sample gives signal without capture antibody - No capture antibody but samples give high signal compared to blank (Feb/19/2010 )

Hello,
I am optimizing a sandwich ELISA to detect the amount of antigen in CSF samples. I coated wells with capture antibody and added CSF samples and added detection antibody and secondary antibody. Also included negative controls without capture antibody and added CSF samples and added detection antibody and secondary antibody. There was signal in wells without capture antibody with CSF samples but recombinant protein was not detected.
The amount of antigen detected was similar with and without capture antibody in CSF samples.
Please let me know what could be the problem.

Just curious, have you repeated and still get the same result? Could you change the plate or wells that you for ELISA.

Good Luck !!!

How do you block the plates ??? and what plates do you use ?

ics on Feb 20 2010, 02:13 PM said:

I tried different blocking buffers with 1% casein, 2.5%gelatin with 0.05%tween -20, 5%Goat serum, 3%BSA.
none seem to reduce the background for CSF samples without capture antibody.I use 384 well plates

shi on Feb 21 2010, 08:52 AM said:

Hi shi

As I read you post you are optimizing the assay, which means that it has worked…have you change anything… It appease from what you are writing that you have a problem with detection antibody (as it reacts with CSF without the antigen in)…perhaps if you have an other detection antibody you could try that.. it may also be the secondary antibody that reacts with CSF.. Have you tried having an other matrix that CSF with you target (say PBS)

Did you block the wells without the capture antibody? And place also a brief setup from the elisa on the forum.

Please answer a few questions. Was this assay working properly before...are you truly optimizing it or is this a new assay you are developing? ICS asked earlier if you changed something in the assay?

You also indicated that you are getting signals without the capture antibody. So either the sample, antigen, detection or secondary antibodies...are sticking.

Are you completetly filling the wells when blocking? I would remove the tween from the blocking solution and make sure you fill the wells and block for 1-2 hours at room temperature. You can leave the tween in the wash buffer and make sure you wash each well completely 3-4X between steps.

The matrix as discussed by ICS may also be an issue. Does CSF without antigen (true negative sample) also produce + result? You can dilute your samples in your assay buffer to reduce any matrix effects.

Gerard on Feb 21 2010, 10:46 AM said:

Yes i blocked the wells without adding capture antibody.