ELISA SANDWICH PROBLEM helppppppp pleaseeee - (Dec/09/2010 )

Hi!! i am trying to standarize and ELISA sandwich for the detection of an allergen. I have problems because i got the same results using or not the antigen.

The protocol is the following:

Capture antibody (polyclonal rabbit antibody) 70ul Over Night at 37°C in PBS buffer
3 Washes with PBS tween 0,05%
Block with milk 5% during 2 hours at 37°C in PBS buffer
2 washes with PBS tween 0,05%
Antigen at different concentrations in PBS
7 washes with PBS tween 0,05%
Recognition antibody (polyclonal chicken antibody) 70ul over night at 37°C in PBS milk 2%
7 wasshes with PBS tween 0,05%
Anti-chicken Promega 1:1000 1 hour 37°C in PBS milk 2%
7 washes with PBS tween 0,05%
TMB 25 mins - HCl 1N
Read: 450 nm

I really appreciate any help! Thank You

Use less amout of 1st antibody.

linistrespa on Thu Dec 9 18:46:34 2010 said:

Maybe it will be easier to help, if you tell us, what type of signal you obtained - was the result negative for both or positive for both?

Lets not make any assumptions. Make some strips coated with antigen and check your primary antibody for binding.

This may not be the cause, however in my experience with chicken antibodies I have found that they have higher than normal non-specific binding to polystyrene and consequently a higher background signal. I had to do a whole side project on blocking conditions just to minimize the signal-to-noise ratio.

antibodymania on Wed Jan 19 17:51:37 2011 said:

I will make the assumptions that you have ALREADY checked for binding of your polyclonal rabbit to antigen and your chicken antibody to antigen and your anti chicken conjugate and they react specifically and concentrations used do not create significant backgrounds in your screening test. Also, you have screened plate blocking reagents and milk creates the lowest background. I would suggest getting a copy of a commercial allergen elisa kit and compare their steps to what you are doing. See comments below:

Capture antibody (polyclonal rabbit antibody) 70ul Over Night at 37°C in PBS buffer (that is really a long time; 1 hr 37C should be fine or 2 hr rt or overnight at 4C..make sure plates are sealed )
Washes with PBS tween 0,05% (tween unnecessary)
Block with milk 5% during 2 hours at 37°C in PBS buffer (blocking at RT should be fine)
2 washes with PBS tween 0,05% (tween unnecessary)
Antigen at different concentrations in PBS (ok how long are you allowing for binding and at what temperature? 1 hr 37C; do you shake the plates? )
7 washes with PBS tween 0,05% (7 washes probably overkill)
Recognition antibody (polyclonal chicken antibody) 70ul over night at 37°C in PBS milk 2% (this is also real long time at high temp)
7 wasshes with PBS tween 0,05% (7 washes probably overkill)
Anti-chicken Promega 1:1000 1 hour 37°C in PBS milk 2%
7 washes with PBS tween 0,05% (7 washes probably overkill)
TMB 25 mins - HCl 1N
Read: 450 nm

mdfenko on Wed Jan 19 19:34:23 2011 said: