I 'm performing a lot of indirect ELISA assays to assess IgG in human blood on DBS. In general, I have to use 0.1% Tween 20 in PBS buffer to elute my DBS. This is going great. But I recently received DBS made ​​with the same paper (Whatman 3M), on which drop of blood does not elute although I used the same protocol (even incubated for more than 48 hours).

However, it's important to tell that this DBS were stored for a long time at room temperature, but just under the air conditioner (instead of at 4°C as usually).

Please, could anyone with experience on elution of DBS give me any ideas?