human complement sera  (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: Communications Biology

Article Title: Antibodies targeting the glycan cap of Ebola virus glycoprotein are potent inducers of the complement

doi: 10.1038/s42003-024-06556-0

Figure Lengend Snippet: Antibody-dependent complement deposition (ADCD) assay. a Dose–response curves. b Relative C3 deposition (normalized to mAb binding with beads). GP-coated beads were incubated with mAbs and complement, and mAb binding and C3 deposition were quantified by flow cytometry. The results were expressed as a ratio of area under the curve values for C3 deposition and mAb binding to beads. c Complement-dependent cytotoxicity (CDC) assay. 293F cells expressing EGFP, EBOV GP, and the chimeric CCR5-SNAP tag protein were labeled with AF647 and incubated with mAbs (10 µg ml −1 ) and complement (10%). The cytotoxicity was determined as percentage of EGFP – AF647 + cells by flow cytometry. ( b ) and ( c ) also include schematic representations of ADCD or CDC assays, respectively. Representative dot plots are shown. a , c : mean ± SEM of triplicate samples are shown.

Article Snippet: Plates were washed four times in PBST, two-fold serial dilutions of human complement sera (Sigma-Aldrich) in PBST-0.5% BSA from 1:1 to 1:2048 were added, and plates were incubated for 20 min at 37 °C.

Techniques: Binding Assay, Incubation, Flow Cytometry, CDC Assay, Expressing, Labeling

Journal: Communications Biology

Article Title: Antibodies targeting the glycan cap of Ebola virus glycoprotein are potent inducers of the complement

doi: 10.1038/s42003-024-06556-0

Figure Lengend Snippet: a Specificity of CDC assay. Complement was pre-treated with zymosan at 20 mg ml −1 (left) or 1E2 antibody at 0.1 mg ml −1 (middle) and added to cells, or 293F cells were treated with 1 µg ml −1 TFPI and incubated with complement (right). As controls, cells were mock-treated (no mAb), or treated with absolute ethanol (cell death control). * p < 0.001; ns, not significant (unpaired t-test). Representative dot plots are shown. b Difference in complement activation by BDBV223 isotypes (10 µg ml −1 ) validated by ELISA. Antigen-bound mAbs were incubated with serially diluted intact or heat-inactivated complement and the results were expressed as a ratio of C3-specific OD signals (OD intact compl./OD heat-inact. compl.). Mean ± SEM of triplicate samples are shown.

Article Snippet: Plates were washed four times in PBST, two-fold serial dilutions of human complement sera (Sigma-Aldrich) in PBST-0.5% BSA from 1:1 to 1:2048 were added, and plates were incubated for 20 min at 37 °C.

Techniques: CDC Assay, Incubation, Control, Activation Assay, Enzyme-linked Immunosorbent Assay