Anti-human IgG phosphatase give negative results - (Mar/06/2012 )
Hi everyone, i am in great trouble since last week . My secondary antibody that is Goat Antihuman IgG alkaline phosphatse labbled gave me always positive results even without antigen coating or use og primary human IgG , i tried many times and got same results . My trainer said to me its your handling mistake but i dont think so , can any body have some idea whats wrong ? Please help me
You better give us more information that what you sent. A numerical step wise procedure as to what you are doing and how the assay initially worked. Then, we can point you in right direction or give you more specific questions.
. I use the 1ug/ml (antihuman IgG in PBS) to coat the plates; 3%BSA in 0.1 % PBSt as a blocking buffer, 0.1% Tween 20 PBS as a wash solution, 2nd ab for human IgG is peroxidase labeled goat anti-human IgG. (1x1000 in PBS)
I always got the same result for the coated wells and non coated wells, high read. Even some time only 2nd ab gave signals in non coated wells, without adding primary ab that is IgG??
I also tried with 1% BSA but got same results ? I dont know where is the problem , is that because of my poor washing or technique???
Sorry for my poor English please help me out from this situation
3%BSA in 0.1 % PBSt; if this contains tween(t) take it out, block with BSA/PBS overnight.
Are you vigorously washing 4-5X with blotting between steps allowing the wash solution to remain in the wells for 1 min before decanting and blotting?
Your antihuman conjugate could be too concentrated so you can dilute it to maximize the delta between negative and positive.
A quick check to see if any of your reagents are contaminated is to put a small volume in tubes and add substrate...if you get color your reagent is contaminated.
Thankyou very much
i will try and then upload my results for you??
what about the contamination of antibodies , how can i check ?? with the same process as you mentioned
wash solution, antibodies, etc can be checked for enzyme contamination just add small volume to tubes...then substrate...if you get color they are contaminated with your conjuagate. I suspect your plates are not blocked because of surfactant, the concentration of the conjugate may be too high, or the wash steps are not performed properly.
i tried with more vigrous washing 4 to 5 times , my negative control looks good but i used my cell medium as my samples, they are still giving high background ??
what should i do next increase blocking time ???
Make sure you block the plates with bsa buffer that does not have surfactant. You can block overnight at 4C without any worry.
Check your conjugate concentration. vary the dilutions from 1:10 to 1:1000 and run with an abbreviated dose response curve (human IgG that you purchase from Sigma or other source...probably 3 positive levels in the range you believe your samples will be in and dilute this in fresh media to match your sample matrix).
You will be varying 2 things: concentration of human IgG against concentration of conjugate. Select the best concentration of conjugate that provides the best discrimination.
Check your second detection Ab if it has cross reaction with BSA and block with casein or gelatin (1%) 2h 37oC. Make a gradient of conjugate Ab concentration.
alkaline phosphatse is inhibited from phoshate of PBS. Use tris