Trouble with Elisa calibration curve - ??? (Mar/14/2007 )
I 've made Elisa to determine HIV virus titer in cultured lympocytes ( antigen: inner membrane core protein HIV p24) . when i did calibration with recomb p24 ( e-coli produced), in 20% FBS i got a good calibration curve. But when I tried to do it with 20% human serum I did'nt get calibration. The absorbance was observed - values as in the middle of calibration curve with FBS ( near 0.5 abs). The scheme of ELISA : plate sorbtion first mAB ( mouse source) then antigen then second biotinylated mAB ( goat origin) . How can this fact may be explain?
Did you get any absorption with the human sera? I have experienced much higher background when working with human serum.
yes, hi enough ( 0,34). Well so I need increase time and number of wash steps . What is about optimal composition of wash buffer for 20% human serum
You could play with your Tween percentage. I used to use a PBS based buffer with 1%BSA and 0.05% Tween. Going higher on the Tween percentage could keep the serum proteins from sticking, but I don't remember if it made that big of a difference. Could you use 5-10% human serum? Or is that cheating? Have you tried increasing your coat antibody concentration or your blocking %?
Yes I've used PBST 0.05% WITH 1% BSA in exp. Really we use 20% HS to cultivate lymphocytes ( but I think we can decrease to 10% )
About coating - I will experience this point. And question about blocking sol: may be change it to BSA 5% or casein or gelatin or another one?
Thank you for reply!
I have only used up to 2% BSA, so I do not know if going higher or using a different blocker could help. I bet you that 10% human serum will help a lot. I have seen in some cases that increasing the coat concentration up to 25ug/ml can help reduce background.