Elisa falses positives
Posted 17 August 2010 - 08:04 AM
I'm kind of embarrased for disturbing everyone, I hope you can help me a little or point where to go. English isn't my natural language so I'm doing by best to make me understand.
Excuses to everyone (first of all).
Now, my problem:
I'm getting a lot of ELISA (sandwich) false positives., which I know exactly the cause, or most probably... It's a terrible state (or condition) of the samples.
Though, as I'm doing a thesis, it was unacceptable to just say "bad condition of the samples" as the cause to get those false positives.
My teachers need me to say "WHY" a sample in bad condition gives a false positive... as, what is esentially giving the "positiviness".
I'd tried searching a lot but I can't find any reason that I use for to settle this once for all.
Could anyone point me a paper/publication/issue/troubleshooting guide/ ... or anything... where to look?.
I'm getting desperate...
Thanks in advance and excuse me for using your time.
Posted 17 August 2010 - 12:54 PM
I've never done an ELISA but posting what your samples are from (even cell type/organism) and what you are specifically (in protein terms) looking for may help.
Also, your english is fine so far, we see a lot of worse posts on here.
Posted 17 August 2010 - 10:27 PM
FlockChek* AI MultiS-Screen Antibody Test Kit (IDEXX)
TMB as sustrate (included) and HRP as conjugate.(included too)
(I think that is the all the information I can get from the site).
The other ingredients are defined in the instructions I have only as
"Solucion de frenado" (Stop Solution?) <- Using google translator here
I have not the name of the component.
The original samples were from 2 species of Gulls.
I'm comparing the results between AGID and ELISA (<-- This is the topic of my thesis (Studying Medicine Veterinary))
A lot (near 95%) of positives in ELISA / Negatives in AGID.
(They are considered 100% Negatives from prior AGID test, so they are ELISA false positives)
This is all the information I'm available to get from this point. =(
Hope is some kind of enough? probably?.
Sorry for my english. =(
Posted 18 August 2010 - 01:26 PM
If you decide to go with the false positive explanation; it could be that degradation of the samples releases protein sequences that are similar to the viral antigen thus leading to the flase positive results. I don't have any papers or references confirming that for you though.
Posted 18 August 2010 - 02:16 PM
Because when the samples "arrived", they were put into ELISA and AGID both (and probably PCR too... not sure about this one)
and they were clean.
They were put in the freezer and remained there until I used them.
Mmm... could be a good reason, the one that you said, but... how could I "backup" it?...
Posted 19 August 2010 - 01:13 PM
Posted 20 September 2010 - 10:59 AM
If this kit is designed for one type of animal, bird, etc and you are testing another type then the cut-off level may be entirely different. You would need to run a panel of negatives and positives to determine the cut off point.
Since you are using a 'commercial' kit you should inquire with the manufacturer for guidance with respect to sample type, condition, etc and your false results.