Anti-Mal binds AnWj-positive red cells, but not AnWj-negative cells. (A) Red cells sequentially incubated with either mouse polyclonal anti-Mal (ab167376, Abcam; 1 in 10) or mouse monoclonal anti-AnWj (H86; 1 in 5) followed by goat anti-mouse immunoglobulin G show agglutination in AnWj-positive red cells (top row) but not in AnWj-negative red cells (lower 3 rows). Two representative examples of inherited AnWj-negative phenotype (P1 and P2_F1), both carrying homozygous MAL deletions, and 1 example of suspected AnWj antigen suppression, without MAL mutation (P5) cells, are shown. Negative (secondary antibody only) and positive (anti-GPA; BRIC256) controls were included (data not shown). Scale bars are 40 μm. Cells were imaged using a Leica DM750 microscope (Leica Microsystems) at 100× magnification and imaged using a Pixera Penguin 600CL camera (Digital Imaging Systems). (B) Flow cytometry with anti-Mal (ab167376) and anti-AnWj (H86) on control RBC (top row) and 4 representative examples of AnWj-negative cells, 2 with MAL deletion (P1 and P4), and 2 lacking the MAL deletion (P5 and P6). No detectable expression of Mal or AnWj is observed in any AnWj-negative samples. (C) Red cell membranes prepared from 1 AnWj-positive control and 3 AnWj-negative samples (P1, inherited; P5 and P6, suppression) were immunoblotted using mouse monoclonal anti-Mal antibody E1 (sc-390687; Santa Cruz). A band consistent with full-size Mal (15 kDa) was present in the control sample and absent in all AnWj-negative samples tested. The anti-protein 4.2 (BRIC273) control demonstrates consistent protein loading. A further 2 AnWj-negative samples showed the same results (supplemental Figure 1A). Multiple commercially available anti-Mal antibodies were tested (supplemental Table 3) but only 1 worked in our hands by red cell serology/flow cytometry (ab167376) and 2, E1 (shown here and supplemental Figure 1A) and 6D947 (supplemental Figure 1A-B), by immunoblotting.
