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 IgG2 Kappa (clone AbD18705_hIgG2); starting concentration: 0.01 mg/mL , Bio-Rad , Cat#HCA193.

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 IgG2 Kappa (clone AbD18705_hIgG2); starting concentration: 0.01 mg/mL , Bio-Rad , Cat#HCA193.

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 IgG2 Kappa (clone AbD18705_hIgG2); starting concentration: 0.01 mg/mL , Bio-Rad , Cat#HCA193.

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 IgG2 Kappa (clone AbD18705_hIgG2); starting concentration: 0.01 mg/mL , Bio-Rad , Cat#HCA193.

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 IgG2 Kappa (clone AbD18705_hIgG2); starting concentration: 0.01 mg/mL , Bio-Rad , Cat#HCA193.

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

Journal: The Veterinary Quarterly

Article Title: Evaluation of the safety and immunogenicity of a peptide vaccine against canine leishmaniosis: a double-blind, multicenter, controlled clinical trial in dogs

doi: 10.1080/01652176.2025.2591396

Figure Lengend Snippet: Serum IgG subtypes response to recombinant vaccine antigens in vaccinated dogs. (A) Shows IgG2a and (B) shows IgG1 titers in response to the recombinant vaccine proteins HisDTC and protein Q, as detected by ELISA at multiple time points post-vaccination. Data are presented as median ± interquartile range. Statistical significance is denoted by asterisks (** p < 0.01) compared with the His-PLGA group.

Article Snippet: HRP-conjugated anti-dog IgG2a (sheep) and IgG1 (goat) (Bethyl Laboratories, Montgomery, TX, USA), diluted 1:45000, were used for detection.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Infection and Immunity

Article Title: Rickettsia rickettsii inactivated whole cell antigen vaccine protects against Rocky Mountain spotted fever independent of the adjuvant used

doi: 10.1128/iai.00412-25

Figure Lengend Snippet: Development of antibodies by vaccinated dogs. Plasma samples collected from all dogs several days following vaccinations and infection challenges were assessed by ELISA for the presence of R. rickettsii -specific IgG ( A ), IgG1 ( B ), and IgG2 ( C ). All dogs receiving WCAV developed antigen-specific IgG responses following the primary vaccination, which increased following the booster vaccination. R. rickettsii -specific total IgG and IgG subclass; IgG1 and IgG2 in different groups of vaccinated dogs were compared by two-way ANOVA test followed by multiplicity-adjusted post hoc comparisons (Tukey). P -value < 0.05 was considered statistically significant, which was observed for total IgG and IgG 2 for Quil A compared to Montanide and Alhydrogel from day 37 onward with P -values of ≤0.0001. Vertical dotted blue lines refer to the days of booster vaccination.

Article Snippet: One hundred microliters of a 1:50,000 dilution of anti-canine total IgG (PA1-29738, ThermoFisher, USA), 1:10,000 anti IgG1 (AHP947P, Bio-Rad, Hercules, CA, USA), or 1:5,000 of anti IgG2 (AHP948P, Bio-Rad, Hercules, CA, USA) peroxidase-labeled antibodies was added to each well, and plates were incubated for 45 min at 37°C.

Techniques: Clinical Proteomics, Infection, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Quantifying maternal antibody transfer to colostrum and cord blood reveals virus-specific selectivity in dogs

doi: 10.1101/2025.11.24.690260

Figure Lengend Snippet:

Article Snippet: Following washing with PBS-T, anti-canine IgG (H+L) conjugated to horse radish peroxidase (HRP) (Invitrogen, CAT#PA1-29738) or anti-canine IgG2 (Bethyl Laboratories CAT#A40-121P) was added for 1 hour.

Techniques: Virus, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: bioRxiv

Article Title: Quantifying maternal antibody transfer to colostrum and cord blood reveals virus-specific selectivity in dogs

doi: 10.1101/2025.11.24.690260

Figure Lengend Snippet: End point titers of IgG specific for A) canine parvovirus (CPV) and B) canine distemper virus (CDV) were quantified in clinical samples collected from dams and umbilical cords during cesarean sections. Transfer ratios of IgG between dam serum and colostrum/cord for puppies are shown in C. Dotted lines in A/B represent lower limit of quantification. Dashed line in C represents a 100% transfer efficiency (equivalent IgG levels between samples). Significance was assessed using Kruskal Wallis tests with multiple comparisons. Asterisks denote statistical significance: p <0.01 (**) , p <0.001 (***), and p <0.0001 (****).

