Journal: STAR Protocols

Article Title: FlowLITE: A protocol to characterize and quantify total antibody isotypes in human plasma using flow cytometry

doi: 10.1016/j.xpro.2025.104200

Figure Lengend Snippet: Schematic of 8-bead FlowLITE assay (A) FlowLITE consists of fluorescent yellow beads coated with anti-human isotype antibodies (monoclonal mouse IgG1). These antibodies are specific to the isotype variations in the heavy chain constant region. The bound antibodies from the sample are revealed by a fluorescently conjugated antibody specific to the light chain of the captured human antibody. (B) FlowLITE can be used to distinguish human antibody isotypes. Isotypes are distinguished by color and structure in the schematic.

Article Snippet: Purified Recombinant Human IgG3 Kappa (clone AbD18705_hIgG3); starting concentration: 0.01 mg/mL , Bio-Rad , Cat#HCA194.

Techniques:

Journal: STAR Protocols

Article Title: FlowLITE: A protocol to characterize and quantify total antibody isotypes in human plasma using flow cytometry

doi: 10.1016/j.xpro.2025.104200

Figure Lengend Snippet: Analysis of human plasma using 3-bead FlowLITE assay (IgM, pan-IgG, and pan-IgA) (A) Gating strategy for discrimination of anti-type bead species based on intensity of yellow fluorescence, detected in the V5 channel on the Cytek Aurora. (B) Titration of purified human IgM, IgG, and IgA. A 2-fold dilution series of the purified antibody was performed, starting from a concentration of 0.01 mg/mL. Background subtraction was performed using a control bead incubated with flowLITE media. (C) Linear range of detection (9.8 to 5,000 ng/mL) and ordinary least squares regression output (performed in Excel). Antibody concentration is determined using the linear regression calculated for each anti-isotype bead. The regression is adjusted based on the starting concentration of antibody in the standard curve (0.01 mg/mL). This value is represented by a constant, C =– log 2 (0.01 mg / ml )–1. (D) 2-fold dilution series of healthy human plasma starting from a concentration of 1:250. (E) Inter-assay variability (across days and runs) of quantified concentrations of antibody in mg/dL of the same donor at an optimal dilution of 1:64000 (dilution step 9 as indicated by the red box in D) was assessed in triplicate. The concentrations of IgM, IgG, and IgA were calculated and presented as the geometric mean and standard error for the three trials.

Article Snippet: Purified Recombinant Human IgG3 Kappa (clone AbD18705_hIgG3); starting concentration: 0.01 mg/mL , Bio-Rad , Cat#HCA194.

Techniques: Clinical Proteomics, Fluorescence, Titration, Purification, Concentration Assay, Control, Incubation, Inter Assay

Journal: STAR Protocols

Article Title: FlowLITE: A protocol to characterize and quantify total antibody isotypes in human plasma using flow cytometry

doi: 10.1016/j.xpro.2025.104200

Figure Lengend Snippet: Optimal bead saturation determined by titration of anti-human isotype capture antibodies (A) Schematic diagram of bead loading as the concentration of anti-isotype monoclonal mouse IgG1 antibody monomers decreases. The concatenated plot illustrates the saturation of the bead as the capture antibody concentration decreases. The dashed line indicates the base fluorescence of the bead in the YG1 channel when the coated bead is incubated in FlowLITE media. (B) Anti-IgE bead load titration was performed using yellow peak 9 (Y9) from the 12-set yellow-labeled beads (Spherotech). MFI values plotted against anti-IgE antibody concentration starting from a molarity of 1.6 μM (40 pmol/25 μL). (C) Bead load titrations of bead species Y3 (IgG1), Y4 (IgG2), Y5 (IgG3), Y6(IgG4) conjugated to the denoted anti-IgG subtype antibodies.

Article Snippet: Purified Recombinant Human IgG3 Kappa (clone AbD18705_hIgG3); starting concentration: 0.01 mg/mL , Bio-Rad , Cat#HCA194.

Techniques: Titration, Concentration Assay, Fluorescence, Incubation, Labeling

Journal: STAR Protocols

Article Title: FlowLITE: A protocol to characterize and quantify total antibody isotypes in human plasma using flow cytometry

doi: 10.1016/j.xpro.2025.104200

Figure Lengend Snippet: Application of FlowLITE in 8-bead assay and quantification of IgG subclasses (A) Readout of an 8-bead FlowLITE assay for detection of isotype subclasses. (B) Pie chart representation of the MFI ratios of the antibody isotypes and subclasses in the human plasma of a healthy donor. (C) Linear regressions for the calibration curves of the different IgG subclasses utilizing purified kappa IgG antibodies (Bio-Rad). (D) IgG subclass calibration curve regression output and quantification of IgG subclasses in human plasma.

Article Snippet: Purified Recombinant Human IgG3 Kappa (clone AbD18705_hIgG3); starting concentration: 0.01 mg/mL , Bio-Rad , Cat#HCA194.

Techniques: Clinical Proteomics, Purification

Journal: STAR Protocols

Article Title: FlowLITE: A protocol to characterize and quantify total antibody isotypes in human plasma using flow cytometry

doi: 10.1016/j.xpro.2025.104200

Figure Lengend Snippet: Reporting the absolute count of antibody molecules with QuantiBrite (A) PE QuantiBrite beads were used for calibration. A linear regression was performed to correlate fluorescence with the number of PE molecules. (B) The QuantiBrite regression was used to calculate the concentration of IgG molecules in a purified antibody sample. A 2-fold dilution of purified human IgG (starting at 0.1 mg/mL) was assessed using an antigen-coated bead (FlowBEAT format) and a pan-IgG reveal. Fluorescence was converted to PE copies using the formula derived in (A), assuming a conjugation of 1 PE per reveal antibody.

