Analysis of anti-D
Anti-D quantification was performed using flow cytometry. To increase
sensitivity erythrocytes were pretreated with the enzyme papain. All
dilutions were made in ID-CellStab (Bio-Rad Laboratories Inc) with 0.5%
BSA (A7906, Sigma-Aldrich) as additive. The calibration standard was the
2nd BRITISH STANDARD 1992 Anti-D (Rho) Antibodies (code: 73/515, NIBSC,
Potters Bar, UK). In short, 50 µL 0.8% erythrocytes (R0r) and 100 µL
standard or sample was incubated for 30 min at 37°C. After washing with
3 x 200 µL PBS, 50 µL mouse anti-human IgG-PE (Southern Biotech) diluted
100-fold was added and incubated for 30 min at RT. After an additional
wash as above, the erythrocytes were dispersed in 250 µl PBS and
analysed on Cytomics FC500 MPL. The mean fluorescense intensity (MFI) on
10,000 gated cells were measured and imported to SoftMaxPro (Molecular
Devices) which was used for constructing the calibration curve and
calculation of anti-D concentration. The LOD was 1 IU/L (0.2
µg/L).