Competetive ELISA - (Oct/30/2006 )

Hi Everybody,

I have been doing Sandwich ELISA for the past few months and got good absorbance values.
Now I am doing competetive ELISA using same antigen, antibodies, substrate and also the conditions. But the absorbance values which i am getting now are jumping around and are not following any trend which they are supposed to be. Can any of you help me regarding this.


Thanks in advance,
Vani

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What is different between your setup before and now?

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My previous setup was coating the ELISA plate with ovalbumin10ug/ml (protein) incubate it overnight at 30deg C then adding PBST with 1%BSA(Blocking step), ConA 5ug/ml (antigen), Anti-ConA 1ug/ml (primary antibody) , Anti-RabIGg 0.1ug/ml(secondary anti body) and finally Nitrophenyl phosphate 0.1mg/ml(substrate).
With the exception of first step I incubated all other steps at 37deg C for 1hr.
After each step I washed thrice with PBST containing 0.05% Tween.

In competetive ELISA setup, instead of adding ConA it is pre-incubated 2hrs with serially diluted dendrimer(Carbohydrate which binds to Antigen) and then added to Ovalbumin. The intension of doing this pre-incubation is that ConA binds to dendrimer and this ConA-dendrimer binding inhibits the binding of ConA to ovalbumin. Finally I would like to know the inhibitory concentration of dendrimer. Other than this step all the steps are same as above. I hope this would make some sense.


Thanks WAstate,
Vani.

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Do you get rid of the ConA-dendrimer complex before adding to the ovalbumin?
If not, the anti-ConA may binds to the ConA in the ConA-dendrimer complex, instead of free ConA
or the ConA may dissociate from the complex and becomes free ConA.

I hope this may help.

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Do you incubate your antigen-antibody complex at RT or do you use an incubator? I am just thinking that if you do the reaction at RT maybe your RT actually fluctuates throughout the day? This happened to me before.

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