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total protein S trouble shooting

elisa protein S measurement trouble

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#1 marcusbock1987

marcusbock1987

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Posted 14 April 2013 - 03:57 AM

Hello!
I do not have a lot of experience in doing ELISA test, so i try to find some help. I did test of Total protein S using Asserachrom Elisa test. I have use plasma on trisodium citrate , which were stored in -70 degrees Celsius. I thawed it at 37 degrees for 45 min. Temperature in room was about 24+/- 2.

I prepared two plates. results first was fine, as described in literature,but for all values of samples ​​of the second plate were much higher than before (20-40% of the absorbance). I used the same samples for the first and for the second time(the same object, but new plasma samples ). Terms of thawing and testing were identical. Standards and quality control in the first and second plate were the same and have the correct values​​. Repeats between samples on the same assays are fine(less than 5% SD). Every time new reagents were used. The only reagent, which was prepared by me is sulfuric acid. On the first and the second plate was used the same solution from the same bottle. My question is, is it possible that the second time there was no stopping the reaction and the absobrance came out high, despite good value for standards and control??
Maybe another idee why the results are critcal not the same??
Marcus





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