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. I’ll describe the system below. If anyone could posit a potential problem I’d 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.
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.
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.
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!?!?