Secreted and intracellular HBsAg levels were estimated using MONOLISA HBsAg Ultra ELISA kit (BioRad) as described earlier23 (link),28 (link). Cells transfected with HBV construct and treated with DMSO (0.5% final concentration) were used as controls. Percentage Inhibition was calculated using absorbance value of DMSO as control. ELISA was performed in triplicates and data are represented as Mean ± SD and the plots were generated using GraphPad Prism software version 8.4.3. Nonlinear regression curve fitting was performed and IC50 values were calculated using log(inhibitor) vs response-variable slope (four parameters) equation.
Monolisa hbsag ultra elisa kit
Manufactured by Bio-Rad
Sourced in France, United States
The Monolisa HBsAg ULTRA Elisa Kit is a laboratory diagnostic tool used for the detection of hepatitis B surface antigen (HBsAg) in human serum or plasma samples. The kit employs the enzyme-linked immunosorbent assay (ELISA) technique to identify the presence of HBsAg, which is a key indicator of hepatitis B virus (HBV) infection.
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6 protocols using monolisa hbsag ultra elisa kit
1
Quantification of HBsAg Secretion and Inhibition
2
Anti-HBV Activity of Pure Compounds
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The HepG2.2.15 cells were seeded in flat-bottom 96 well plates (0.5 × 105/well in triplicate) and incubated overnight. Based on the cytotoxicity results, and previously reported assays, the treatment dose of 10 μg/ml was chosen for all pure compounds in anti-HBV experiments. Lamivudine (2 μM) prepared in DMSO and RMPI, was included as positive control (Parvez et al., 2006 (link)) while RPMI with 0.1% DMSO served as untreated control. Next day, the old media were replaced with fresh media containing compounds and controls, and the culture was incubated for 5 days as described elsewhere (Arbab et al., 2017 (link)). Culture supernatants were replaced with fresh dose every alternate day and samples collected on day 1, 3, and 5 were saved at −20 °C. The viral HBsAg production in the culture supernatant was analyzed using Monolisa HBsAg ULTRA Elisa Kit (BioRad, USA) as per the manufacturer’s instructions. All data were presented as % inhibition of HBsAg expression in relation to the untreated control. The experiment was repeated to confirm the reproducibility.
3
Serological Markers for Hepatitis B
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Monalisa ELISA kits produced by BIO-RAD laboratories with sensitivity and specificity greater than 99% were used to qualitatively determine the different HBV serological markers. Monolisa HBsAg ULTRA ELISA kit was used to test for the presence of HBsAg, Monolisa Anti-HBs PLUS ELISA kit (BIORAD, Marnes- La-Coquette-France) was used to test for the presence of anti-HBs, Monolisa Anti-HBc PLUS ELISA kit was used to test for the presence of anti-HBc while Monolisa HBe Ag-Ab PLUS ELISA kit (BIORAD, Marnes- La-Coquette-France) was used to test for the presence of anti-HBe.
4
Sensitive HBsAg Detection by ELISA
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HBsAg was tested using the Monolisa HBsAg ULTRA ELISA kit (Bio-Rad, Marnes-La-Coquette, France), a fully multivalent assay with high sensitivity in detecting HBV mutants to determine those who were positive for HBsAg.6 (link) ELISA assay was performed following the manufacturer’s instructions.
5
Antiviral Effects of AM Fractions on HBV
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The HBV cell line, HepG2.2.15, was seeded in 96-well plates (0.5 × 105/well in triplicate), including naïve HepG2 cells as negative control. Next day, the old media were replaced with 100 μL each of four doses (31.25, 62.5, 125 and 250 μg/mL prepared in culture media) of the five AM fractions, including Lamivudine (2.0 μM), and the culture was incubated for 2 days. Culture supernatants of each sample (triplicates) were collected and analyzed for the viral HBsAg and HBeAg using Monolisa HBsAg ULTRA Elisa Kit (BioRad, USA) and HBeAg/Anti-HBe Elisa Kit (DIASource, Belgium), respectively, as per the manufacturer's manual.
6
HBV Screening in Blood Donors
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The blood bank’s screening algorithm identifies a blood donor as HBV-positive using HBsAg rapid diagnostic test (RDT) kit and a conventional semi-automated enzyme-linked immunosorbent assay (ELISA). The Bio-Check Rapid kit test (Bio-Check, San Francisco, California, United States) employs lateral chromatographic flow, while the Monolisa™ HBs Ag ULTRA ELISA kit (Bio-Rad, Marnes-la-Coquette, France) employs a qualitative one-step solid-phase enzyme-linked immunoassay technique of the ‘sandwich’ type. Each test kit came with positive and negative controls used in each assay. First, the rapid test was administered to all donors pre-donation. Donors who were HBsAg-negative by the rapid test were further tested with the semi-automated ELISA test. Then, using one of the two aliquots of sera stored, all samples were tested further for anti-HBc using the HBcAb ELISA Kit (Melsin Medical Co., Ltd, Changchun, China). The anti-HBc assay measures the total anti-HBc, including the IgG and IgM HBc antibodies.
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