Can bacteria lysate be used to test a serum ab titer by ELISA? - Need suggestion (Jul/26/2005 )
I used a kind of bacteria to immune mice and found there is a specific antibody produced in mouse serum. I confirmed that this antibody is specific to a certain protein of the bacteria. Now I am wondering if I can use the whole bacteria protein lysate as the antigen to test the titer of the specific antibody by ELISA. I know it will be easier and give better result if I have the purified antigen for the ELISA. But I don't think that I will get the purified antigen very soon.
Also if you think I can use the whole bacteria protein, could you address anything I should pay attention to? Since I've never done an ELISA before, I will appreciate it if you would recommend a protocol to me.
Thanks.
yes, you can use bacterial lysate for ELISA... You should thing of controls, such as pre-immune serum and/or a negative controll serum....
we do antibody titre measurement by limited dillution endpiont ELISA assay:
1) coat 100-300ng of antigen in each well of a medium binding capacity 96 well ELISA plate (we use Greiner's F-Bottom and 0.2 carbonate/hydrocarbonate buffer at pH 9.5, 100µl /well overnight at 4°C)
3)wash and block your plate (we wash three times with >200µl PBS, 0.05%Tween 20 well, and block with 200µl PBS, 3%FCS, 2%Tween 20 for 1 h at 37°C)
4) dillute your sera (important here to have a negative serum like from PBS only immunised mice or, better, a pre-immunisation serum of the immunised animal) 1:50 in approprate buffer (we use blocking buffer) and make serial dillutions of 1:2 from there on for all sera (in the 16th dillution you will be at 1:1.6 million, which is quite good for my sera, at least)
5) wash your plate again (we do 3 times)
6) add 100µl/well of your dilluted sera (we incubate for 2h at 37°C)
7) wash plate (yes, three times again)
8) add appropriate secondary antibody in appropriate dillution (we use BioRads goat-anti mouse HRP 1:3000 in PBS, 3%FCS, 2%Tween 20), incubate (we do 1h at 37°C)
9) wash (3x)
10) add substrate for enzyme (TMB 100µl/well, incubated for roughly 10-15 minutes in the dark, stopped then by addition of 50µl 2N H2SO4)
11) readout your OD values (at our case 450nm)
put the ODs of the negative control(s) next to the ODs of your positive serum, and compare your ODs from the highest dillution on "downwards" (1:1.6E6 to 1:50). The lowest dillution that has a 3 times OD than the respective dillution of the negatice control serum is considered positive and yield yout titre (eg. 1:800.000).
if you have further questions, just ask
mike
we do antibody titre measurement by limited dillution endpiont ELISA assay:
1) coat 100-300ng of antigen in each well of a medium binding capacity 96 well ELISA plate (we use Greiner's F-Bottom and 0.2 carbonate/hydrocarbonate buffer at pH 9.5, 100µl /well overnight at 4°C)
3)wash and block your plate (we wash three times with >200µl PBS, 0.05%Tween 20 well, and block with 200µl PBS, 3%FCS, 2%Tween 20 for 1 h at 37°C)
4) dillute your sera (important here to have a negative serum like from PBS only immunised mice or, better, a pre-immunisation serum of the immunised animal) 1:50 in approprate buffer (we use blocking buffer) and make serial dillutions of 1:2 from there on for all sera (in the 16th dillution you will be at 1:1.6 million, which is quite good for my sera, at least)
5) wash your plate again (we do 3 times)
6) add 100µl/well of your dilluted sera (we incubate for 2h at 37°C)
7) wash plate (yes, three times again)
8) add appropriate secondary antibody in appropriate dillution (we use BioRads goat-anti mouse HRP 1:3000 in PBS, 3%FCS, 2%Tween 20), incubate (we do 1h at 37°C)
9) wash (3x)
10) add substrate for enzyme (TMB 100µl/well, incubated for roughly 10-15 minutes in the dark, stopped then by addition of 50µl 2N H2SO4)
11) readout your OD values (at our case 450nm)
put the ODs of the negative control(s) next to the ODs of your positive serum, and compare your ODs from the highest dillution on "downwards" (1:1.6E6 to 1:50). The lowest dillution that has a 3 times OD than the respective dillution of the negatice control serum is considered positive and yield yout titre (eg. 1:800.000).
if you have further questions, just ask
mike
For the first coating step, do you need to use capturing antibody to capture the antigen? Or the F-Bottom plate you mentioned doesn't need these capture step? What is 0.2 carbonate/hydrocarbonate buffer? Do you have the recipe for it?
Thank you very much for your information.
Ok, 1st, you don't need no capture AB, since you coat the antigen (lysate) directly to the plate....
2nd, 0.2 carbonate/hydrocarbonate should read 0.2M carbonate/hydrocarbonate, meaning 0.1M Na2CO2 mixed with 0.1M NaHCO3 so that the resulting pH is 9.5 ...sorry for the missing "M"!
mike