Mouse IgG detection - Blocking ? - (Oct/24/2012 )

Hi everyone,

I am doing elisa to measure the mouse IgG.
I use mouse sera 1:25 dilution; second antibody at 1:2000 dilution; blocking is with 5% BSA 37 deg for 2 hr. Detection is with TMB substrate

The problem is - I am never able to get negative control samples values below 0.2. I tried different dilutions of sera and secondary antibody but If i use more dilutions - the positive sera readings also drop.

I tried FBS, Skim powder and BSA - all give similar readings -

I also tried sera from - new mice - 0 day - but still same thing happens

I am wondering If I should change the seconday Ab ? Is it possible that BSA still allows my current secondary Ab to diffuse througn ?

Please help me on this Thank you for your time

To add - even control mouse will non-specific Ab in blood - How can I minimize the reaction with these antibodies. Another thing is TMB substrate is highly sensitive - will changing the substrate have any effect ?

you can try serial dilution. I used one sample as standard and did serial dilution from X5000 to X32000