Article Snippet: Purified Recombinant Human IgG3 Kappa (clone AbD18705_hIgG3); starting concentration: 0.01 mg/mL , Bio-Rad , Cat#HCA194.

Techniques: Fluorescence, Concentration Assay, Purification, Derivative Assay, Conjugation Assay

Journal: Vaccines

Article Title: FLU-v, a Broad-Spectrum Peptide-Based Influenza Vaccine, Induces NK Cell Activating IgG1 and IgG3 Subclass Antibodies in Humans

doi: 10.3390/vaccines13111084

Figure Lengend Snippet: FLU-v IgG1 ( A ) and IgG3 ( B ) concentrations across all groups on days 0, 42, and 180. Data from the following groups are shown: adjuvanted FLU-v ( n = 52), non-adjuvanted FLU-v ( n = 58), adjuvanted placebo ( n = 26), and non-adjuvanted placebo ( n = 32). Box and whisker plot showing all individual values, 25th and 75th percentiles, and median. Whiskers represent ranges. The Mann–Whitney U test was used to compare different groups at each time point. The Wilcoxon signed-rank sum test was used to compare differences between time points within each group. Only significant differences are shown.

Article Snippet: Wells in the standard rows were coated with human IgG1 or IgG3 reference antibodies (16-16-090707-1/16-16-090707-3, Athens Research, Athens, GA, USA), performing double decreasing serial dilutions starting at 1000 ng/mL up to 1.95 ng/mL.

Techniques: Whisker Assay, MANN-WHITNEY

Journal: Vaccines

Article Title: FLU-v, a Broad-Spectrum Peptide-Based Influenza Vaccine, Induces NK Cell Activating IgG1 and IgG3 Subclass Antibodies in Humans

doi: 10.3390/vaccines13111084

Figure Lengend Snippet: Fold increase in FLU-v IgG1 ( A ) and IgG3 ( B ) concentrations across all groups from day 0 to days 42 and 180. Data from the following groups are shown: adjuvanted FLU-v ( n = 52), non-adjuvanted FLU-v ( n = 58), adjuvanted placebo (n = 26), and non-adjuvanted placebo ( n = 32). Box and whiskers plot showing all individual values, 25th and 75th percentiles, and median. Whiskers represent ranges. Fold change in IgG1 concentrations on day 42 and 180 is defined as the ratio of day 42 to day 0 and the ratio of day 180 to day 0, respectively. The Mann–Whitney U test was used to compare different groups at each time point. The Wilcoxon signed-rank sum test was used to compare differences between day 42 and 180 within each group. Significant differences are shown.

Article Snippet: Wells in the standard rows were coated with human IgG1 or IgG3 reference antibodies (16-16-090707-1/16-16-090707-3, Athens Research, Athens, GA, USA), performing double decreasing serial dilutions starting at 1000 ng/mL up to 1.95 ng/mL.

Techniques: MANN-WHITNEY

Journal: Vaccines

Article Title: FLU-v, a Broad-Spectrum Peptide-Based Influenza Vaccine, Induces NK Cell Activating IgG1 and IgG3 Subclass Antibodies in Humans

doi: 10.3390/vaccines13111084

Figure Lengend Snippet: Antibody-mediated NK cell activation after adjuvanted and non-adjuvanted FLU-v vaccination. For this analysis, a subset of paired samples ( n = 50) from day 0 and day 42 was selected based on high IgG1 and IgG3 fold increases. Data from the following groups are shown: adjuvanted FLU-v ( n = 32), non-adjuvanted FLU-v ( n = 16), and non-adjuvanted placebo ( n = 2). Antibody-dependent NK cell activation, or percentage of GFP + CD107a + CD16 (V176) NK-92 cells by flow cytometry, was measured in response to antibodies immobilized by plate-bound FLU-v ( A ) or non-related peptides used as negative controls ( B ). Medians ± 95% confidences intervals are shown. The Wilcoxon signed-rank sum test was used to compare differences between time points within each vaccine group. A Bonferroni test was used to correct for multiple comparisons, with a p value < 0.025 considered significant.

Article Snippet: Wells in the standard rows were coated with human IgG1 or IgG3 reference antibodies (16-16-090707-1/16-16-090707-3, Athens Research, Athens, GA, USA), performing double decreasing serial dilutions starting at 1000 ng/mL up to 1.95 ng/mL.

Techniques: Activation Assay, Flow Cytometry

Journal: Vaccines

Article Title: FLU-v, a Broad-Spectrum Peptide-Based Influenza Vaccine, Induces NK Cell Activating IgG1 and IgG3 Subclass Antibodies in Humans

doi: 10.3390/vaccines13111084

Figure Lengend Snippet: Correlation between antibody-mediated NK cell activation and FLU-v-specific IgG ( A ), IgG1 ( B ) and IgG3 ( C ) on days 0 and 42. Spearman rank order correlations were performed between the antibody-dependent NK cell activation assay, measuring the percentage of GFP + CD107a + CD16 (V176) NK-92 cells by flow cytometry, and total IgG, IgG1, and IgG3 with correlation coefficients (r) and p -values shown. IgG data for this analysis were obtained from a previous report .

Article Snippet: Wells in the standard rows were coated with human IgG1 or IgG3 reference antibodies (16-16-090707-1/16-16-090707-3, Athens Research, Athens, GA, USA), performing double decreasing serial dilutions starting at 1000 ng/mL up to 1.95 ng/mL.

Techniques: Activation Assay, Flow Cytometry