Introduction

Hepatitis B caused by infection of hepatitis B virus (HBV) remains a serious global disease, which may eventually lead to liver cirrhosis and hepatocellular carcinoma (HCC)1,2,3. To date, hundreds of HBV sequences have been reported and are grouped into 10 genotypes from A to J4,5,6 with varied geographical distributions7,8. For example, genotype A is prevalent in Western Europe, the United States, Central Africa, and India9, whereas genotypes B and C are mainly found in East Asia, including China, Japan, Korea, etc10,11,12. Genotype D is distributed in Central Asia, Africa, the Mediterranean and India13, while genotype E is in West Africa14. Genotypes F to I together account for less than 2% of global HBV infections8,15. Moreover, it has been noted that countries with a large population of immigrants are more likely to show multiple HBV genotypes16. In China, genotypes B and C dominate together with roughly 90% prevalence7,17.

The virulence of HBV depends on the genotype. Genotypes A and B are more responsive to interferon treatment than genotypes D and C, respectively18,19,20,21. The individuals with genotypes B and C exhibit distinct metabolic fingerprints in serum, suggesting that these two genotypes have different effects in hosts22,23. Genotype C is more virulent with more active replication and frequent mutations, and thus is more likely to develop HCC than genotype B24,25,26,27. Consistently, clinical isolates of genotype C show higher in vitro transmission efficiency than genotype B isolates24. Although genotype B patients exhibit more severe lobular necroinflammation, they turn to undergo earlier seroconversion and suffer less portal inflammation and fibrosis than genotype C patients28. In contrast, genotype C is highly correlated with the development of HCC29.

The genotype classification of HBV is based on the difference of whole HBV genome > 7.5% or the difference of surface protein (S) gene ≥ 4%4,5,30. Hence existing methods for HBV genotyping mainly include polymerase chain reaction (PCR)-based whole genome sequencing and S gene sequencing, nested PCR31, quantitative real-time PCR32, as well as PCR-restriction fragment length polymorphism (PCR-RFLP). The PCR-based sequencing method is time-consuming. PCR-RFLP requires special digestion of PCR-amplified fragments33,34,35. The genotype-specific linear probe detection is a new approach with high specificity36, but improper for large-scale application.

Some scientists have developed rapid protein-based genotyping methods by leveraging antigen-antibody recognition, such as enzyme-linked immunosorbent assay (ELISA)37,38, flow-through reverse dot blotting39, fluorescent lateral flow immunoassay (LFIA)40,41, etc. These antigen-based approaches may have advantages over DNA-based genotyping methods in patients under nucleos(t)ide analog treatments or after seroconversion42.

In this work, we have developed two monoclonal antibodies (mAbs) specific for genotypes B and C of HBV, two major genotypes in China. Then, a sandwiched ELISA kit that can distinguish genotypes B and C of HBV have been established using two pairs of genotype-specific mAbs (Fig. 1a). One pair is commercial 16D12 mAb40 and home-made B19 mAb for genotype B, and the other pair is commercial 6H3 mAb40 and home-made C04 mAb for genotype C. A thorough study has been conducted to compare our ELISA kit with the gold standard of DNA sequencing and a commercial PCR-based genotyping kit. The results demonstrate that ELISA-based genotyping is a reliable and more convenient method for HBV genotyping.

Fig. 1
figure 1

(a) Schematic illustration of the sandwiched ELISA for HBV genotyping. (b-c) Verification of B19 (b) and C04 (c) mAbs by SDS-PAGE. M: protein marker; Lane 1: reduced by DTT (2 ug); Lane 2: without DTT. (d-e) Verification of purified P-B (d) and P-C (e) controls by SDS-PAGE. M: protein maker; Lane 1: unpurified proteins; Lane 2: purified protein. (f) The specificity verification of B19 and C04 mAbs. The original uncropped SDS-PAGE images for (b-e) are provided in Figs. S1-S4.

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Results

Preparation of mAbs and control proteins

The envelope of HBV consists of three surface proteins, i.e. HBV surface (HBS) protein, medium HBV surface (MHBS) protein and large HBV surface (LHBS) protein43,44. MHBS contains one extra segment (pre-S2 region) than HBS, while LHBS contains another segment (pre-S1 region) beyond MHBS. The pre-S1 region unique to LHBS forms the spikes of HBV and is responsible for binding and entering host cells43,45. It has been reported that the serum levels of antibodies in chronic hepatitis B (CHB) patients against the amino acid sequence of 94–117 in pre-S1 region can be used to predict the responsiveness of interferon therapy46. It is also known that CHB patients of genotype B is more responsive to interferon treatment than patients of genotype C47. We have speculated a correlation between these two findings and hypothesized that genotype-specific mAbs can be generated with the peptides corresponding to the amino acids of 94–117 in the pre-S1 regions of HBV genotypes B and C.

