Very inconstant ELISA - or why one day and not the other one... (Apr/05/2006 )

Hi,
I established an ELISA since couple of months. It's a sandwich one, to mesure the concentration of my protein in human serum. It works fine, generally...
My big big problem, is that on some days, nothing work, meaning absolutely no yellow color in any wells, even the positive controls.
Its seem to me to happen randomly. Monday-tuesday did'nt work, wednesday yes. I used the same reagents, the same protocol. I tried all the reagents individually (coat each one and detect simply with the appopriate conjugate) and everything is fine.
It's not manipulation error, it's happen too much time for it. The reagents works, I followed the exact same protocol, use the same incubator and have no more ideas!!

By the way, when it works great, I generally a big discreanpcy on day-to-day results. The highest point of curve can be sometimes at OD 0.4 or 1.5!!

Thanks a lot if someone could help me! If not, soon I will monitoring the moon cycles!!
Mélanie

Another few questions to add to Swanny's,

when you say "same incubator" is this for the ELISA or for cell culture?

Are you using the same samples and standard each time?

Have you aliquoted your abs and standard so you're not freeze-thawing the same reagents each time?

All the best,
Ceri

Hi,
please try not to confuse people around, if u say ur ELISA does not work on monday and tuesday IT DOES NOT MEAN ANYTHING, what i want to say is there is no connection between days of a week and ur ELISA to compare.
u must be doing same mistaque on these days, moreover since the coincedence was high ur assuming that ELISA works only other than monday and tuesday.

coming to solving the problem, every time when u perform ELISA do u check whether substrate solution preparation is fine or not? because it really makes a big difference.
u can proceed in this way to get answer for your problem

1-prepare secondary antibody solution little more thatn necessary and keep it till the last step of ELISA, once u prepare substate solution add the left over secondary antibody with HRP conjugate to substate solution around 500ul or so, separately in a tube and u must observe the yellow color
RESULT-HRP ENZYME IS WORKING FINE

2, if you donot get any color in ur assay(positive ctrl)- doubt ur second Ab whether the HRP enzyme is still conjugated with sec Ab ? u can check this in two ways
a- coat secondary antibody and look for yellow color this give hint, whether ur sec Ab and HRP are still conjugated.
b- usee diff Ab-HRP source whihc u know for sure it is working.
RESULT- HRP & SEC Ab ARE IN CONJUGATED FORM

3,second step is fine, then doubt your primary antibody
a, use either fresh aliquote or use known working antibody

i hope i could convey you wat i want to say,

gud luck

Hi,
Scuse me, I didn't want to confuse anybody. It was just an image to mean that is seem to seem to me that happen very randomly, even if I followed the exact same protocol.

Answers :
I tried fresh and stored plates (kept at 4°C in a tupperware with a wet towel)

I used the same incubator to make the incabation at 37°C.

I used an aliquot of Ab kept at 4°C. I also tried a new one .
I use the same samples. The recombinant protein is in small aliquot, and I tried different batches. The serum are freeze-thaw, but I dont really have choice, I cannot keep a lot of tubes. Anyway during my set-up, I re-use the serum kept at 4°C for couple of days and it was ok.

Yes, I tried the substrate. It become colored with the 2nd Ab.

I tried all the combinations of Ab.
Coat the Ab + HRP Ab -> fine
Coat the 1st coating Ab + HRP Ab -> fine
coat the protein + Ab+ HRP -> fine
But on this experiment, the positive control and the curve also become yellow.

In an other experiment where nothing worked, I tried a different HRP antibody, and it's also didn't work like my habitual one.

By the way, I just have this 2 antibodies, and I cannot switch them.
I'm confident that my reagents are ok, I'm sure it's something subtil and probably very stupid, but I dont know what!!
Thanks a lot
Mélanie

Melanie,
Do you seal your plates after coating and in between steps with a plate seal ideally? This should stop evaporation when you store it in the fridge and prevent spill over beween wells. Also how do you block your plates and how what are your washing steps (manual/plate washer, %Tween, no. of times etc.)?

"I tried all the combinations of Ab.
Coat the Ab + HRP Ab -> fine
Coat the 1st coating Ab + HRP Ab -> fine
coat the protein + Ab+ HRP -> fine
But on this experiment, the positive control and the curve also become yellow.
In an other experiment where nothing worked, I tried a different HRP antibody, and it's also didn't work like my habitual one."

I'm guessing fine means a colour change. In which case the coating ab plus the HRP Ab alone shouldn't give you a colour change.

What species are the coating and 2ndary abs from? What species are you HRP-conjugated abs raised in and what species Ig are they against?

Sorry for all the questions,
Ceri

Hi,
I didn't seal the plates, because I put in a tupperware with a wet towel to prevent evaporation. I could try this also, but just remind you that with fresh plates I also have the same problem.
I block with 4%BSA 0.5% Tween20. Wash manually 4x 2min with PBS/tween 20 0.5%.
All incubations 1h @37°C.

I tried the coating ab + the appropriate 2nd, not with the one in this essay. (just to see if it have Ab that was coated)

It's a coating polyclonal IgG home-made rabbit + an polyclonal commercial IgG goat + a rabbit anti-goat IgG HRP.

It's ok for all the questions!
Mélanie

hi
is ur sample fuse protein? maybe some problem,u can use westblot detect ur sample

May be something wrong with your multi-channel pipette.

Ok. 0.5% Tween seems a bit high we use 0.05% in our ELISAs (1ml in 2L PBS). There's no possibility of azide contamination which would inhibit your HRP activity? After that I'm out of ideas.

All the best,
Ceri