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concentration of samples with ELISA


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#1 Tielli

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Posted 10 October 2014 - 11:44 AM

I’m having a problem with my sandwich ELISA. I have a sample that I do the measure every three months since 2012. Until the early of this year, this sample have 4 ug/mL of protein. To July until now, my ELISA tests give results of 14 ug/mL of protein. All the other samples keep going the same results, only this sample is “wrong”. This sample, obviously, don’t increase the amount of protein. The kit that I use is the same (same bath), the washing buffers are the same (not same bath), and the reader is the same. That is confusing because only this sample gives this result. The standard curve is always good. The negative controls are good. I don’t know if this is important, but this sample has the smaller quantity of protein of all my samples. My other samples has between 30 to 50 ug/mL of protein.

Has someone a idea for what is going on?

 

Sorry about my english!

Thanks



#2 bob1

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Posted 11 October 2014 - 12:19 PM

Welcome to the forums.

 

It is possible that your sample is outside the range of sensitivity of your assay. In other words you need a higher concentration of protein for your ELISA to measure the concentration accurately.



#3 Tielli

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Posted 13 October 2014 - 09:34 AM

Hi, I checked the sensitivity of the test, and it is ok. All my samples are between the ranges. I don’t know why changed for only two samples showed different results. I use the Indoor Kits for allergens to house dust mite.



#4 mdfenko

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Posted 14 October 2014 - 03:58 AM

is the sample in single use aliquots or do you freeze-thaw several times?

 

or is it stored at 4C?

 

are you sure neither dust has entered nor something isn't growing in the sample?

 

have you tried with a fresh 4ug/ml sample?


Edited by mdfenko, 14 October 2014 - 03:58 AM.

talent does what it can
genius does what it must
i used to do what i got paid to do


#5 Tielli

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Posted 17 October 2014 - 04:31 AM

Hello!

I use aliquots, despite this, the samples are 50% glycerin, ie, they don’t freeze. They are stored at -20ºC. The package is sterile and sealed and contain 0,5% of phenol to inhibit the microorganisms growing. Furthermore, when I test these samples with others protocols for quantity of total protein and other allergens and nothing change. The SDS-PAGE is equal, the Bradford is equal, etc. I don’t know what thinking anymore. I study a lot of papers, books, protocols and I don’t have a clue.



#6 mdfenko

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Posted 17 October 2014 - 04:51 AM

have you tried a fresh preparation of the 4ug/ml sample? you can make a dilution from one of the higher concentration samples which gave proper results.


Edited by mdfenko, 17 October 2014 - 04:53 AM.

talent does what it can
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#7 Tielli

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Posted 17 October 2014 - 05:50 AM

My aliquots are pure and every time that I do ELISA, I make a new dilution. I don’t make a 4ug/mL aliquot, I make a dilution like 1:100 of the pure to see how many allergens the sample still have. Every time, since 2012, the results showed about 4ug/mL. Since july of this year, the results are showing 14ug/mL, 12 ug/mL. The concentrations of allergen don’t increase. It’s impossible. The other allergens that I have tested seems equal, nothing changed.



#8 mdfenko

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Posted 19 October 2014 - 07:50 AM

are you sure that your pipette is operating properly and calibrated?


talent does what it can
genius does what it must
i used to do what i got paid to do


#9 Tielli

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Posted 21 October 2014 - 03:25 AM

I’m sure. The other tests are OK. No one did show different results. Even the other ELISAs. It’s only this sample and only in this ELISA. Other tests with this sample, even ELISA for other allergens give me the same results than before.



#10 sgt4boston

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Posted 14 February 2015 - 05:49 AM

Dilute your 30 sample 1:2. do you still see the same problem?






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