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sample gives signal without capture antibody


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7 replies to this topic

#1 shi

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Posted 19 February 2010 - 09:19 PM

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.

#2 scolix

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Posted 20 February 2010 - 11:09 AM

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

Good Luck !!!

#3 ics

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Posted 20 February 2010 - 01:13 PM

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

#4 shi

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Posted 20 February 2010 - 11:52 PM

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

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

#5 ics

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Posted 21 February 2010 - 09:26 AM

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

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


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)

#6 Gerard

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Posted 21 February 2010 - 10:46 AM

Did you block the wells without the capture antibody? And place also a brief setup from the elisa on the forum.
Ockham's razor
Pluralitas non est ponenda sine necessitate
-- "You must assume no plural without necessity".

#7 sgt4boston

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Posted 22 February 2010 - 11:03 AM

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.

#8 shi

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Posted 23 February 2010 - 03:41 AM

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

Yes i blocked the wells without adding capture antibody.




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