alpha-fetoprotein ELISA problem - (Jan/24/2007 )

Hi eveyone, I have a problem with my alpha-fetoprotein ELISA. I am only trying out the standard curve. Yet, my blank and standard all colorized. I purchased my antibodies from abcam and used bicarbonate buffer for coating and 5% BSA for blocking. I still have no idea how to solve my problem. My antibody dilution and procedures as attached. Thank you for your reply!!
1. Alpha 1 fetoprotein antibody (ab3980) – 1mg/ml
stock (1mg/ml  2g/ml)
2 l original antibody + 998 l coating buffer
Working solution (2g/ml  0.2g/ml)
80 l antibody stock (2g/ml) + 720l coating buffer
2. alpha 1 fetoprotein antibody (ab3969) – 1.00mg/ml
Stock concentration (1mg/ml  4g/ml)
4l original antibody + 996 l blocking buffer
Working solution (4g/ml  0.4g/ml)
100 l antibody stock (2g/ml) + 900l blocking buffer
3. secondary antibody – sheep anti-mouse IgG antibody (ab6808)
Stock concentration (2mg/ml  5g/ml)
2.5l original antibody + 996 l blocking buffer
Working solution (5g/ml  0.5g/ml)
100 l antibody stock (2g/ml) + 900l blocking buffer
4. Antigen (ab38189) – 0.10mg/ml
Stock ( 0.1mg/ml  0.5g/ml)
5l original antigen + 995 l blocking buffer
Working solution (0.5g/ml  400ng/ml)
100 l antigen stock (0.5g/ml) + 700l blocking buffer

1. Coating: dilute Alpha 1 fetoprotein antibody (ab3980) from antibody stock solution. 80 l antibody stock (2g/ml) + 720l coating buffer. Add 100l diluted final working antibody solution (0.2g/ml) to each well.
2. incubate overnight, 4oC.
3. PBS wash 2 times, 200l/well
4. Blocking: add 200l 5% BSA blocking buffer/ well
5. incubate at room temperature, 4 hours
6. PBS wash 2 times, 200l/well
7. add properly diluted sample, 100l/well
8. incubate overnight, 4oC
9. PBS wash 3 times
10. dilute Alpha 1 fetoprotein antibody (ab3969) from antibody stock solution. 100 l antibody stock (4g/ml) + 900l blocking buffer. Add 100l diluted final working antibody solution (0.4g/ml) to each well.
11. incubate at room temperature for 2hrs
12. PBS wash 3 times
13. dilute secondary antibody (ab6808) from antibody stock solution. 100l antibody stock (5g/ml) + 900l blocking buffer. Add 100l diluted final working antibody (0.5g/ml) solution to each well.
14. incubate, room temperature, 2 hrs
15. 2.2l SA-HRP + 1.1ml blocking buffer, 100l/ well
16. incubate, room temperature 30min
17. ABT, 100l/well
18. incubate, room temperature 15mins
19. OD reading at = 405nm

1. your antibody coating concentration is too low (I think). I always stayed between 5-25 ug/ml.
2. I also think your secondary concentration is too high. I use about 0.1 ug/ml.
3. dumb question, but what do you use as your blank? blocking solution, right?
4. also dumb question, but is your secondary biotinylated? what species?

5. all those things aside--the reason for your background (and that's what it is) is that your coat antibody is the same species as your probe antibody. You need to either have, e.g., a rabbit coat and a mouse probe or vice versa.

You tried to get around that by having a biotinylated secondary (I think that was your motivation), but it needs to be conjugated to your probe antibody because otherwise, no matter how well you block the plate, my experience is that your secondary is finding the coat antibody somehow.

So, either vary your coat and probe antibody species from each other, or use a probe-biotin conjugate. If you use that, then you shorten your protocol by one step because you won't have to use the secondary!