What can I do with blood sample from immunized rabbit? - we want to know whether there's antibody or not. (Oct/20/2006 )

Hi,

I am making a polyclonal antibody. We send protein sample to immunize rabbit, and soon, the first blood smaple will be sent to us. It is good, but what can I do with this sample?

I want to know whether this antibody is produced or not.. Do I have to spin down the blood first? how much blood do I need for one test?...

Thanks.

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for a first test, you need a little drop of blood.
Let it coagulate, and centrifuge, and take the serum.

now you can test by direct ELISA.
coat your protein (50-100 ng per well) in PBS, 2-3 hours à 37°C
saturate with BSA 1% 1 hour at room temperature.
wash three times with PBS + 0.05% tween-20
incubate with your serum diluted 50 times, and do also sequential dilution twice , so you will test 1:50 ; 1:100 ; 1:200 ; 1:400 ; 1:800 ; 1:1600 ; 1:3200 diliutions. 2 hours at RT
wash three times
incubate with secondary antibody, like goat anti rabbit coupled to HRP or AP, 2 hours at RT
wash three times
incubate with the appropriate substrate, like PNPP if you used AP, until the color appears and read in a spectrophotometer.
The aim is to see how much you can dilute your serum until you still see a signal.
this last point will give you the titrate of your serum.
let's say you see a signal at 1:400, but no more at 1:800 : your serum titrate 1:400

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to prepare serum is right; if you will not affinity purify your Ab you will routinely use serum;

the question is if Ab is directed against denaturized or native protein; ELISA is a good idea by missele for native protein (more faster is dot blot) but may fail if Ab is directed against denaturized protein; here use dot blot after SDS-sample buffer treatment or Westernblot to show specificity;

important is to optimize pH of buffer; try which is better, PBS and TBS

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Thank you for the suggestions. Getting clear now.

Missele, I think we have ELISA in the lab... however, I don't have purified protein.... I only do transient transfection and WB.. Can I use total lysate as target for my antibody? ....
Kodmodrom. Maybe I will purify my antibody afterwards. but now I will use serum and try denatured dot blot.

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to test your Ab you can take lysate or enriched fractions of subcellular compartments where you assume your protein of interest;

BUT lysate is not to recommend for ELISA or dot blot as in case of positive signals you will not be able to decide if these are specific or unspecific signals;

you CAN take ELISA or dot blot for lysates if you have enough of your immunogenic/antigenic protein or peptide; in a control, you pre-incubate your Ab with the antigen and then apply the pre-incubated in parallel to the not pre-incubated Ab to ELISA or dot blot;

in the case you have not enough of your antigen, use Western and qualify the intensity and specificity of your immunosignal

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True. I didn't think about the unspecific binding..

Then western first.. Thanks, Kosmodrom. Do you have suggestions about the dilution ratio of the serum (when I incubate with blot), or a range people normally do?

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