?
Hi, firstly, I would not recommend reducing the volumes of the reagents. This invalidates the assay as you have noted and is considered off-label (i.e. not to manufacturer instructions). Diluting the 10 µL serum sample/s to 50 µL is your best option but please consider the following:
a) dilution matrix - ensure this does not interfere with the assay - confirm with manufacturer; and
b) limits of detection/measurability - while you can adjust for the dilution factor, the read maybe below limits of detection therefore will not register a read producing incorrect results; and
c) Confirm a pre-dilution is not required according to the IFU. Usually serum samples are diluted (at least 1:50) in order to minimise backgrounds caused by non-specific antibody binding.
Hope this helps.
Aus.
Dear Eugine Mukhaye,
It seems that you are using ELISA kit purchased from manufacturer but you did not mention whether the company has provided diluting buffer for standards with the kit or mentioned the diluting medium or standards have been provided in ready to use condition. If company has provided standard diluting buffer or mentioned the diluting medium in the company kit manual, then you take the advantage of that and dilute your sample (10 µL serum sample +40 µL standard diluting buffer or mentioned medium), vortex mix and then load in the microplate provided in the ELISA kit.
The results obtained can be normalized by multiplying the results by diluting factor (here x5)
In case you have got the ready to use standards, you can contact manufacturer for dilution matrix in order to avoid any problem that could be generated due to different matrix on the OD obtained in your case.
Hope this may help you
Best
Shail K Chaube
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