Dual-Genotype Protein Yields Broader ELISA for Hepatitis B preS1 Antibodies

Hepatitis B virus (HBV) remains one of the most consequential human pathogens, and in China it is a leading driver of hepatocellular carcinoma, the result of persistent viral replication in hepatocytes and the chronic immune responses it provokes. The virus’s envelope carries three surface proteins, and the largest of them, the large surface protein, bears a unique preS1 region that forms the viral protrusion and mediates binding and entry into host cells. Antibodies directed against this preS1 region can neutralize HBV and block infection of new cells. More strikingly, higher serum levels of preS1 antibodies have been linked to faster clearance of hepatitis B surface antigen, raising the possibility that measuring these antibodies could serve as an early serological indicator of infection resolution. Compared with conventional quantitative measurements of HBsAg and HBV DNA, quantifying preS1 antibodies may capture the direct immune response against the virus while also offering information about disease stage and prognosis.

A major complication, however, is viral genetic diversity. Ten HBV genotypes, designated A through J, have been identified, and genotypes B and C dominate in East Asia. In China, more than 95 percent of hepatitis B patients carry genotype B or C virus, with genotype B more prevalent in the south and genotype C in the north. The preS1 sequence varies considerably across genotypes, producing genotype-specific immune responses in patients. Unfortunately, previously available kits for preS1 antibody detection did not account for this genotypic variation, which can yield false-negative outcomes and compromised accuracy when the assay confronts mismatched genotypes. A research team led by investigators at Jilin University has now reported in the journal iScience a quantitative ELISA built on a dual recombinant strategy designed to close this diagnostic gap, covering patients infected with either of the two major Chinese genotypes.

The core of the innovation is a fusion protein the authors call B-C-preS1. Starting from viral DNA isolated from the sera of patients infected with genotype B or genotype C HBV, the researchers amplified the DNA fragments encoding each genotype’s preS1 region by PCR, each yielding a band of roughly 1,200 base pairs on gel electrophoresis. Overlapping PCR then joined the two fragments into a single construct of approximately 2,400 base pairs. This DNA was cloned into a pcDNA3.1(−) vector, amplified in E. coli DH5α cells, and verified by restriction digestion, which released the expected vector and insert bands. The recombinant plasmid was then transfected into HEK293F cells for protein expression. After two rounds of affinity chromatography purification using Ni-NTA and Strep-Tactin resin, purified B-C-preS1 appeared as a single band of roughly 100 kilodaltons on SDS-PAGE. Western blotting with two previously reported genotype-specific antibodies confirmed that the fusion protein cross-reacts with antibodies raised against the preS1 regions of both genotype B and genotype C virus.

With the antigen in hand, the team needed a quantification standard: an antibody that recognizes preS1 epitopes from both genotypes. They immunized Balb/c mice with the B-C-preS1 fusion protein and monitored preS1-specific antibody levels by ELISA against plates coated separately with genotype B preS1, genotype C preS1, or the fusion protein. One mouse produced serum with high and comparable reactivity on all three plate types even after dilution to one part in a hundred million. Peripheral blood mononuclear cells from this animal were subjected to flow cytometric sorting using FITC-labeled B-C-preS1 and a PE-conjugated anti-mouse CD19 antibody to isolate antigen-specific B cells. The variable region genes from these cells were reverse-transcribed, amplified, and cloned into vectors carrying human IgG1 Fc for expression in HEK293F cells. Screening of the expressed antibodies identified one, named HB2, that cross-reacts with both genotype B and C preS1. SDS-PAGE showed a single band for the purified chimeric antibody, which separated into roughly 50- and 25-kilodalton bands after reduction, and size-exclusion chromatography confirmed that the monomer accounted for more than 95 percent of the preparation.

The binding properties of HB2 were characterized in detail. In ELISA, the half-maximal effective concentrations for detecting B-C-preS1, B-preS1, and C-preS1 were 177.27, 177.71, and 179.86 nanograms per milliliter, respectively, indicating essentially equivalent recognition of all three antigens. Surface plasmon resonance across a concentration range of 12.5 to 6,400 nanomolar demonstrated concentration-dependent binding to B-C-preS1, with a dissociation constant of 1.90 × 10⁻⁷ M and a slow dissociation rate of 1.25 × 10⁻³ per second, signifying a strong and stable interaction. These properties made HB2 an ideal calibrator for the assay. The team then systematically optimized every stage of the ELISA protocol through checkerboard titrations: the optimal coating antigen concentration was 2 micrograms per milliliter, the HRP-conjugated anti-human IgG detection antibody worked best at a 1:200,000 dilution, phosphate-buffered saline at pH 8.0 gave the highest positive-to-negative ratio as coating buffer, and overnight coating at 4 °C was optimal. Blocking with a mixture containing 0.2 percent bovine serum albumin, 0.25 percent Tween 20, 4 percent sucrose, and 8 percent goat serum for two hours at 37 °C maximized signal over background, and a 20-fold dilution of patient serum placed most readings within the linear range.

