Hello,

here some suggestions:

"For now, I wasn't able to detect anything but (probably) artifacts. " To check this, I would use the secondary antibody without incubation with the first one. On the ohter hand, patient sera might include antibodies recognizing more than one protein or you have some degradated proteins in your final cell lysate (here use additional protease inhibitors and work on ice properly ) ...

"should I try out different serum / 2nd antibody dilutions?" Yes, the antibody concentration might depend on the patient and the last exposure time point to the mould so the titer is too low for detection or the serum dilution is too high..

If you are interested in a special protein try purified proteins to test for antibody binding of the patient sera...

"is it possible that there are no longer any IgG against mold present in the blood?" this depends on the last exposure time of the patient to the mould... (see above) may be you could use an secondary antibody which recognizes IgG (H+L) heavy and light chain... This might also detect other Ig´s not only IgG...

Maybe you should reduce the NFDM to 1 or 3% for the serum or the blot of the proteins is not sufficient or too long...

Good luck

Fungal antigens and allergens can be difficult to extract and detect, but in order to trouble shoot your Western you will need a mold positive human sera.  You do not say what size comb you are using for your gel, but 15 ug of total protein in your extract is probably too little depending on the quality of your extract. You will also need to optimize the dilution of your secondary antibody, for that you will need a positive patient sera.