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Strange results using sandwich Elisa - All antigen concentrations show same result - same optical density! (Dec/12/2008 )

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Hi everyone, I am carrying out sandwich ELISA. I am coating wells with NaHCO3/Na2CO3 buffer and monoclonal mouse antibody. I then apply blocking buffer followed by the antigen at varying concentrations, rabbit antibody, goat antibody, TMB, stopping reagent.

I prepared a blank on same plate where I place everything in above sequence excluding mouse antibody and antigen.

Results: blank and ALL sample wells with VARYING concentrations of antigen give the same colour - no variation in optical density...no gradient in colour. I have repeated expt more than once yet i get the same results.

A person who works abroad using same protocol obtains good results using same antigen concentrations, same antibodies, same supplier for reagents and antibodies. Can anyone imagine what could possibly be going wrong?

Thanks everyone,

Racla

-Racla-

One thing you may think about is washing step, how is your washing buffer.



QUOTE (Racla @ Dec 12 2008, 02:22 PM)
Hi everyone, I am carrying out sandwich ELISA. I am coating wells with NaHCO3/Na2CO3 buffer and monoclonal mouse antibody. I then apply blocking buffer followed by the antigen at varying concentrations, rabbit antibody, goat antibody, TMB, stopping reagent.

I prepared a blank on same plate where I place everything in above sequence excluding mouse antibody and antigen.

Results: blank and ALL sample wells with VARYING concentrations of antigen give the same colour - no variation in optical density...no gradient in colour. I have repeated expt more than once yet i get the same results.

A person who works abroad using same protocol obtains good results using same antigen concentrations, same antibodies, same supplier for reagents and antibodies. Can anyone imagine what could possibly be going wrong?

Thanks everyone,

Racla

-microlight-

QUOTE (Racla @ Dec 13 2008, 06:22 AM)
Hi everyone, I am carrying out sandwich ELISA. I am coating wells with NaHCO3/Na2CO3 buffer and monoclonal mouse antibody. I then apply blocking buffer followed by the antigen at varying concentrations, rabbit antibody, goat antibody, TMB, stopping reagent.

I prepared a blank on same plate where I place everything in above sequence excluding mouse antibody and antigen.

Results: blank and ALL sample wells with VARYING concentrations of antigen give the same colour - no variation in optical density...no gradient in colour. I have repeated expt more than once yet i get the same results.

A person who works abroad using same protocol obtains good results using same antigen concentrations, same antibodies, same supplier for reagents and antibodies. Can anyone imagine what could possibly be going wrong?

Thanks everyone,

Racla

If every single well in your assay has the same OD, I think your blocking solution is stuffed! I presume the OD is very high, too.
Get some blocking solution from someone else and try again.
BTW, what's your protocol?

-swanny-

Hi microlight,

Thanks for your reply. My washing buffer is:

  • 450ml distilled water
  • 50ml D-PBS
  • 250┬Ál TWEEN


After the addition of coating buffer (with 1st antibody) and the blocking buffer I wash 3x and from then on i wash 4x.




QUOTE (microlight @ Dec 12 2008, 01:20 PM)
One thing you may think about is washing step, how is your washing buffer.



QUOTE (Racla @ Dec 12 2008, 02:22 PM)
Hi everyone, I am carrying out sandwich ELISA. I am coating wells with NaHCO3/Na2CO3 buffer and monoclonal mouse antibody. I then apply blocking buffer followed by the antigen at varying concentrations, rabbit antibody, goat antibody, TMB, stopping reagent.

I prepared a blank on same plate where I place everything in above sequence excluding mouse antibody and antigen.

Results: blank and ALL sample wells with VARYING concentrations of antigen give the same colour - no variation in optical density...no gradient in colour. I have repeated expt more than once yet i get the same results.

A person who works abroad using same protocol obtains good results using same antigen concentrations, same antibodies, same supplier for reagents and antibodies. Can anyone imagine what could possibly be going wrong?

Thanks everyone,

Racla


-Racla-

Hi Swanny,

I am doing Sandwich ELISA, detection for HSP Protein. Hence I am using mouse monoclonal antibody (anti-HSP) in coating buffer, then I add the blocking buffer. I then add a gradient of concentrations of HSP-protein (at concentrations used previously). The second antibody is the rabbit anti-HSP polyclonal antibody and finally the goat anti-rabbit polyclonal antibody. I then add TMB and then 2N sulphuric acid as stopping agent.

Between each step i wash 3-4x with washing buffer (0.05% TWEEN 20).



QUOTE (swanny @ Dec 14 2008, 09:37 PM)
QUOTE (Racla @ Dec 13 2008, 06:22 AM)
Hi everyone, I am carrying out sandwich ELISA. I am coating wells with NaHCO3/Na2CO3 buffer and monoclonal mouse antibody. I then apply blocking buffer followed by the antigen at varying concentrations, rabbit antibody, goat antibody, TMB, stopping reagent.

I prepared a blank on same plate where I place everything in above sequence excluding mouse antibody and antigen.

Results: blank and ALL sample wells with VARYING concentrations of antigen give the same colour - no variation in optical density...no gradient in colour. I have repeated expt more than once yet i get the same results.

A person who works abroad using same protocol obtains good results using same antigen concentrations, same antibodies, same supplier for reagents and antibodies. Can anyone imagine what could possibly be going wrong?

Thanks everyone,

Racla

If every single well in your assay has the same OD, I think your blocking solution is stuffed! I presume the OD is very high, too.
Get some blocking solution from someone else and try again.
BTW, what's your protocol?

-Racla-

Hi Racla,

This is an interesting problem as I am experiencing exactly the same problem as you have only am I trying to measure an other antigen than you. Indeed high absorbances in all the wells. One thing that is striking though is that we both use antibodies produced in the same species, i.e. I also use a monoclonal mouse AB, a polyclonal rabbit AB and a goat-anti-rabbit-IgG. So there is a definite resemblance, if it means anything I don't know but it is striking though.

One thing that did OK for me was coating the antigen in different concentrations, then blocking (BSA), followed by AB, antiAB-HRP and TMB. This gave perfect curves (but with high background) but it is not what I want to use in measuring my clinical samples, I really want the sandwich. This might suggest that there is some sort of interaction between the different antibodies????

In the next few days I will try using different blocking reagents.

So we are on the same ride here and any suggestions from you or from other readers are more than welcome.

Thanks

-Bertrand-

Hi Bertrand,

Glad to know im not alone smile.gif I doubt it is interaction between the antibodies since the person who trained me using ELISA carries out the procedure in the exact same way as I said before....and he gets good results (and has been getting good results for a very long time). I am going to try increase the Tween concentration from 0.05% to 0.1%. I am having my doubts on the blocking buffer...I am using SuperBlock T20 (PBS) Blocking buffer. I will considering ordering a different Blocking buffer however this would take time until it is delivered...unless I try out the one with milk!?!

Let me know how it goes with the different buffers. Will let you know if I manage to get any good results.

Thanks.


-Racla-

why don't you have the person who trained you evaluate your procedure and technique (ie- watch you)?

you may find it is something as simple as timing of the development.

-mdfenko-

Hi mdfenko

The procedure didnt work for the person who trained me while he was here, I have also carried out the procedure myself before him and again got same results...but it works ok at his laboratory abroad. He suggeted the coating buffer, however we are using the same grade of sodium carbonate/sodium hydrogencarbonate, in same ratios, same monoclonal antibody - so i doubt thats it. Now i am going through every step alone, breaking it down to try find the problem.

If you have any ideas please let me know.

-Racla-

may check the pH of the coating buffer

-Minnie Mouse-

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