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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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QUOTE (Racla @ Dec 15 2008, 06:21 PM)
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 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,


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?

Ok. First of all, what OD are you getting? 0.1? 3.0? What is your blocking solution? Details, please!

What does the data sheet for the goat antibody say about the blocking and washing solutions? Does it mention the use of inactivated goat serum?

Go to and look at their assay development section (, among other sections - it's a great website!!! In it, you'll find their data sheets for about 55 gazillion antibody reactions. See how they use different coating, blocking and washing reagents.

All the best!


QUOTE (Racla @ Dec 15 2008, 05:59 PM)
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 suggested 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.

is there anything that you are using that is different? the other antibodies? substrate brand?

how does your water quality compare?


Sounds like the conjugate is sticking to the wells OR you titer of conjugate is too low thus signal is very high.

Without having to run an entire test just block some wells and run conjugate, wash, substrate etc...does it (conjugate) stick? You can compare just the blocking portion of your assay and work backwards.


Swanny: I am getting OD values ranging between 0.739 and 0.841 (randomly not according to concentration).

Blocking solution is SuperBlock T20 (PBS) Blocking buffer (a protein based blocker formulation in phosphate buffered saline containing 0.05% Tween 20.

Washing buffer is a solution of 450ml deionised water (also tried with distilled) + 50ml PBS (x10) and 500µl Tween 20 to obtain 0.1% Tween washing buffer (I also tried with 0.05% but got same results).

The data sheet of all three antibodies say nothing about the washing and blocking buffers and neither does it mention anything about inactivated goat serum. I will not have a look at the site you gave me. Thank you.

Minni: pH is 9.3 at a room temperature of 18 degree celcius

mdfenko: Everything is the same (as far as I know)! As regards water quality, when the trainer was here we tried both deionised water and also distilled water.

Sgt4boston: A coincidence happy.gif just yesterday I carried out the experiment you said…I worked backwards and carried out a normal sequence (working forwards) on the same plate.

Please find the results attached. I have formulated some vague ideas and also 2 other tests I should have done. But I would not like to give them here so you are not biased when looking at results.

Hope I can get to the bottom of this!

Thank you everyone.


Hi Racla,

Looking at your working protocol I see nothing strange, will also look at you results a bit later.

I promised to come back to you. Today I tried 3 different blocking solutions. I tried BSA, fetal calf serum (FCS)and fat free milk, all in 3 different concentrations. Unfortunately I still get the same OD's when using different concentrations of antigen instead of the expected calibration curve, no matter what blocker I use. The results are below. What is interesting is that when I lower the concentration of blocking reagent the OD gets higher (suggesting bad blocking?) except for the milk-block where the OD's remain the same regardless of the milk concentration.

All antibodies are used at 1 ug/ml. Coating and blocking was both done overnight at 4C. Washing 4x PBS-Tw0.05%.

Pipetting order:

BSA 5% : OD 0.725
BSA 1% : OD 0.825
BSA 0,2%: OD 1.000
Milk 5% : OD 1.100
Milk 1% : OD 1.250
Milk 0,2%: OD 1.200
FCS 10% : OD 0.650
FCS 2% : OD 1.100
FCS 0,4%: OD 1.500

I don't mention the OD's at different concentrations of antigen as they are always the same, no matter what conc I use.

I find it amazing that even at the highest conc of blocking (which I think is really high) I get these high OD's. All places on the plastic must surely be covered at these conc, so something sticking to the BSA or tho the Mab?

This experiment does not help either of us.


Hi Racla,

Just looked at you results.

The results in column 8 are not as expected. I expect blanc values here, but instead the OD is about 0.5. Is there an interaction between AB1 and AB3 here? I say this also because of column 7 where you left AB1 out and found low OD's

Did you block columns 10/11. If not I would expect real high OD's as the AB's would certainly attach to the wall of the well.

I am also interested in what would happen if you switched AB1 and AB2, you would only need another AB3 then?

