human male ab plasma as a source of human complement  (Millipore)

Journal: Analytical and Bioanalytical Chemistry

Article Title: A homogeneous immunoassay technology based on liposomes and the complement system enables one-step, no-wash, rapid diagnostics directly in serum

doi: 10.1007/s00216-025-05882-4

Figure Lengend Snippet: Optimization of the liposome composition to generate stealth liposomes. a Homogeneous complement assay of 10 mM SRB-encapsulating liposomes (10 µM total lipids; batches –6) showing the cholesterol dependency of liposome stealthiness in active serum (10 vol% PHS). Statistical analysis (two-sample independent t-test) revealed a significant difference between active and inactive serum samples at a cholesterol content of 10 mol% (p = 0.026) and above (p < 0.001). In contrast, no significant difference was observed at a cholesterol content of 5 mol% (p = 0.881). b Liposome stealthiness in active serum depending on SRB and cholesterol content (batches –10). 10 vol% human serum was used as a complement source (IRS41174). Statistical analysis (two-sample independent t-test) showed no significant difference between active and inactive serum samples of liposomes containing 10 mM SRB and 5 mol% cholesterol (p = 0.714), while in the other cases either a statistically significant amount of complement lysis (p < 0.001) or general serum stability was observed. Fluorescence measurements were carried out for 1 h at 37 °C in LCB, iaS, aS, and 30 mM OG + aS. Fluorescence intensities were normalized to the endpoint fluorescence of the positive control. λ Ex = 565(5 or 8) nm and λ Em = 585(5 or 8) nm; gain 150. T = 37 °C. n = 3. c Heterogeneous complement assay of biotin-modified chemiluminescence liposomes (30 mM m -carboxy luminol; batches CL1–2). Statistical analysis (two-sample independent t-test) showed a significant difference between active and inactive serum samples of high-cholesterol liposomes. Low-cholesterol liposomes remained stealth and showed no statistical difference in 5 and 10 vol% serum, whereas 25 vol% led to minor lysis in active serum. Liposomes (50 μM total lipids) were immobilized overnight on a streptavidin-coated MTP and incubated for 1 h at 37 °C in either inactive serum or active serum (5, 10, or 25 vol% PHS in LCB). Chemiluminescence measurements were performed by adding 50 μL of 4 μM hemin and 50 μL 40 mM H 2 O 2 in 0.01 M CBS, pH 10.5, 2 s integration time, gain 80, RH 1 mm, T = 25 °C, n = 3. d Flow chart of the homogeneous complement assay procedure for SRB-encapsulating liposomes

Article Snippet: Pooled human complement sera (batches 31,758, 33,478, 35,577, 41,174 and 45,270) were purchased from Innovative Research (Novi, MI, USA); ARMS peptide (peptide sequence: IHTELCLPAFFSPAGTQRRFQQPQHHLTLSIIHTAAR) was purchased from ProteoGenix (France).

Techniques: Liposomes, Complement Assay, Lysis, Fluorescence, Positive Control, Modification, Incubation

Journal: Analytical and Bioanalytical Chemistry

Article Title: A homogeneous immunoassay technology based on liposomes and the complement system enables one-step, no-wash, rapid diagnostics directly in serum

doi: 10.1007/s00216-025-05882-4

Figure Lengend Snippet: Homogeneous complement assay of 10 mM SRB-liposomes using antibodies as complement trigger. Time-resolved normalized fluorescence intensities of 0.5 mol% ARMS-modified liposomes (10 µM total lipids; batch ) a without or d with 0.5 mol% complement-triggering anti-ARMS antibody (A626). ARMS-liposomes were incubated with the A626 antibody for 1 h at 300 rpm. 10 vol% human serum was used as complement source (IRS35577). Time-resolved fluorescence intensities of biotinylated liposomes (1 µM total lipids; batch ) b without or e with 0.5 mol% complement-triggering anti-biotin antibody. Biotin-liposomes were incubated with the anti-biotin antibody for 1 h at 300 rpm. 5 vol% human serum was used as complement source (IRS45270). Time-resolved fluorescence intensities of PEGylated liposomes (1 µM total lipids; batch ) c without or f with 0.05 mol% complement-triggering anti-PEG antibody (clone RM105). PEG-liposomes were incubated with the anti-PEG antibody for 1 h at 300 rpm in 5 µL HSS. 5 vol% human serum was used as complement source (IRS45270). Fluorescence measurements were carried out for 1 h at 37 °C in LCB, iaS, aS and 30 mM OG + aS and normalized to the endpoint fluorescence of the positive control. λ Ex = 565(8) nm and λ Em = 585(8) nm; gain 150. T = 37 °C. n = 3