Performance metrics were rigorous. A serial dilution of HB2 produced a linear standard curve with an R² of 0.9939, and the quantitative detection range was set at 10 to 100 nanograms per milliliter. The qualitative cut-off, calculated from healthy donor sera as the mean plus two standard deviations, was 0.0775 optical density units, corresponding to a preS1 antibody concentration of 7.62 nanograms per milliliter, below the lower limit of the quantitative range. Intra-assay coefficients of variation across three independently manufactured kit batches ranged from 5.89 to 8.88 percent, while inter-assay variation ranged from 7.77 to 8.53 percent, both comfortably within the 15 percent precision requirement of Chinese guidelines. Recovery experiments, in which known amounts of HB2 were spiked into patient sera representing high, medium, and low antibody levels, yielded recovery rates between 90.80 and 102.58 percent, confirming good accuracy, particularly for medium-to-high antibody concentrations.

The validation panel comprised 156 patient sera confirmed positive by a commercial qualitative kit, plus 66 sera from healthy individuals. Genotyping by PCR amplification and sequencing of HBV DNA classified 29 sera as genotype B and 127 as genotype C. The home-made ELISA scored all 156 patient sera as positive and all 66 healthy sera as negative, producing an area under the ROC curve of 1. Cross-reactivity testing against hepatitis A virus, hepatitis C virus, and HBV genotypes A and D returned readings below the cut-off, indicating high specificity. Most revealing was the head-to-head comparison with a commercial quantitative kit that does not account for genotype. That kit correctly quantified the genotype C sera, correlating well with the new assay at an R² of 0.9519, but it returned negative results for all 29 genotype B sera, roughly 19 percent of the panel. In other words, the existing commercial assay systematically misses nearly one in five Chinese patients.

Stability testing addressed the practical demands of commercial deployment. Sealed kits stored at 4 °C for a full year retained 91.07 to 98.33 percent of their day-zero performance at month 12, well above the 80 percent threshold required in China. Accelerated testing at 37 °C for ten days showed readouts of 87.2 percent or better, with week-long values between 91.5 and 98.1 percent. Opened kits held at 4 °C retained 90.6 to 100.1 percent of baseline through seven days of use, and simulated transportation involving seven days of shaking followed by repeated drop tests produced no significant change in measured antibody levels. Taken together, the results describe an assay robust enough for real-world clinical logistics, with the authors noting that Beijing Abace Biotechnology has already received approval from China’s Food and Drug Administration for the kit.

Beyond hepatitis B, the authors frame the dual recombinant strategy as a general answer to a persistent problem in virology: rapid mutation and genotypic diversity undermine both nucleic acid detection, which depends on conserved primer and probe binding sites, and protein-based detection, which depends on conserved epitopes. By tandemly expressing key antigenic regions from multiple genotypes in a single molecule, the approach physically assembles characteristic epitopes into one broad-spectrum reagent, simplifying production compared with mixing separate genotype-specific proteins and widening patient coverage in a single reaction. The authors acknowledge limitations, including the need to know genotype sequences in advance, the requirement that mutation rates not be overwhelming, the need to optimize linker design and epitope spacing, and the small number of genotype B sera in this study, which came from northern China where genotype C predominates. Still, they argue the concept could extend to detection reagents, vaccines, and therapeutic antibodies for other highly variable viruses such as influenza, HIV, hepatitis C virus, and SARS-CoV-2, offering a template for keeping viral diagnostics ahead of viral evolution.

Subject of Research: Development and validation of a dual-genotype recombinant ELISA for quantifying hepatitis B virus preS1 antibodies in patients infected with genotype B or C virus

Article Title: A dual recombinant strategy to quantify PreS1 antibody in hepatitis B patients with genotype B or C virus by ELISA

Article References: Li, Y., Wen, X., Zhan, J., Cao, T., Chi, X., Cheng, H., Huang, Q., Zhang, W., Niu, J., Du, H., & Liu, Z. (2026). A dual recombinant strategy to quantify PreS1 antibody in hepatitis B patients with genotype B or C virus by ELISA. iScience, 29(11), Article 117857. https://doi.org/10.1016/j.isci.2026.117857

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Keywords: hepatitis B virus, preS1 antibody, ELISA, genotype B, genotype C, recombinant protein, chimeric antibody, diagnostics, viral genotypes, serology, China, antibody quantification