Indirect ELISA false positives? - (Mar/06/2008 )
Hi All,
I recently started doing indirect ELISAs for my lab (we suddenly lost our biochemist.. boo politics) and have come across a very confusing result.
We are studying collagen I production of human bladder smooth muscle cells by adding TGFB1 to cell cultures. In a recent experiment, we also added TGF Antibody to neutralize the TGFB. When I tried to perform the ELISA, I got a false positive of collagen I for the samples that contained the TGF antibody. I'm pretty sure the antibody is not bad bc realtime PCR showed it neutralized the TGFB effectively. This also happened when we did an experiment using CTGF antibody.
I don't really understand why this is happening, can someone help me out? Could the antibodies added in cell culture be binding to the Collagen? I feel like that's unlikely. How about binding to the primary antibody when they are incubating O/N?
I can write out the process:
1. Coat plate with 1:1000 collagen I antigen in carbonate buffer (incubate O/N at 4C)
2. Standard curve of serial dilutions of collagen I antigen in PBS/0.05% T20
3. add sample to tube with PBST20
4. 1:5000 monoclonal Col I antibody in PBS to all tubes, incubate at 4C O/N
5. Add all samples to plate, incubate 1 hr in 4C
6. Wash plate in PBST20 x2
7. Add biotinylated 2ndary antibody (1:2000) to all wells, incubate at RT for 1hr on shaker
8. Add OPD substrate for 20 min.
I've been scouring papers for the entire day and just have more questions than answers. Help! 
to get your answer you should have two controls :
supernatants of cells treated only with anti-TGF
medium with anti-TGF (only the medium, not a supernatant of cells)
You know what, I just realized the anti-TGFb is a chicken IgY... I bet that has something to do with it, sneaky chickens... Gonna try to find a column (i guess protein L?) to run it through or maybe someone has some IgY purification system...
We'll see what happens; if this works, it would be eggggsellent.
We'll see what happens; if this works, it would be eggggsellent.
hey are you sure to use PBS-T... twin in coating step too.....!! i coat my Ag in carbonate buffer, and i guess after adding MAb you should incubate in 37*c for 1 or 2 hours unless ur coated protein does not degrade at that temperature?.... i do it for most of my ELISA.
If you uncertain about ur Ab u can do direct ELISA (coating Ab) and check out what's happening.