concentration of samples with ELISA - (Oct/10/2014 )

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

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.

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.

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?

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.

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.

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.

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

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.

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