does 2nd antibody concentration matters? - (Mar/07/2007 )
hi~~
I have been trying to coating my own ELISA for alfa-fetoprotein. if i use 1microg/ml (coating antibody), 1 microg/ml (mono anti poly ab) and 0.1 microg/ml (secondary ab), the resultant standard curve looks like a saturation curve. therefore, my boss decided to change the concentration of all the antibody to 2/4 microg/ml, 1/2micro/ml and 1/2 microg/ml respectively. However, the result was horrible as the blank chromized and all well looks like same color. As I have been posted my protocol on the forum before and got a suggestion of 0.1microg/ml for secondary antibody. I am wondering if the secondary antibody concentration that contribute such result?
my antigen concentration ranges from 3.125ng/ml-200ng/ml. my protocol as follows: Thank you for the helps!!
1. Coating: dilute rabbit polyclonal to alpha 1 fetoprotein (ab8201) to a final concentration of 2 and 4 g/ml respectively. Add 100l to each well
2. incubate 2hr, Room temperature.
3. PBS wash 2 times, 200l/well
4. Blocking: add 200l 5% BSA blocking buffer/ well
5. incubate at room temperature, 6 hours
6. PBS wash 4 times, 200l/well
7. add properly diluted sample (alpha 1 fetoprotein, ab38189), 100l/well ( 200ng/ml, 100ng/ml, 50ng/ml, 25ng/ml, 12.5ng/ml, 6.25ng/ml, 3.125ng/ml and 5% BSA as blank)
8. incubate at room temperature, 2hrs
9. PBS wash 6 times, 200l/well
10. dilute mouse monoclone to alpha 1 fetoprotein (ab3969) to a final concentration of 1 and 2g/ml respectively. Add 100l to each well
11. incubate at room temperature, 2hrs
12. PBS wash 8 times, 200l/well
13. dilute secondary antibody (sheep polyclonal to mouse IgG H&L (HRP), ab6808) to a final concentration of 0.1, 1 and 2g/ml respectively. Add 100l to each well
14. incubate, room temperature, 2 hrs
15. PBS wash 5 times, 200l/well
16. ABT, 100l/well
17. incubate, room temperature 15mins
18. OD reading at = 405nm
I have been trying to coating my own ELISA for alfa-fetoprotein. if i use 1microg/ml (coating antibody), 1 microg/ml (mono anti poly ab) and 0.1 microg/ml (secondary ab), the resultant standard curve looks like a saturation curve. therefore, my boss decided to change the concentration of all the antibody to 2/4 microg/ml, 1/2micro/ml and 1/2 microg/ml respectively. However, the result was horrible as the blank chromized and all well looks like same color. As I have been posted my protocol on the forum before and got a suggestion of 0.1microg/ml for secondary antibody. I am wondering if the secondary antibody concentration that contribute such result?
my antigen concentration ranges from 3.125ng/ml-200ng/ml. my protocol as follows: Thank you for the helps!!
1. Coating: dilute rabbit polyclonal to alpha 1 fetoprotein (ab8201) to a final concentration of 2 and 4 g/ml respectively. Add 100l to each well
2. incubate 2hr, Room temperature.
3. PBS wash 2 times, 200l/well
4. Blocking: add 200l 5% BSA blocking buffer/ well
5. incubate at room temperature, 6 hours
6. PBS wash 4 times, 200l/well
7. add properly diluted sample (alpha 1 fetoprotein, ab38189), 100l/well ( 200ng/ml, 100ng/ml, 50ng/ml, 25ng/ml, 12.5ng/ml, 6.25ng/ml, 3.125ng/ml and 5% BSA as blank)
8. incubate at room temperature, 2hrs
9. PBS wash 6 times, 200l/well
10. dilute mouse monoclone to alpha 1 fetoprotein (ab3969) to a final concentration of 1 and 2g/ml respectively. Add 100l to each well
11. incubate at room temperature, 2hrs
12. PBS wash 8 times, 200l/well
13. dilute secondary antibody (sheep polyclonal to mouse IgG H&L (HRP), ab6808) to a final concentration of 0.1, 1 and 2g/ml respectively. Add 100l to each well
14. incubate, room temperature, 2 hrs
15. PBS wash 5 times, 200l/well
16. ABT, 100l/well
17. incubate, room temperature 15mins
18. OD reading at = 405nm
Total colouring of all wells has one of a few possible causes; incomplete blocking (but your protocol seems to be OK for that), or secondary Ab left in the wells. The most embarrassing one is if the secondary antibody isn't totally washed out (but if that happens, it's possibly due to a platewasher error).
You are totally correct in thinking that too much secondary Ab can cause excess colour development. If you run an assay using the three different Ab concentrations, when does the over-development of colour start?
To start, keep your secondary ab concentration below 0.1ug/ml. Also, cut your primary and secondary incubation times in half. I have had good linearity success with cutting the secondary antibody incubation time to 20-30 min, but no longer than 1 hr at RT.
Also, it will help if you incubate your standards overnight at 4C.
And do you use the 5% BSA buffer that you have in your blank to dilute all of your samples?
Also, it will help if you incubate your standards overnight at 4C.
And do you use the 5% BSA buffer that you have in your blank to dilute all of your samples?
thank you for your help!!
Yes, I use the 5% BSA buffer to dilute all my sample and prepare all the antibody (except the coating antibody).
I'll try your suggestions and hopefully everything workout!!!