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Guest Srikanth

assay diluent blank is fine but standards and samples readings are high

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Guest Srikanth

Hi all,

 

I am doing ELISA where my assay diluent blank looks fine with a reading of 0.07 but the standards and samples readings are too high. can someone kindly suggest me the solution to it.

 

Thanks

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Dear Srikanth, would be great if you could describe your experimental setup a bit more. Are you using a Mabtech ELISA? Which one? Is it ALP or HRP? Is this serum samples or is it cell supernatants you are analyzing. 

 

Having readings that are too high could be numerous things, but come back with some details and we will do our best to help you out!

 

best,

Christian

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Guest Srikanth

Hello Sir,

 

I am using Mabtech Human IFN-α2 ELISA development kit (HRP) and i am performing the experiment as per the given protocol with the kit. i am analyzing the cell supernatants.

 

I have used the new ELISA plate (Nunc Maxisorp) for coating the primary antibody and also the freshly prepared solutions. Previously everything was going fine and suddenly encountered with this problem. If standard would have come fine but samples readings are too high i can think i have increase my dilution or if all the wells of ELISA plate are uniformly blue then there would be problem in the handling or the solutions used but in my case blank reading was fine i.e 0 pg/ml but standard readings are saturated.

 

Regards

srikanth katla

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Just to be sure that we don't misunderstand anything I am afraid we will need some further information from you. However, as I understand it, the problem is primarily with the IFN-alpha standard that does not titrate as you expect but gives high values throughout the series of dilutions. This together with the fact that your background control i.e. just a parallel sample to which no IFN-alpha has been added is very low seem to indicate, and as you also suggest that there might be something wrong with the standard itself.  Normally though, if something is wrong with a standard, it seems more likely that it would generate a too low signal because of e.g. the standard protein being unstable. Here you find the opposite which is more difficult to explain unless it is due to some simple error when diluting the standard. However, this seems unlikely given that you have previously run the assay successfully.

 

As we cannot offer a good explanation we will send you a new standard to try but at the same time it would be good if you could provide some more details on how you performed the assay as well as the results you obtained.

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Guest Guest

Thanks for your reply sir,

 

i have performed the assay as per the protocol given with the kit. I have diluted accordingly the standard as said the manual and after which i have aliquoted into several vials to avoid the freeze thaw cycles. i take one vial for eg. which has 2ul standard sample for which i add 998ul diluent and from which i take 200 ul and add 800 ul diluent to get final conc. of 400 pg/ml. From the 400 pg.ml sample i take 500 ul and add 500ul diluent to make 200 pg/ml and so on till 6.25 pg/ml.

 

To the primary antibody coated ELISA plate i add 100 ul of samples and standard after blocking the plate for 1 hr with incubation buffer(PBST+0.1% BSA). Rest all the steps are as per the protocol given.

 

Regards

Srikanth katla

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