Keep you posted of my future experiments, will probably be next year.


Yes that is the first thing i thought of also. And when i showed these results to the person who trained me he said the same thing. What I find very strange though is that he gave me these antibodies, so he should be experiencing the same interaction between the two antibodies, but he is not!

No I added no antibody in columns 10 and 11 and so I also found it strange that the antibodies did not attach to the wells!!!

The only time I am getting a reasonable detection is when the 1st Ab is present. Would be interesting to try out the Ab2 first and then Ab1 BUT i do not have a 3rd Ab that gives colour indication to Ab1.

Tomorrow I am going to run another test (the last one before next year). So far I have the following tests in mind, please tell me if you think I should run some other combination:

1) Blocking Buffer, 1st Ab, Antigen, 2nd Ab, 3rd Ab, TMB
2) 1st Ab, Blocking Buffer, Antigen, 2nd Ab, 3rd Ab, TMB
If the above two give same readings then we can say BB is not doing its job.

3) Blocking Buffer, 3rd Ab, TMB (a combination i didnt do before)

4) 1st Ab, 2nd Ab, 3rd Ab, TMB
5) 1st Ab, Ag, 2nd Ab, 3rd Ab, TMB
(to see if the presence of Ag is making any difference)
5) 1st Ab, 3rd Ab, TMB
(to see if the presence of Ab2 makes any difference)

Anything else?


HI Bertrand,

I didnt see this message...

Your results are interesting though. See the below 2 sites:
(shows that we need to use a protein-free Blocking Buffer to prevent cross-reactivity with the antibodies)
(indicates that lower concentrations of blocking buffer may be best to prevent masking antibody-antigen interaction)

So your results do make sense. But still the same OD values were obtained. I will try dilute my blocking buffer by 5x (max dilution can be up to 10-fold) and see how I go. Therefore I will do the same trials I listed before 2x (once with undiluted blocking buffer and once with diluted blocking buffer) - since the error in our results may be due to more than one factor. Will send the results tomorrow or late this evening.


I think you might be running yourself ragged for no good reason. Something's just not right. Some questions:-

Why do you use a non-protein blocker? Have you tried BSA in PBS? The R&D Systems website could give you some ideas for alternate blocking solutions. Dumb question, but are you blocking with 300ul? How long?

The comment about the conjugate being to concentrated is certainly worth considering.

Also, what plates are you using? See if the manufacturers have any data on the plates, or if anyone else using them has had problems. You will need to give them the lot number.


Hi again, I carried out the test I mentioned above...will send results tuesday since I left them at work and wont be there until tuesday.

But basically results showed:

1) Blocking buffer is not a is blocking wells perfectly...I also tried to half the concentration of blocking buffer and I also tried different timings of application of blocking buffer...both of which gave no particular difference in OD.

The protocol I have been trained to use is 200ul for 1.5 hrs.
The protocol on the data sheet of the blocking buffer I am using says 300ul repeated for 3x (ie fill wells and empty immediately - repeated three times).

Came to this conclusion since when I add blocking buffer before 1st Antibody very low signal (practically no signal), when added after 1st antibody a signal was obtained.

2) Whether or not I add the antigen and/or 2nd antibody, a signal is obatained.

3) Whenever there is 1st and 3rd antibody in plate there is a signal (Even when i added just 1st antibody, 3rd antibody, TMB) must be smthg between the two.

And as you are saying...trying lowever concentrations of 3rd antibody might be a good idea. I am using this blocking buffer simply because it is the one i found available in the other reason (and also it is the one being used by my trainer abroad). I will check what plates I am using when I go in on Tuesday - however I have tried using a plate from my lab and one given to me by the trainer and still got same results.

Thank you very much everyone for your ideas...I really appreciate it. I will get back to you all with my results on Tuesday. I will try run a plate with varying concentrations of 3rd Ab and will let you know how it goes also.

Thanks once again!!


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