Journal: Pathogens (Basel, Switzerland)

Article Title: Development of Low-Cost In-House Assays for Quantitative Detection of HBsAg, HBeAg, and HBV DNA to Enhance Hepatitis B Virus Diagnostics and Antiviral Screening in Resource-Limited Settings.

doi: 10.3390/pathogens14030258

Figure Lengend Snippet: Figure 1. Optimization of in-house quantitative sandwich ELISA for detection of HBsAg and HBeAg. Standard curve depicting the optimal dilution of detection antibody for the detection of (A) HBsAg and (B) HBeAg. Primary antibodies used for antigen capture were mouse monoclonal α-HBsAg (Fitzgerald, 10-H05H) and mouse monoclonal α-HBeAg (Fitzgerald, 10-H10M). Detection was achieved using HRP-conjugated α-HBsAg goat polyclonal antibody (Fitzgerald, 70-HG15S) and α-HBeAg mouse monoclonal antibody (Fitzgerald, 61-H10K). OD450 values were normalized against BSA control wells. The assay was optimized by adjusting antibody concentrations to achieve a strong signal-to-noise ratio while maintaining linearity. Data were normalized by subtracting the optical density reading at 450 nanometers (OD450nm) from bovine serum albumin (BSA) control wells. (C) Summary table of ELISA data for HBeAg and HBsAg. Assay quality was assessed based on linearity of the standard curve R2 and Z′ factor. Data from 3 biological replicates.

Article Snippet: Detection was achieved using HRP-conjugated α-HBsAg goat polyclonal antibody (Fitzgerald, 70-HG15S) and α-HBeAg mouse monoclonal antibody (Fitzgerald, 61-H10K).

Techniques: Sandwich ELISA, Control, Enzyme-linked Immunosorbent Assay, HBsAg Assay

Journal: Pathogens (Basel, Switzerland)

Article Title: Development of Low-Cost In-House Assays for Quantitative Detection of HBsAg, HBeAg, and HBV DNA to Enhance Hepatitis B Virus Diagnostics and Antiviral Screening in Resource-Limited Settings.

doi: 10.3390/pathogens14030258

Figure Lengend Snippet: Figure 1. Optimization of in-house quantitative sandwich ELISA for detection of HBsAg and HBeAg. Standard curve depicting the optimal dilution of detection antibody for the detection of (A) HBsAg and (B) HBeAg. Primary antibodies used for antigen capture were mouse monoclonal α-HBsAg (Fitzgerald, 10-H05H) and mouse monoclonal α-HBeAg (Fitzgerald, 10-H10M). Detection was achieved using HRP-conjugated α-HBsAg goat polyclonal antibody (Fitzgerald, 70-HG15S) and α-HBeAg mouse monoclonal antibody (Fitzgerald, 61-H10K). OD450 values were normalized against BSA control wells. The assay was optimized by adjusting antibody concentrations to achieve a strong signal-to-noise ratio while maintaining linearity. Data were normalized by subtracting the optical density reading at 450 nanometers (OD450nm) from bovine serum albumin (BSA) control wells. (C) Summary table of ELISA data for HBeAg and HBsAg. Assay quality was assessed based on linearity of the standard curve R2 and Z′ factor. Data from 3 biological replicates.

Article Snippet: The α-HBsAg goat polyclonal detection antibody used in the sandwich ELISA (70-HG15S, Fitzgerald, Acton, MA, USA) was prepared in advance by conjugating HRP at 4:1 (antibody-to-HRP ratio) using a commercial kit (LNK002P, Bio-Rad, Hercules, CA, USA).

Techniques: Sandwich ELISA, Control, Enzyme-linked Immunosorbent Assay, HBsAg Assay

Journal: Pathogens (Basel, Switzerland)

Article Title: Development of Low-Cost In-House Assays for Quantitative Detection of HBsAg, HBeAg, and HBV DNA to Enhance Hepatitis B Virus Diagnostics and Antiviral Screening in Resource-Limited Settings.

doi: 10.3390/pathogens14030258

Figure Lengend Snippet: Figure 1. Optimization of in-house quantitative sandwich ELISA for detection of HBsAg and HBeAg. Standard curve depicting the optimal dilution of detection antibody for the detection of (A) HBsAg and (B) HBeAg. Primary antibodies used for antigen capture were mouse monoclonal α-HBsAg (Fitzgerald, 10-H05H) and mouse monoclonal α-HBeAg (Fitzgerald, 10-H10M). Detection was achieved using HRP-conjugated α-HBsAg goat polyclonal antibody (Fitzgerald, 70-HG15S) and α-HBeAg mouse monoclonal antibody (Fitzgerald, 61-H10K). OD450 values were normalized against BSA control wells. The assay was optimized by adjusting antibody concentrations to achieve a strong signal-to-noise ratio while maintaining linearity. Data were normalized by subtracting the optical density reading at 450 nanometers (OD450nm) from bovine serum albumin (BSA) control wells. (C) Summary table of ELISA data for HBeAg and HBsAg. Assay quality was assessed based on linearity of the standard curve R2 and Z′ factor. Data from 3 biological replicates.

Article Snippet: Next, 100 μL of the appropriate HRP-conjugated detection antibody diluted in blocking buffer was added to each well, either α-HBsAg goat polyclonal antibody (70-HG15S, Fitzgerald, Acton, MA, USA) or α-HBeAg mouse monoclonal secondary antibody (61-H10K, Fitzgerald, Acton, MA, USA).

