This also happened to me once during my diploma thesis and I really got mad finding the reason. But I used a kit, which had some problems. After calling the company, it was replaced and everyting was fine again. As far as I see, you don't use a kit, right?

Nope, no kit. All of my stocks are the same as the ones I used last week for the successful ELISA, so I'm hoping it's just a fluke. We store all reagents in -20 freezers except the antibodies, which stay at 4 degrees, so I'm not sure what is different about this run, but I'll try again. Thanks for the help!

One of the probable reason for that is change in pH of buffers, especially the reaction buffer and the buffer in which you prepared your substrate.

you are welcome in the world of ELISA assay!! When Revelation step is slowered, it is first because Enzyme tracer is destroyed (generally it is the normal lysis between antigene and enzyme) or inhibited (one of the more frequent and less diagnosed is the water quality. A pb in the water tubes/pipes in the campus and during sometimes you have earth, rust mixed with water and despite purification steps you could lead to very "nice" enzyme inhibition!! In your case, because substrate as kepts at room temperatuire, you have to teste it!. If a part of your revelation system include H2O2, be careful because, if walls or tubes are not not correctly closed, the H2O2 concentration inside could be modified and reaction slowered. Good luck.

What samples are you detecting? I've had something similar (but with TNfa)?. I would probably use new standard protein to check your other steps are fine. Which substrate do you use?

I think the pH in my buffers are off. I checked this morning, and the pH was almost 8.5, so I am trying my test again with pH 7.4 buffers today, and I will also check the secondary enzyme antibody and the substrate reaction before I use them. Thanks for your help everybody! This was my first time using this site for help with a conundrum, and I think it was very helpful!

-Alex

Dear Alexandra,

If you use a commercial kit, please test another kit peharps the kit do not works well.

I would recommend to add Hydrogen peroxide to your substrate. Only 9 uL H2O2 to 50 mL aprox. can work.

The incubation temperature must be checked too, if it is very low the signal could be faint.

Another important aspect is the pH of your solutions that already you have checked.

Good luck!!!

Thanks,we'll see shortly. The moment of truth in 20 mins... I'm hopeful the pH was just off, and I don't have to troubleshoot anymore, but if I do at least I have a whole bunch of great suggestions!

It's not the pH, I'm going to try again tomorrow with all new room temp solutions and hope for the best. Thanks again for all the help!

Suggestions made by other workers as above have genuinity.It is difficult to pin point now.Please use freshly prepared reagents and use plate of another brand.These may solve your problem.

add 1 ul of your secondary to 300 ul of substrate. The chromogenic reaction should be virtually instantaneous with deep color development. Is your secondary Ab an HRP conjugate or an AP. If it's HRP what substrate are you using? If you are making up your HRP substrate yourself the hydrogen peroxide may have decomposed and what you have is a bottle of water. try a new bottle of H2O2!

I use a commercial single reagent TMB substrate in my HRP ELISAs. This substrate contains a stabilized peroxide source and has never caused me any problems!

The pH optimum of HRP is in the range of 6.0 to 6.5; activity at 7.5 is 84% of the maximum. The enzyme is most stable in the pH range of 5.0 to 9.0

@Ramesh, Do you have a reference for HRP enzymatic activity over broad pH range? I've encountered a similar problem to this and found the pH of my solutions to be around 8.1-8.5. I haven't had a problem since I stopped using the community PBS and made my own.

Evan, try this. Clinical Chemistry

December 1982 vol. 28 no. 12 2423-2426

Evan, figure 1 is of interest to you I would think.

20 mM citrate buffer at pH 7 is the best buffer to use as the enzyme activity remains high if the pH wanders off. Not true of PBS.

Did you try Optimizing the enzyme loading and incubation time? if you did, try to incease the second antibody concentration and its incubation time. It is also recomended that all Ab dilutions should be done in the blocking buffer (with 3% BSA/PBS).

The assay was working and suddenly stopped. Unless new reagent lot numbers were used it's not an optimization issue its a reagent gone bad issue

I read she used newly made reagents and a new substract. If it is a reagent issue she should start by changing the substract as suggested by Jain

Yes newly made working reagents not new stock reagents. If the hydrogen peroxide stock solution has decomposed her stock reagent is nothing but water.

I would agree with fresh reagents. Also, did you try a positive control with your samples? In the past I used home made reagents but after similar problems with pH changes etc `i decided to go with the commercial reagent TMB substrate in my HRP ELISAs and i haven't changed since. It does worth the extra pennies.

Has the problem been solved Alexandra? Did you get a chromogenic reaction when you added your HRP conjugate to some substrate? If not did you do it again with oPD containing fresh hydrogen peroxide?

We have been doing ELISA almost regularly.We also encountered problems,but we were able to solve it out. Dr./Mr.Ramesh Nayak have made various suggestions.I am happy to learn about it. The Scientist has not replied, whether his/her problem has been solved with these suggestions.If so, which one has worked out? We shall be glad to learn about it.

I checked out most of your answers, and it turns out our plates are bad. We ordered from "evergreen" instead of the costar plates we normally use. When my colleague tested the plates with our positive IL-6 control, we noticed the evergreen plates did not undergo the same enzymatic reaction. We think the coating on the wells must be insufficient. Moral of the story: be careful who your vendor is! Thanks for all of your great suggestions! I hope string of answers helps troubleshooting in the future!

-Alex

Ha! The only thing that wasn't considered. Buy the best quality. Cheaper goods are not cheaper if they don't work. A lot of time and money can be wasted on failed experiments.

Well I guess it falls into the intent of this statement "The assay was working and suddenly stopped. Unless new reagent lot numbers were used it's not an optimization issue its a reagent gone bad issue"

Happy to know that you resolved the problem. It seems like your assay needed to be optimized for the evergreen plate after all.....

My impression from Alexandra's update is not that they needed to optimize the assay for the evergreen plate but to stop using the evergreen plate and return to using the costar product!

check it step by step, first check the substrate with fresh HRP-Ab，if no problem, consider coating Ab, maybe you can use Carbonate buffer in stead of PBS

We checked everything, only the plate was bad (not binding primary antibody very well), so we stopped using them.

Dear Alexandra

there are many potential reasons.

1) your secondary antibody was coupled to HRP(peroxidase) ? how was stored your secondary antibody at 4°C , frozen , pure or stored already diluted at 4°C

2) What about your IL-6 ? storage at 4°C or frozen highly concentrarted?

3) primary antibody same questio that above

4) substrate issue... that is easy to check you take your secondary antibody coupled to HRP and your diluted 1/10000 and until 1/10000000 dilution and you just add directly in your well wiout any washing 10ul of your subsrate ..the color should appears immedialtly..if not then your subsrate is not working-

your substrate is ready to used or you have to mix 2 components?

to summarize you may have antigen, antibody stability issues or poor coating the easier is first to check if your subsrate is working

From my experience，Coster plate 2592 is always work for elisa.However,coating buffer is very important,usually Cp buffer is better than PBS.of course depending on different Abs.So IL-6 ab is work not means other Abs will work.