Article Snippet: Pooled human complement sera (batches 31,758, 33,478, 35,577, 41,174 and 45,270) were purchased from Innovative Research (Novi, MI, USA); ARMS peptide (peptide sequence: IHTELCLPAFFSPAGTQRRFQQPQHHLTLSIIHTAAR) was purchased from ProteoGenix (France).

Techniques: Complement Assay, Liposomes, Fluorescence, Modification, Incubation, Positive Control

Journal: Analytical and Bioanalytical Chemistry

Article Title: A homogeneous immunoassay technology based on liposomes and the complement system enables one-step, no-wash, rapid diagnostics directly in serum

doi: 10.1007/s00216-025-05882-4

Figure Lengend Snippet: Dose–response curves of liposome lysis dependent on the antibody concentration. Corrected lysis values of a Biotin- (batch ) or b , c PEG-biotin-liposomes (batch ) (10 mM SRB, 1 µM total lipids) in a homogeneous complement assay performing an anti-biotin or anti-PEG (clone RM105 or clone 6.3) antibody titration. d Different combinations of these antibodies (0.03 mol% clone RM105, 0.1 mol% clone 6.3, 0.3 mol% anti-biotin antibody) with PEG biotin-liposomes (batch ). A one-way ANOVA, including a post hoc Tukey test, was performed for statistical analysis (p < 0.05) of the antibody combinations. Liposomes were incubated with the respective amount of antibodies for 1 h at 300 rpm. 5 vol% human serum was used as a complement source (IRS45270). Fluorescence intensities were adjusted by the iaS signal and normalized to the endpoint fluorescence of the positive control. λ Ex = 565(8) nm and λ Em = 585(8) nm; gain 150. T = 37 °C. n = 3

Article Snippet: Pooled human complement sera (batches 31,758, 33,478, 35,577, 41,174 and 45,270) were purchased from Innovative Research (Novi, MI, USA); ARMS peptide (peptide sequence: IHTELCLPAFFSPAGTQRRFQQPQHHLTLSIIHTAAR) was purchased from ProteoGenix (France).

Techniques: Lysis, Concentration Assay, Liposomes, Complement Assay, Titration, Incubation, Fluorescence, Positive Control

Journal: Analytical and Bioanalytical Chemistry

Article Title: A homogeneous immunoassay technology based on liposomes and the complement system enables one-step, no-wash, rapid diagnostics directly in serum

doi: 10.1007/s00216-025-05882-4

Figure Lengend Snippet: Screening of anti-PEG antibodies in human sera. Corrected lysis values of PEGylated 10 mM SRB-liposomes (1 µM total lipids; batch ) in a homogeneous complement assay performing a serum titration of different commercial sera (IRS batches 31,758, 35,577, 41,174, and 45,270). 0.1–10 vol% of human sera were used. Fluorescence intensities were adjusted by the iaS signal and normalized to the endpoint fluorescence of the positive control. λ Ex = 565(8) nm and λ Em = 585(8) nm; gain 150. T = 37 °C. n = 3

Article Snippet: Pooled human complement sera (batches 31,758, 33,478, 35,577, 41,174 and 45,270) were purchased from Innovative Research (Novi, MI, USA); ARMS peptide (peptide sequence: IHTELCLPAFFSPAGTQRRFQQPQHHLTLSIIHTAAR) was purchased from ProteoGenix (France).

