Elisa falses positives - Need bibliography of the real reason. =( (Aug/17/2010 )
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.
What you are probably looking for is ... "what component of the degraded samples is causing the false positives?" It may be that there is a short protein sequence that is binding non-specifically, or something that is reacting with the enzyme causing it to react with the substrate.
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.
I used this one:
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. =(
So you are looking at immunodetection of avian influenza... I would have said that ELISA is more specific and more sensitive than AGID, so I would be very careful about calling these ELISA results as false positives without further tests that can confirm either way. You could do a PCR on the samples to look for the presence of viral DNA as another confirmation of the presence or absence of the virus.
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.
Oh, yes, I'm going full for the "false positives" one.
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?...
I would look at a lot of ELISA papers (try google scholar or pubmed) and see if there is anything in there that would be helpful. Unfortunately it is a lot of work and there isn't really any shortcut for the process.
The test in question is a test for antibodies to virus not virus particles. You indicated in first email that samples were in bad condition. In a later email you indicated samples were frozen until you conducted your test. Frozen serum samples should be fine for storage and testing of antibodies. If the samples were going through multiple freeze thaws then the immunoglobulins might be degraded and values negative. OR, if you are testing for IgM then, perhaps, the M broke into fragments and the signal you have is above the negative cut off...positive. this would only occur if the conjugate recognizes both IgM and IgG antibodies.
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.