The HCDR3 sequences of CLL69 homologs isolated from the MAA-BSA panned umbilical cord Fab phage display library.

<p>Sequence analysis of the IGHV1-69 homologous segment in six PCR clones isolated from the MAA-BSA panned umbilical cord blood Fab library. Determination of IGHV, IGHD and IGHJ usage and nucleotide sequence of HCDR3 in IGHV1-69.</p

Binding pattern of CLL69C and CLL69A Ig heavy chains paired with non-native light chains to OSE.

<p>CLL69C or CLL69A rAb Ig heavy chains were paired with Ig light chains derived from other CLL69 subsets. Chemiluminescent immunoassays were used to determine the binding of each of the CLL69 rAbs (2 µg/ml) to the indicated plated antigens (5 µg/mL) for 1.5 hour at RT. Data are expressed as RLU/100 msec and are from three independent experiments (mean ± SD), with each value determined in triplicate.</p

MAA-peptide mimotopes are recognized by CLL69C rAb.

<p>(<b>A</b>) Amino acid compositions of MAA-mimotopes P1 and P2 as recently reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065203#pone.0065203-Amir1" target="_blank">[20]</a>. (<b>B</b>) Chemiluminescent ELISA for the binding of increasing concentrations CLL69C rAb to peptide mimotopes P1 and P2, MAA-BSA, or an irrelevant control peptide (pep-ctrl). Data are expressed as RLU/100 msec, and are from three independent experiments (mean ± SD), each value determined in triplicate. (<b>C</b>) Competition immunoassay demonstrates specificity of the CLL69C rAb to mimotope peptide P1. A fixed and limiting concentration of CLL69C Ab was incubated in the absence or presence of increasing concentrations of indicated competitors, and extent of binding to P1-coated plates was determined and expressed as B/B0 from triplicate determinations (mean ± SD) as explained in legend to Fig. 1B.</p

Expression and purification of active UL10 Fab Ab.

<p>(<b>A</b>) The UL10 Fab was produced in <i>E. coli</i> and purified by affinity chromatography from soluble fraction of <i>E. coli</i> lysate using nickel resin column. SDS-PAGE analysis of purified fractions under non-reducing condition and stained with Coomassie Blue (left three lanes), and blotted with anti-HA tag Ab (right three lanes). Lane 1, protein size standards (in kDa) shown at left; lane 2, <i>E. coli</i> extract expressing UL10 Fab; lane 3, purified UL10 Fab. (<b>B</b>) Binding pattern of CLL69C homologous UL10 Fab to OSE. Chemiluminescent ELISA for the binding of UL10 Fab (1 µg/ml) to each of the plated antigens (5 µg/mL) for 1.5 hour at RT. The data are expressed as RLU/100 ms and represent values from three independent experiments (mean ± SD), each value determined in triplicate.</p

Binding of CLL69 Abs to OSE on apoptotic cells and atherosclerotic lesions.

<p>(<b>A</b>) Flow cytometry analysis of CLL69 rAb binding to apoptotic cells. Representative flow cytometry contour plots of apoptotic murine thymocytes and histogram plots for the binding of rAb to apoptotic cells. Murine thymocytes were induced to undergo apoptosis by incubation with 10 ng/ml PMA for 16 hours. Apoptotic cells were stained with rAbs CLL69 A–D, isotype VH3-21, or no primary Ab, followed by detection with a FITC-conjugated anti-human IgG1 and staining with PE-labeled Annexin-V and 7-AAD to identify the viable and apoptotic cells. The contour plot (upper left panel) identifies the viable cells (Q3, PE-Annexin-V^−/7-AAD⁻), early apoptotic (Q4, Annexin-V⁺/7-AAD⁻) and late apoptotic cells (Q2, Annexin-V⁺/7-AAD⁺). Histogram panels Q3 (bottom left), Q4 (bottom right) and Q2 (upper right) represent rAb staining of viable cells, early apoptotic cells, and late apoptotic cells, respectively. Relative cell fluorescence of rAbs CLL69 A–D and IGHV3-21 control rAb are indicted by colored lines, and staining with the secondary Ab alone is shown in grey. (<b>B</b>) Immunofluorescence microscopy of CLL69C rAb binding to apoptotic cells. Deconvolution microscopy showing the binding of CLL69C rAb to late apoptotic Jurkat cells (FITC green color, the cell on the left panel) with dense fragmented nucleus detected by Hoechst staining (blue color), and apoptotic cells detected by PE-Annexin-V (red color), but not to a cell with intact nucleus (Panel A). The vector control and secondary Ab only (anti-IgG1-FITC) do not bind the apoptotic cells (panel B and C, respectively). (<b>C</b>) Inhibition of MAA-LDL binding to macrophage scavenger receptors by CLL69Ab. Competition assays for the inhibition of biotinylated-MAA-LDL binding to macrophage J774 by CLL69C rAb, UL10 Fab, or an irrelevant control VH3-21 rAb, as indicated. A fixed and limiting amount of biotin-MAA-LDL (5 µg/ml) was added to J774 macrophages in the absence or presence of increasing concentrations of indicated competitors. Extent of binding was determined in RLU/100 msec as described for chemiluminescent ELISA assays. Data shown represent the ratio MAA-LDL binding in the presence or absence of competitors (B/B₀) as described in legend to Fig. 1B. Results are representative of three independent experiments, each point determined in triplicate. (<b>D</b>) Immunohistochemical staining of human carotid endarterectomy (CEA) specimens. Roughly parallel sections of human carotid endarterectomy tissue were stained with rAbs of CLL69C (panel C), CLL69A (panel B), or a secondary Ab alone (panel A). MAA-epitopes recognized by CLL69C or CLL69A are indicated by red color.</p

Binding pattern of CLL69 rAbs to OSE.

<p>(<b>A</b>) Chemiluminescent ELISA of CLL69 rAbs (1 µg/mL) binding to plated BSA, MAA- and MDA-modified BSA, MAA- or MDA-modified LDL, copper oxidized LDL (OxLDL), phosphocholine-modified BSA (PC-BSA), or native LDL (LDL), each at 5 µg/mL, for 1.5 hour at RT. Binding was determined as in methods, expressed as relative light units (RLU) per 100 milliseconds (ms) and represent at least three independent experiments in triplicate determinations (mean ± SD). (<b>B</b>) Competition immunoassay for the specificity of CLL69C rAb binding to MAA- epitopes. A fixed and limiting dilution of CLL69C rAb was incubated in the absence and presence of increasing amounts of indicated competitors and the extent of binding to plated MAA-LDL (left panel), MAA-BSA (middle panel) or MDA-LDL (right panel) was determined. Data are expressed as a ratio of binding in the presence of competitor (B) divided by absence of competitor (B₀). Data are representative of three independent experiments, each determined in triplicate.</p