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Mouse IgG detection - Blocking ?

ELISA control

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2 replies to this topic

#1 @bhijit

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Posted 24 October 2012 - 06:25 PM

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 [before vaccination] 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

#2 @bhijit

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Posted 24 October 2012 - 06:44 PM

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 ?

#3 hj_chris

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Posted 12 November 2012 - 10:40 PM

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





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