Techniques: Lysis, Liposomes, Complement Assay, Titration, Fluorescence, Positive Control

Journal: Analytical and Bioanalytical Chemistry

Article Title: A homogeneous immunoassay technology based on liposomes and the complement system enables one-step, no-wash, rapid diagnostics directly in serum

doi: 10.1007/s00216-025-05882-4

Figure Lengend Snippet: Long-term storage stability study of 10 mM SRB-liposomes. Normalized fluorescence intensities of SRB-liposomes (10 µM total lipids) with different lipid compositions: a low-cholesterol, carboxylated (batch ); b low-cholesterol, PEGylated (batch ); and c high-cholesterol, biotinylated (batch ) in LCB, aS, or iaS throughout the long-term storage stability study up to 40 months at 4 °C. 10 vol% human serum was used as a complement source (PHS for 0–10 months and IRS45270 for the 40 months datapoint). Fluorescence intensities were normalized to the endpoint fluorescence of the positive control. λ Ex = 565(5) nm and λ Em = 585(5) nm; gain 150. T = 37 °C. n = 3. d Summary of the liposome storage stability at 4 °C, RT, and 37 °C

Article Snippet: Pooled human complement sera (batches 31,758, 33,478, 35,577, 41,174 and 45,270) were purchased from Innovative Research (Novi, MI, USA); ARMS peptide (peptide sequence: IHTELCLPAFFSPAGTQRRFQQPQHHLTLSIIHTAAR) was purchased from ProteoGenix (France).

Techniques: Liposomes, Fluorescence, Positive Control

Journal: Clinical Cancer Research

Article Title: KLRG1 Cell Depletion as a Novel Therapeutic Strategy in Patients with Mature T-Cell Lymphoma Subtypes

doi: 10.1158/1078-0432.CCR-23-3504

Figure Lengend Snippet: Anti-KLRG1 monoclonal antibodies exert no apoptosis but induce ADCP, ADCC, and complement-mediated cytotoxicity (CDC). A, SMZ1-WT and SMZ1-KLRG1 cells were incubated with the indicated antibodies (mAb208, 13F12F2, or its isotype control mIgG2aκ) at increasing concentrations from 1 to 100 μg/mL or 0.2 or 1 μmol/L staurosporine (positive control) for 24 hours, and the percentage of apoptotic cells was quantified by flow cytometry. Three independent experiments were conducted. Shown are the mean values of technical triplicates with error bars representing SEM from one representative experiment. B–C, CFSE-labeled SMZ1-WT and SMZ1-KLRG1 cells were incubated with human monocyte-derived macrophages (hMDM) from three healthy donors and bone-marrow–derived murine macrophages (mBMDM) from different lots of mice in the presence of indicated antibodies for 2 hours. Anti-CD47 mAb, MIAP410-mediated phagocytosis of Jurkat cells relative to its isotype control, mIgG1κ, was used as a positive control. The percentage of CFSE + macrophages (of human CD14 + or murine CD11b + cells) was determined by flow cytometry and plotted. D–E, CFSE-labeled SMZ1-WT and SMZ1-KLRG1 cells were incubated with human-derived PBMCs from three healthy donors in the presence of indicated antibodies at increasing concentrations for 4 hours. The percentage of propidium iodide–positive (% PI pos; of CFSE + ) cells was determined by flow cytometry and plotted. Rituximab and Raji cells were used as a positive control. F–G, ADCC reporter assay measuring luminescence with murine FcγRIIIA-expressing Jurkat cells was performed in triplicate in three independent experiments at an effector:target ratio of 25:1 with indicated antibodies. Fold induction is relative to isotype control (mIgG2aκ for mAb208 and 13F12F2). Human anti-CD20 mAb and Raji cells were used as positive control along with its isotype control, hIgG1. Representative results from one experiment are shown. H–K, CDC assay with 30% murine and human complement was performed in triplicate in three independent experiments, and % PI pos cells were reported. Rituximab and Raji cells were used as positive controls. Shown are the mean values of technical triplicates, with error bars representing SEM from one representative experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by two-sided Welch t test.

Article Snippet: Human male AB plasma as a source of human complement was obtained from Sigma-Aldrich.

Techniques: Flow Cytometry Complement-Mediated Cytotoxicity Assay, Incubation, Positive Control, Flow Cytometry, Labeling, Derivative Assay, Reporter Assay, Expressing, CDC Assay