Journal: Scientific reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression.

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: Figure 1. Evidence for domain-specific and concentration dependent associations between C1q and CD33 using purified proteins in slot blot assays. Unlabeled proteins were immobilized on membranes; biotin labeled proteins were in solution. (A–C) Dose related binding of immobilized whole C1q and gC1q, but not CLR, to biotin-CD33M (5 ug/ml). (D) Biotin-whole C1q (10 ug/ml) binds to immobilized CD33m (lacking the extracellular V-like domain and expressing the C2-like domain) in a dose specific manner. (HSA = human serum albumin control showing no reactivity. Bound proteins were detected using streptavidin conjugated Infrared 800 (LI-COR). Pixel density was determined by densitometry. (C,D) Bar graphs represent the mean ± SE of pooled data; one-way ANOVA followed by Tukey’s pairwise multiple comparison was used to determine significance. N ≥ 3.

Article Snippet: Recombinant (r) hu proteins purified from cDNA transfected HEK293 T cells were: full length CD33 (CD33M, Novoprotein Scientific Inc., Short Hills, NJ), CD33m (TP317716, Origene technologies, Rockville, MD), biotin-labeled CD33M (CD3-HB22R; ACRO Biosystems, Bethesda, MD).C1q derived from normal hu serum (Complement Technology Inc., Tyler, TX) was used either as unmodified or biotin labeled, or cleaved into gC1q (collagenase digests) or CLR (pepsin digests) as described12, 52.

Techniques: Concentration Assay, Purification, Dot Blot, Labeling, Binding Assay, Expressing, Control, Comparison

Journal: Scientific reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression.

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: Figure 2. C1q binds to CD33M and CD33m isoforms on the cell surface. (A) A representative flow cytometry plot showing binding of biotin C1q (20 ug/ml) to HEK293T cells transfected with plasmids encoding CD33m and CD33M and control mock plasmid. Cells were incubated with C1q as outlined in Materials and Methods. Numbers in graphs = % positive/MFI. Debris/dying cells was excluded on the basis of forward and side scatter. (B) Pooled flow cytometry data illustrating binding of C1q (at comparable levels) on the surface of CD33m/M transfected HEK293T cells and a lack of C1q binding on mock transfected cells. For A and B, N = 3. (C) Representative slot blot showing that C1q binds to cell-associated CD33m, CD33M or LAIR-1 in a dose dependent manner. Protein prepared from whole cell lysates (HEK293T cells transfected with plasmid encoding CD33m, CD33M or LAIR-1) was immobilized on membranes at increasing doses; biotin-whole C1q was in solution at 10 ug/ml. HSA = human serum albumin control. Protein concentration was calculated by the BCA method. Bound proteins were detected using streptavidin conjugated Infrared 800 (LI-COR). (D) Pooled data analyses illustrating dose related interactions between biotin-C1q and cell-associated CD33m or CD33M represented in (C). (B,D), the mean ± SE of pooled data is shown; for all proteins and doses, N = 3–5. *P = < 0.05, **P = <0.01, ***P < 0.001. One-way ANOVA followed by Tukey’s pairwise multiple comparison was used.

Article Snippet: Recombinant (r) hu proteins purified from cDNA transfected HEK293 T cells were: full length CD33 (CD33M, Novoprotein Scientific Inc., Short Hills, NJ), CD33m (TP317716, Origene technologies, Rockville, MD), biotin-labeled CD33M (CD3-HB22R; ACRO Biosystems, Bethesda, MD).C1q derived from normal hu serum (Complement Technology Inc., Tyler, TX) was used either as unmodified or biotin labeled, or cleaved into gC1q (collagenase digests) or CLR (pepsin digests) as described12, 52.

Techniques: Flow Cytometry, Binding Assay, Transfection, Control, Plasmid Preparation, Incubation, Dot Blot, Protein Concentration, Comparison

Journal: Scientific reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression.

