Ability of MB2A4 to detect rituximab in sera and on the surface of cells. (A) MB2A4 was coated to the surface of ELISA plates before blocking nonspecific binding sites and adding various dilutions (1:1-1:10 000) of either pure serum (♦), or serum spiked with 100 μg/mL rituximab (▪), chAT13/5 (X), or chAT80 (□). The amount of human Fc captured to the plate was then detected using anti–human Fc coupled to HRP. (B) The amount of rituximab present in a patients' serum was detected over a period of time during which they had received rituximab. After collection of serum, a number of dilutions of the sample were made and then measured in an ELISA, such as detailed in panel A, along with a standard curve of known rituximab concentrations. Subsequently, reference to the standard curve allowed calculation of the serum rituximab level in the original sample. ▪ indicates when a 375 mg/m² dose of rituximab was given. Error bars represent the standard error of the mean for duplicate points. MFI indicates mean fluorescence intensity. (C) Following binding of various mAbs (10 μg/mL) to EHRB (□) or Raji cells (▪) for 15 minutes at RT, cells were washed twice in phosphate-buffered saline/bovine serum albumin/Azide before staining with FITC-labeled mAb MB2A4 (10 μg/mL) for 15 minutes at RT, washing, and detection by flow cytometry. (D) Rituximab (Ritux), 2B8, or 7D8 (10 μg/mL) was added to whole blood for 15 minutes at RT along with CD19-phycoerythrin before washing and staining with FITC-labeled mAb MB2A4 as in panel C.