Article Snippet: Following washing with PBS-T, anti-canine IgG (H+L) conjugated to horse radish peroxidase (HRP) (Invitrogen, CAT#PA1-29738) or anti-canine IgG2 (Bethyl Laboratories CAT#A40-121P) was added for 1 hour.

Techniques: Virus

Journal: bioRxiv

Article Title: Quantifying maternal antibody transfer to colostrum and cord blood reveals virus-specific selectivity in dogs

doi: 10.1101/2025.11.24.690260

Figure Lengend Snippet: (A) Total IgG titers were quantified in clinical samples by indirect ELISA; dam n=24, colostrum n=18, cord n=21 litters. (B) Transfer ratios of IgG via colostrum (n=18) or cord (n=21) calculated from total IgG titers. The dotted line represents transfer ratio of 1, representing an equivalent concentration of IgG between samples. (C) Transfer ratio of total IgG compared to virus-specific IgG from maternal serum into colostrum. Significance was assessed by a Kruskal Wallis test with post-hoc multiple comparisons (A, C) and a Mann Whitney test (B). Asterisks denote statistical significance: p <0.05 (*) , p <0.001 (***) and p <0.0001 (****).

Article Snippet: Following washing with PBS-T, anti-canine IgG (H+L) conjugated to horse radish peroxidase (HRP) (Invitrogen, CAT#PA1-29738) or anti-canine IgG2 (Bethyl Laboratories CAT#A40-121P) was added for 1 hour.

Techniques: Indirect ELISA, Concentration Assay, Virus, MANN-WHITNEY

Journal: bioRxiv

Article Title: Quantifying maternal antibody transfer to colostrum and cord blood reveals virus-specific selectivity in dogs

doi: 10.1101/2025.11.24.690260

Figure Lengend Snippet: Scatterplots of IgG titers for A) CPV (colostrum n=36, cord n=38 litters) and B) CDV (colostrum n=35, cord n=35 litters), and C) total IgG (colostrum n=18, cord n=22 litters) as determined by ELISA. Non-linear regression curves were fitted to the data. Statistical significance was assessed using Pearson’s correlation coefficient ( r ) for normally distributed data (total IgG colostrum) and Spearman’s rank correlation coefficient ( ρ ) for non-parametric data.

Article Snippet: Following washing with PBS-T, anti-canine IgG (H+L) conjugated to horse radish peroxidase (HRP) (Invitrogen, CAT#PA1-29738) or anti-canine IgG2 (Bethyl Laboratories CAT#A40-121P) was added for 1 hour.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Quantifying maternal antibody transfer to colostrum and cord blood reveals virus-specific selectivity in dogs

doi: 10.1101/2025.11.24.690260

Figure Lengend Snippet: All values were determined by CPV-specific IgG ELISA. (A) End point titers of CPV-specific IgG from dam, colostrum and cord plotted against parity. Horizontal bar represents mean titer of each group. (B-D) End point titers of CPV-specific IgG plotted against dam age, dam weight and litter size respectively. Non-linear regression curves were fitted to the data.

Article Snippet: Following washing with PBS-T, anti-canine IgG (H+L) conjugated to horse radish peroxidase (HRP) (Invitrogen, CAT#PA1-29738) or anti-canine IgG2 (Bethyl Laboratories CAT#A40-121P) was added for 1 hour.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Quantifying maternal antibody transfer to colostrum and cord blood reveals virus-specific selectivity in dogs

doi: 10.1101/2025.11.24.690260

Figure Lengend Snippet: Cord samples from each litter (n=38 litters, 1 – 7 cords per litter) are plotted together, arbitrarily ordered according to mean titer. Error bars represent standard error of the mean. Dotted line represents lower level of quantification of ELISA assay. Dashed line corresponds to an ELISA end point titer of 3239, and hemagglutination inhibition titer (HAI) >1:80. Shaded yellow area represents protective IgG titers.

Article Snippet: Following washing with PBS-T, anti-canine IgG (H+L) conjugated to horse radish peroxidase (HRP) (Invitrogen, CAT#PA1-29738) or anti-canine IgG2 (Bethyl Laboratories CAT#A40-121P) was added for 1 hour.

Techniques: Enzyme-linked Immunosorbent Assay, HI Assay