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 IgG4 Kappa (clone AbD18705_hIgG4); starting concentration: 0.01 mg/mL , Bio-Rad , Cat#HCA195.

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 IgG4 Kappa (clone AbD18705_hIgG4); starting concentration: 0.01 mg/mL , Bio-Rad , Cat#HCA195.

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 IgG4 Kappa (clone AbD18705_hIgG4); starting concentration: 0.01 mg/mL , Bio-Rad , Cat#HCA195.

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 IgG4 Kappa (clone AbD18705_hIgG4); starting concentration: 0.01 mg/mL , Bio-Rad , Cat#HCA195.

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 IgG4 Kappa (clone AbD18705_hIgG4); starting concentration: 0.01 mg/mL , Bio-Rad , Cat#HCA195.

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

Journal: Journal of Advanced Research

Article Title: Molecular and cellular morphology of placenta unveils new mechanisms of reproductive immunology

doi: 10.1016/j.jare.2025.01.025

Figure Lengend Snippet: Increase of IgG4 in pregnant women. (A)Localization of IgG1, IgG2, IgG3 and IgG4 detected with IHC in human placental tissues. The positivity of the four IgG subtypes were shown as brown stain that were similarly distributed on the outer edge of the placental villi and within the blood vessel lumen inside the placenta. It was noted that IgG1 and IgG4 were strongly and similarly distributed mainly along the outer edge of the placental villi (arrows). Positivity of the four antibodies were also observed within the lumen of the blood vessels inside the placenta, and were particularly strong for IgG2 and IgG3. (B) Expressions of IgG4 and GSH in early and term placenta. (C-D) Western blot to examine the capacity of IgG4 binding to IgG1 showed that the Fc-Fc reaction between IgG4 and antigen-bound IgG1 was gradually enhanced as the concentrations of GSH increased. (C) Results of Western blot with commercial IgG1 (2 ng) running on the gel and biotin-labeled IgG4 (2 ng) as the primary antibody (at 50KD). (D) The reaction bands (at 50KD) increased in intensity as the concentrations of GSH increased (from 0-20 mM) while the amounts of IgG1 and IgG4 remained the same, indicating that increased GSH facilitated Fc-Fc reaction between IgG4 and IgG1(The testing samples were run on duplicates.). (E) The results of silver staining showed the increased amount of IgG4 half-molecules (at 75KD) derived from IgG4 whole molecule when it was treated with increasing concentrations of GSH (1––14 mM). (F-K) Changes of IgG4 concentration and IgG4/total IgG level in blood of pregnant women. (F) Total IgG levels in women of different gestational age. (G) IgG4 levels in women of different gestational age. (H) IgG 4/Total IgG (%) levels in women of different gestational age. NP: non-pregnant; D14, 14 days after embryo transfer; 12 W, 12 weeks of gestation; 24 W, 24 weeks of gestation; 39 W, 39 weeks of gestation. (I-K) Serum total IgG and IgG4 levels 14 days after embryo transfer in IVF patients. (I) Total IgG levels in women of different group. (J) IgG4 levels in women of different group. (K) IgG 4/Total IgG (%) levels in women of different groups. NP: non-pregnant; CP: Clinical Pregnancy; IF: Implantation Failure; BP: Biochemical Pregnancy.

Article Snippet: After washing in buffer, the tissue sections were incubated with biotin-labeled IgG4 (Athens research & Technology, 16160907074 M) for 2 h. The biotin was detected with an immunostaining kit (AnaSpec, AS-72057) following the manufacture’s protocol.

Techniques: Staining, Western Blot, Binding Assay, Labeling, Silver Staining, Derivative Assay, Concentration Assay