Techniques: Sandwich ELISA, Control, Enzyme-linked Immunosorbent Assay, HBsAg Assay

Journal: Pathogens (Basel, Switzerland)

Article Title: Development of Low-Cost In-House Assays for Quantitative Detection of HBsAg, HBeAg, and HBV DNA to Enhance Hepatitis B Virus Diagnostics and Antiviral Screening in Resource-Limited Settings.

doi: 10.3390/pathogens14030258

Figure Lengend Snippet: Figure 1. Optimization of in-house quantitative sandwich ELISA for detection of HBsAg and HBeAg. Standard curve depicting the optimal dilution of detection antibody for the detection of (A) HBsAg and (B) HBeAg. Primary antibodies used for antigen capture were mouse monoclonal α-HBsAg (Fitzgerald, 10-H05H) and mouse monoclonal α-HBeAg (Fitzgerald, 10-H10M). Detection was achieved using HRP-conjugated α-HBsAg goat polyclonal antibody (Fitzgerald, 70-HG15S) and α-HBeAg mouse monoclonal antibody (Fitzgerald, 61-H10K). OD450 values were normalized against BSA control wells. The assay was optimized by adjusting antibody concentrations to achieve a strong signal-to-noise ratio while maintaining linearity. Data were normalized by subtracting the optical density reading at 450 nanometers (OD450nm) from bovine serum albumin (BSA) control wells. (C) Summary table of ELISA data for HBeAg and HBsAg. Assay quality was assessed based on linearity of the standard curve R2 and Z′ factor. Data from 3 biological replicates.

Article Snippet: We found that the ideal combination was a mouse monoclonal antibody against HBsAg (Fitzgerald, 10-H05H) and goat polyclonal HBsAg antibody (Fitzgerald, 70-HG15S); however, since neither antibody is available commercially with a horse-radish peroxidase (HRP) tag, advanced antibody preparation was required using the 70-HG15S antibody with a Lynx Rapid HRP Antibody Conjugation Kit (LNK002P, BioRad).

Techniques: Sandwich ELISA, Control, Enzyme-linked Immunosorbent Assay, HBsAg Assay

Journal: Pathogens (Basel, Switzerland)

Article Title: Development of Low-Cost In-House Assays for Quantitative Detection of HBsAg, HBeAg, and HBV DNA to Enhance Hepatitis B Virus Diagnostics and Antiviral Screening in Resource-Limited Settings.

doi: 10.3390/pathogens14030258

Figure Lengend Snippet: Figure 2. Comparison of quantitative in-house ELISA assays for HBsAg (A) and HBeAg (B) detection in plasma samples from HBV-infected individuals (genotypes A–F), evaluated against a commercial ELISA (Abbott Architect). Dilution series (10-, 100-, 500-, 1000-fold) were conducted to determine the minimum plasma volume required for reliable quantification of HBsAg and HBeAg. Statistical analysis via one-way ANOVA with Tukey’s HSD post hoc test revealed significant differences (**) at 1000-fold dilution between groups (p < 0.05), and no significant (ns) differences between other dilution series. Data are presented as mean ± SD from three technical replicates per sample. (C) Summary of 35 samples tested, detailing HBsAg and HBeAg positivity rates across genotypes (A–F), as determined by the Abbott Architect assay. A 10-fold plasma dilution (10 µL in 100 µL of blocking buffer) reliably quantified (D) HBsAg and (E) HBeAg. Notably, HBeAg levels in samples tested from genotypes A and E were below the lower limit of detection (

Article Snippet: We found that the ideal combination was a mouse monoclonal antibody against HBsAg (Fitzgerald, 10-H05H) and goat polyclonal HBsAg antibody (Fitzgerald, 70-HG15S); however, since neither antibody is available commercially with a horse-radish peroxidase (HRP) tag, advanced antibody preparation was required using the 70-HG15S antibody with a Lynx Rapid HRP Antibody Conjugation Kit (LNK002P, BioRad).

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

Journal: Pathogens (Basel, Switzerland)

Article Title: Development of Low-Cost In-House Assays for Quantitative Detection of HBsAg, HBeAg, and HBV DNA to Enhance Hepatitis B Virus Diagnostics and Antiviral Screening in Resource-Limited Settings.

doi: 10.3390/pathogens14030258

Figure Lengend Snippet: Figure 4. Comparison of various DNA isolation methods for determining the EC50 of TDF in HepAD38 cells. HepAD38 cells were seeded in 24-well plates or 96-well plates and treated with TDF for 14 days with media and drug replenishment every 72 h. (A) To determine the toxicity of TDF, HepAD38 cells in the 96-well plate format were incubated with Alamar Blue for 2 h prior to reading the absorbance at 560 nm and 600 nm. Data were normalized according to the manufacturer’s directions to determine cell viability. (B) HBeAg and HBsAg could be quantified from the supernatant of 96-well HepAD38 cells treated with Alamar Blue. Extracellular (cell supernatant, top panel) and intracellular (bottom panel) HBV DNA were determined in (C) 96-well and (D) 24-well plate formats and plotted as % HBV inhibition against log [TDF] to determine the EC50. To investigate if Alamar Blue negatively impacts EC50 determination, HepAD38 cells from the 96-well plate treated with Alamar Blue were also subjected to DNA isolation and quantification. (E) Summary of TDF EC50 values obtained from the treatment of HepAD38 cells in either a 24- or 96-well format. Assay Z′

Article Snippet: We found that the ideal combination was a mouse monoclonal antibody against HBsAg (Fitzgerald, 10-H05H) and goat polyclonal HBsAg antibody (Fitzgerald, 70-HG15S); however, since neither antibody is available commercially with a horse-radish peroxidase (HRP) tag, advanced antibody preparation was required using the 70-HG15S antibody with a Lynx Rapid HRP Antibody Conjugation Kit (LNK002P, BioRad).

Techniques: Comparison, DNA Extraction, Incubation, Inhibition