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: Figure 3. C1q triggers CD33 ITIM phosphorylation and CD33-LAIR-1 physical associations in human monocytes. Monocytes were either untreated (utx), treated with whole C1q, gC1q, C1q CLR or pervanadate (PV) as detailed in Materials and Methods. (A,B) Immunoblot analyses showing increased phosphorylated (p) CD33 after treatment with C1q and gC1q, but not C1q CLR. CD33 was immunoprecipitated with anti-CD33M antibody; tyrosine phosphorylation of CD33 (pCD33M) was detected with anti-phosphotyrosine (4G10) antibody. The membrane was stripped and re-probed with anti-CD33 WM53 antibody (CD33M). Arrows denote molecular weight of 67kD. Bar graphs denote pooled data expressed as fold change relative to total CD33 and corresponding statistical analyses. One-way ANOVA with post-hoc Tukey multiple comparisons was used to determine significance. N = 4 for A; N = 3 for B. (C) The addition of whole C1q (20 ug/ml) prompts concurrent increases in pLAIR-1 and pCD33 in a phospho-immunoreceptor array. (D) C1q CLR “tails” (20 ug/ml) do not elicit increases in pCD33M in the phospho-immunoreceptor array. Bar graphs represent fold change (plus/minus C1q), calculated from the mean pixel density of duplicates, as determined by densitometry analysis. Corresponding C1q minus/plus array data is located below each bar graph in C and D. Data from a typical array are shown. N = 3 for C and D. (E) Proximity ligation assay, performed on freshly isolated human monocytes as detailed in materials and methods, showing that whole C1q is required for CD33-LAIR-1 crosslinking. Red fluorescent dots represent molecular associations between CD33 and LAIR-1; blue represents nuclear staining with DAPI. CLR and gC1q represent the C1q collagen tail and globular heads of C1q, respectively. Original magnification = 60X. One of three representative experiments is shown.

Article Snippet: Recombinant (r) hu proteins purified from cDNA transfected HEK293 T cells were: full length CD33 (CD33M, Novoprotein Scientific Inc., Short Hills, NJ), CD33m (TP317716, Origene technologies, Rockville, MD), biotin-labeled CD33M (CD3-HB22R; ACRO Biosystems, Bethesda, MD).C1q derived from normal hu serum (Complement Technology Inc., Tyler, TX) was used either as unmodified or biotin labeled, or cleaved into gC1q (collagenase digests) or CLR (pepsin digests) as described12, 52.

Techniques: Phospho-proteomics, Western Blot, Immunoprecipitation, Membrane, Molecular Weight, Proximity Ligation Assay, Isolation, Staining

Journal: bioRxiv

Article Title: Rational design of oxidation-resistant antibodies through local electrostatic modulation

doi: 10.1101/2025.06.29.662139

Figure Lengend Snippet: a) Cryo-EM structure of 15G15.3 Fab (gray) bound to CD33 (violet), showing Trp96 forming key interactions with Lys52 (CD33) and Asp101 (Fab). Trp96 oxidizes at 97% under AAPH stress; W96F mutation abolishes binding ( > 1000-fold loss). b) Electrostatic potential of the lead candidate, with 12 mutated residues shown as pink spheres. c) Scatter plot of Trp oxidation vs. Epot for the lead and 13 variants; point color reflects relative KD. d) Summary of 13 engineered variants. S11 and S13 show improved oxidation resistance with preserved binding.

Article Snippet: Briefly, each antibody variant was captured by Protein A sensor chip (Series S) on the different flow cell to achieve approximately 150 response units (RU), followed by the injection of fivefold serial dilutions of human CD33 protein (R&D Systems; 0.16 nM to 100 nM) in HBS-EP buffer.

Techniques: Cryo-EM Sample Prep, Mutagenesis, Binding Assay

Journal: Molecules

Article Title: In Vitro and In Vivo Characterization of 89 Zirconium-Labeled Lintuzumab Molecule

doi: 10.3390/molecules27196589

Figure Lengend Snippet: Radiolabeling of lintuzumab with 89 Zr and its in vitro binding to CD33. ( A ) Binding of Lintuzumab−DFO conjugate to recombinant human CD33 protein is demonstrated via ELISA. ( B ) Binding of Lintuzumab−DFO conjugate to human cancer cell lines that express CD33 is demonstrated via immunofluorescence staining using a flow cytometer. ( C ) HPLC trace chromatograms ran on purified antibody conjugate (upper panel) and radiolabeled conjugate at a wavelength of 280 nm (middle panel UV trace, lower panel radioactivity trace).

