For this type of study, I'd recommend an Indirect ELISA, instead of a western blot. One would simply coat the wells of a 96 well plate with a protein lysate of toxoplasma gondii (or a specific antigen), then probe with serial dilutions of the mouse sera, wash, then detect the presence of mouse antibody in the wells. Your negative control would be the sera of a mouse that had not been exposed to toxoplasma, serially diluted in the same way to your sample mice sera.

I believe you are right to be concerned about the total concentration of antibody in your sera. You would probably need to do multiple western blots with different concentrations of sera to acheive optimal results, whereas the indirect ELISA tests all dilutions on the same plate.

For this type of study, I'd recommend an Indirect ELISA, instead of a western blot. One would simply coat the wells of a 96 well plate with a protein lysate of toxoplasma gondii (or a specific antigen), then probe with serial dilutions of the mouse sera, wash, then detect the presence of mouse antibody in the wells. Your negative control would be the sera of a mouse that had not been exposed to toxoplasma, serially diluted in the same way to your sample mice sera.

I believe you are right to be concerned about the total concentration of antibody in your sera. You would probably need to do multiple western blots with different concentrations of sera to acheive optimal results, whereas the indirect ELISA tests all dilutions on the same plate.

I agree with Christopher, however you can also try western blot provided you run toxoplasma gondi whole cell lysate or crude antigen preps(you can load measured concentration of protein on the (SDS PAGE gel) and then use positive (mouse sera exposed to t.gondi) and negative sera (unexposed mouse sera) and test samples(use different concentrations of sera).it works really well.

I have tried to use an indirect ELISA in the past but struggled to get it to work. Since this work is for an undergraduate dissertation and I'm bound by time, I found it would be better for me to try a different tactic and I have had some good results with a Western so far. I just wondered how I would go about using the actual serum as a probe, I have already got the Western working.

You can use serum without any problems. It's not necessary to know the serum protein concentration (in mice it's between 70 - 90 g/L. The important concentration is the one of the specific antibody. This is a very small part of the IgG fraction (normal concentration in mice is at 10 g/L). Therefore a titration is helpful. Normally a dilution of 1:50 - 1:5000 (in immune sera) is working fine. You should use only the same dilution for both, positive and negative (control) samples.

I have no idea about the presence of Toxoplasma in normal mice. If you need a serum which comes from a defined negative murin population. SPF animals are fine for this approach. Here is the IgG level mostly much lower (2 - 5 g/L). Thats can be taken into account.

Controls when using serum are a common problem. Your best control would be serum from the same animal prior to exposure to the antigen, but that is not always feasible. You can buy pooled mouse sera- the laboratory mice should be free of Toxoplasma. The use of pooled sera gets away from some of the problems of individual variation. Make sure to use mouse serum as a control, some people will try to use purified mouse IgG, but this is only a good control if you are purifying the immunoglobulin fraction out of your sera. As Stephan said, you would use the same dilution of the negative serum as the positive serum, or the same range of dilutions.

I think you can dilute your serum between 1:100 and 1:1000, but it would depend as well of your conjugate dilution. Maybe with serum at 1:500 and a dilution of 1:10000 for the conjugate it could work. If not, you can adjust modifying any of these dilutions.

I agree that the most likely is that you make an indirect ELISA, where you must cover plate wells with a lysate of the parasite, and then determine the presence of anti-Toxoplasma, adding serial dilutions of immune serum.

The negative control should be a serum that was not in contact with the parasite.

You can also perform the Western blot, electrophoretically separated total extract and then transferring it to parsito paper, make strips and each strip test a serum dilution.

I send an article where he made this type of test, oajala serve you.

Article Specific antibody immune response against the parasitic port...

You can do the western blotting using serum, but the serum dillution factor depons on the WB kit sensitivity. For example with the high sensitive chmiluminescence Amersham kit, you may dilute the serum by a factor of about 200000 times. You also need to optimize the secondary antibody as well as the first one (serum). An alternative to serum is the IgG fraction. You can precipitate the IgG fraction of both control serum and the positive srum, dialyze it in PBS 1N at 4C, and normalize the two IgG fraction protein concentration by doing protein assay and use them instead of serum in WB assay.

I have been using serum in WB for a long time. I just do an ELISA test before use that in WB just to confirme the negative sorology for toxoplasmosis. After that I do a pradronization in WB with differential dilution of this serum sample to choose the better dilution for the test.

Hi Bianca,

I am planning to set up a western blot using serum at 1:100 dilution and antihuman IgG at 1:5000 dilution. i am using purified protein. can you please send me the protocol you are following in your lab?