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Sandwich ELISA Optimization HELP!?

Sandwich ELISA Optimization HELP!? - ELISA Assay Forum

Sandwich ELISA Optimization HELP!? - ELISA Forum. Discuss and post questions including optimization, troubleshooting, protocols and theory of ELISA assays using monoclonal, and polyclonal antibodies or peptides.


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  #1  
Old 11-21-2009, 08:42 PM
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Default Sandwich ELISA Optimization HELP!?



Hi out there,

I am trying to develop a sandwitch-type ELISA to quantify protein content. I have not done this before and have no one with experience (except for with kits) in my department. I've done a lot of reading on the subject so hopefully what you'll find below is a decently well planned system.

I ran an initial run of the system that I am trying to design and got extremely high OD in capture antibody-negative wells and protein blank wells. Ill describe the system below. If anyone could posit a potential problem Id be so grateful!

Capture Antibody: Rabbit-raised polyclonal antibody against N-terminus of the protein.
Secondary Antibody: Hen-raised polyclonal antibody against C-terminus of the protein. Labeled with biotin.
Enzyme label: Streptavidin conjugated to Alkaline Phosphatase (Invitrogen)
Substrate: pNPP (AbD Serotec Ready-to-use)
Both work great for Western Blots.

Wanted to first titrate Capture Antibody against a dilution series of purified target-protein.
1. Dilute Capture Ab 1:250, 1:500, 1:1000, 1:2000, 1:4000, 1:8000. In PBS with 1% BSA. Rotating overnight at 4C. Each column of the plate used had a dilution. Plus a capture-antibody negative column. 50 uL per well.
2. In morning, wash 3X with PBST + 1X DDI H2O.
3. Block with PBS with 5% BSA for 1 hour on a rotator. 200 uL per well. Room temp.
4. Wash
5. Dilute purified protein (~100 kDa from Arabidopsis) in PBS: 8, 4, 2, 1, 0.5, 0.25, 0.125 ng/uL. 50 uL per well. Each row had a particular dilution with the last row being a 50 uL PBS only. Rotate at room temp. for 2 hours.
6. Wash
7. Keep secondary (with biotin label) at constant dilution across plate to optimize capture dilution. Used 1:1000 dilution in PBS. 50 uL per well. Rotate at room temp. for 2 hours.
8. Wash
9. Keep Streptavidin-AP dilution constant. Used a 1:1000 dilution (supplier recommends between 1:1000-1:2500) in TBST, pH 7.6. 50 uL per well. Rotate at room temp. for 1 hour.
10. Wash (2 times in PBST, then in TBST, finally in water)
11. Add 100 uL pNPP substrate (as per supplier instructions). Rotate at room temp. for 15 minutes.
12. Read at 405 nm after 15 min, 30 min and 1 hour.

The result was that I got OD(protein sample) > OD(blank) only sometimes. There was no clear winning dilution. Only after 1 hour did I get a single replicate of the 1:4000 dilution to show even remotely linear increase in OD with protein concentration. But the other replicate column with 1:4000 did not show this.

I think this means that I have successfully detected protein, but there is something very wrong with the protocol thus far. Help!?!?

Thank you!!!!

~M
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Old 12-02-2009, 04:30 PM
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Default Re: Sandwich ELISA Optimization HELP!?

Dear WolfMD,

The only thing that looks suspicious to me is the presence of 1% BSA in the coating solution. This is likely preventing getting much of your coating antibody onto the plate, and I would recommend PBS or pH 9.5 carbonate buffer (no BSA in either) instead. Overnight at 4C is fine for this but 1 hour at RT will probably work, as well.

Everything else looks fine, although I think you'll probably need some lower concentrations of your target protein--you might want to consider a 3x or 4x dilution series for the initial experiments, so you can get down to low ng/mL concentrations.

Best Regards,
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Old 12-02-2009, 05:46 PM
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Default Re: Sandwich ELISA Optimization HELP!?

1st and foremost, clear up the buffer system. If you want to use AP, switch to TBS. You can bind capture Ab in PBS or carbonate, but after that only use TBS, so when it is detection time all the PBS is completely gone.

2nd get rid of BSA in the Antibody coating step.

3rd plate selection, use high quality polystyrene plates, they are good for binding.

4th for capture Ab you can need as little as 1ug/ml to as much as 10ug/ml, make sure your dilution series is in this range.

5th run a blocking assay to test the biotin-strep-AP system on wells that were blocked with different concentrations of your blocking agents, run different incubation times and temps to find the most effective.
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Old 12-05-2009, 06:28 AM
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Default Re: Sandwich ELISA Optimization HELP!?

*Instead of washing after the blocking step try aspirating and letting the plate dry for at least an hour. Sometimes stabilizing the plate with a 5% sucrose solution helps too.
*Definitely don't put BSA in your coating buffer...ever. Is it possible for you to try a monoclonal capture? This tends to work better as capture in sandwich ELISAs.
*You can try putting BSA in your assay/sample diluent.
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