Article Snippet: ELISA: Nunc MaxiSorp flat-bottomed 96-well plates were coated with 100 ng/well of human recombinant CD33 His-Tag protein (R&D Systems) in PBS and incubated overnight at 4 °C.

Techniques: Radioactivity, In Vitro, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Flow Cytometry, Purification

Journal: Molecules

Article Title: In Vitro and In Vivo Characterization of 89 Zirconium-Labeled Lintuzumab Molecule

doi: 10.3390/molecules27196589

Figure Lengend Snippet: 89 Zr-lintuzumab Selectively Accumulates in CD33 Expressing Tumors. ( A ) PET/CT imaging of mice administered with 5.55 MBq 89 Zr-DFO-Lintuzumab only ( n = 4). Images were taken on Days 1, 2, 3, and 7 post administration. The radioconjugate was cleared from blood and other organs except for the tumor after Day 1 and accumulated in CD33-positive OCI-AML-3 tumors. ( B ) Cohort of mice ( n = 4) pre-blocked with cold Lintuzumab 24hr prior to administration of radioconjugate. PET/CT images were taken on Days 3 and 7 post radioconjugate administration. The clearance of radioconjugate from blood and other organs in mice pre-blocked with Lintuzumab was not as effective as in non-blocked mice: radioactivity was detected in various organs by PET/CT imaging even on Day 7 post administration. ( C ) Standardized uptake values (SUV) analysis of PET/CT images taken on Days 3 and 7. Tumor volumes of interest (VOI) were drawn, and SUV were calculated. 89 Zr-DFO-Lintuzumab showed a significantly higher uptake in the tumors of non-blocked mice than in the tumors of pre-blocked mice. ( D ) Time–activity curves (TAC) show that the radiolabeled antibody remains mostly constant over the 7-day period after initial uptake for both non-blocked and pre-blocked tumors, with the pre-blocked has significantly less uptake. All images are displayed as maximum intensity projections (MIP). ** and *** mean p = 0.001 and p < 0.0001, respectively.

Article Snippet: ELISA: Nunc MaxiSorp flat-bottomed 96-well plates were coated with 100 ng/well of human recombinant CD33 His-Tag protein (R&D Systems) in PBS and incubated overnight at 4 °C.

Techniques: Expressing, Positron Emission Tomography-Computed Tomography, Imaging, Radioactivity, Activity Assay

Journal: Cells

Article Title: Shape-Shifted Red Blood Cells: A Novel Red Blood Cell Stage?

doi: 10.3390/cells7040031

Figure Lengend Snippet: Protein cell markers for shRBCs. ( a ) RBCs incubated at 25 °C for 3 days were stained with different antibodies and analyzed by flow cytometry. The dot plot shows RBCs (red) and shRBCs (yellow). Flow cytometry results are represented as MRFI (mean relative fluorescence intensity = fluorescence in shRBC/RBC). ( b ) Protein cell markers: erythrocyte marker (glycophorin C [GYPC]), myeloid cell surface antigen (CD33), hematopoietic progenitor cell surface antigen (CD34), and B cell marker (immunoglobulin M [IgM]). Data are displayed as black circles showing the mean ± SD (n = 5). The Mann–Whitney Test was performed for statistical analysis between shRBCs and RBCs. RBCs are represented by the dashed line.

Article Snippet: Myeloid cell surface antigen (CD33) and hematopoietic progenitor cell surface antigen (CD34) antibodies were obtained from CUSABIO Life Science (Houston, TX, USA).

Techniques: Incubation, Staining, Flow Cytometry, Fluorescence, Marker, MANN-WHITNEY