MOLM-1 cells were stained with one of the unconjugated CD164-blocking MoAbs or with the appropriate isotype-matched negative control MoAb before labeling with the second test CD164 MoAb, which differed from the blocking MoAb. After staining these cells with PE-conjugated anti-Ig that specifically reacted with the CD164 test MoAb, they were analyzed on the FACSCalibur and their median fluorescence intensities determined. Percent blocking was calculated from median fluorescence intensities as indicated in Materials and Methods.

Soluble domain deletion constructs, derived from exons 1; 1 and 2; and 1, 2, and 3 were prepared after determination of the intron-exon structure of the CD164 gene,42 subcloned into the pEFBos-Ig mu vector,43 and expressed in 293T cells as soluble recombinant proteins as detailed elsewhere.45 The predicted peptide sequences for these soluble constructs are: Exon 1: DKNTTQHPNVTTLAPISNVTSAPVTSLPLVTTPAP; Exon 2: ETCEGRNSCVSCFNVSVVNTTCFWIECK; Exon 3: DESYCSHNSTVSDCQVGNTTDFCS with exon 1 predicted to contain nine O-linked glycosylation sites on the serine and threonine residues underlined. Potential N-linked glycosylation sites are indicated in bold lettering. The data represent a summary of the results of CD164 MoAb binding to these soluble recombinant proteins as determined by ELISA assays.45