Shreffler:Notebook/Alex Daily/2011/05/25

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Buhlmann Assay pilot 2

Ran Buhlmann assay using Tom Kadlik buffy from 5/25/11. Used five dilutions each of Milk and Peanut allergen (1.00, 0.20, 0.040, 0.0080, 0.0016 µg/mL)

Followed Buhlmann protocol with following differences:

  • Used buffy coat rather than whole blood, diluted with RPMI to 60% of original constitution
  • Incubation step in incubator rather than water bath
  • Included anti-IgE Ab positive control


Clumping was reduced, possibly due to final resuspension in provided wash buffer/rather than our own staining buffer. Also, flow acquisition was done first day (most samples). Acquisition for peanut samples were done next day, with reduced basophil event rate. Basophils were very weakly responding to anti-FCeRI Ab and anti-IgE Ab. Unsensitized to neither milk nor peanut allergen.