Therefore, we prepared two peptides to generate genotype-specific mAbs. One corresponds to the 94–117 amino acids of pre-S1 in HBV genotype B identified in China and is named as P94B. The other represents the 94–117 amino acids of pre-S1 in HBV genotype C identified in China and is denoted as P94C. Then P94B and P94C peptides were coupled to keyhole limpet haemocyanin (KLH) to obtain P94B-KLH and P94C-KLH proteins with enhanced immunogenicity. Splenocytes of Balb/c mice immunized with P94B-KLH and P94C-KLH proteins were extracted and fused with SP2/0 cells. After cross-screening, 9 cell lines specific for P94B peptide and 6 cell lines specific for P94C peptide were identified (Table S1 in Supplementary Information), from which cell line B19 and C04 were selected based on the assessments of specificity and sensitivity. The B19 mAb produced by B19 cells belongs to IgG2b/κ isotype, whereas the C04 mAb from C04 cells is of IgG1/κ isotype (Table S2). The obtained B19 and C04 antibodies were verified by SDS-PAGE, which displayed two clear bands after dithiothreitol (DTT) reduction (Fig. 1b and c and S1-S2). The DNA sequences of variable regions of B19 and C04 mAbs were exhibited in Table S3.

LHBS genes of HBV genotypes B and C were PCR-cloned from patient sera with known genotypes and inserted into the pET-28a vector. Then, two recombinant LHBS proteins were expressed in E. coli and purified as quality controls for the ELISA kit, which were named as P-B (positive control for B) and P-C (positive control for C). Both P-B and P-C proteins display one clear band after purification with His6-tag (Fig. 1d and e and S3-S4) and show high homology with known genotype B and C sequences, especially in the 94–117 region of pre-S1 (Fig. S5). Indirect ELISA was used to verify the specificities of B19 and C04 mAbs on P-B or P-C coated plates (Fig. 1f).

Optimization of the sandwiched ELISA

The sandwiched ELISA involves five major steps (Fig. 1a) including plate coating with first antibodies (i), blocking with sucrose and bovine serum albumin (BSA) (ii), incubation with patient sera (iii), incubation with horseradish peroxidase (HRP)-labeled detection antibodies (iv), and incubation with substrates and plate reading (v). We have focused on steps i, iii and iv to optimize the preparation and detection protocol of the ELISA kit.

Two commercial antibodies 16D12 and 6H3, specific for HBV genotypes B and C respectively, were chosen as the capture antibodies in step i. These two antibodies were diluted in phosphate-buffered saline (PBS, pH = 7.4) and separately coated onto 96-well ELISA plates in an alternating pattern (Fig. 1a) at 4 °C for 16 h. In order to determine the optimal concentrations of capture antibodies and detection antibodies, various antibody combinations of 16D12 with B19 and 6H3 with C04 at different concentrations were used to detect HBV in patient sera with known genotypes (Fig. 2a and b). For genotype B detection, the combination of 4 µg/mL 16D12 and 3 µg/mL B19 yielded the highest ratio of positive readout to negative readout (P/N), while the combination of 3 µg/mL 6H3 and 2 µg/mL C04 gave the best P/N value for genotype C detection. Next, we examined the effects of coating time and temperature on readouts based on these optimal antibody concentrations. For genotype B, 13 sera from genotype B patients (B-1 to B-13) and 2 sera from genotype C patients (C-1 and C-2) were used in detection (Fig. 2c). It is found that the coating at 4 °C for 16 h outperformed the other conditions at 37 °C with time varying from 1 h to 4 h. Overall, the positive readouts for patients B-1 to B-13 increase as the coating time increases at 37 °C, while the negative readouts of C-1 and C-2 remain roughly unchanged. Yet, even at 4 h, the positive readouts at 37 °C are still lower than the counterparts at 4 °C for 16 h. Similar trends have also been observed for the experiment for genotype C (Fig. 2d) with 13 sera from genotype C patients (C-1 to C-13) and 2 sera from genotype B patients (B-1 and B-2). Hence, the coating condition has been determined as 4 °C for 16 h.

Fig. 2
figure 2

(a) Checkerboard titration of 16D12 and B19 mAbs. (b). Checkerboard titration of 6H3 and C04 mAbs. (c-d) Coating time and temperature optimization for genotype B (c) and C (d) detections. (e-f) Dilution optimization for genotype B (e) and C (f) patient sera. (g-h) Optimization of incubation time with patient sera for genotype B (g) and C (h) detections. (i-j) Optimization of incubation time with HRP-labeled detection mAbs for genotype B (i) and C (j) detections. Data presented in (a-f) are the mean values of triplicated experiments. P/N values are calculated from means.

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In order to optimize the dilution factor and incubation time of patient sera for step iii, patient sera of the same known genotypes were pooled together to ensure enough quantity for multiple experiments. First, pooled patient sera of genotypes B and C were diluted in PBS with horse serum (8%) and BSA (0.2%) at ratios varying from 1:9 to 9:1 and then incubated at 37 °C for 1 h in the plates coated at the aforementioned conditions (Fig. 2e and f). The detection with respective mAbs has shown rising positive readouts for both genotype B and C detections, as the dilution factor decreases. However, the negative readouts also exhibit slight increases. Thus, the P/N values of genotype B and C detections display peaks at the dilution of 5:5 (1:1), which is chosen as the serum dilution factor for subsequent experiments. Next, the influence of serum incubation time was investigated in the range of 0.5 h to 2 h (Fig. 2g and h). The positive readouts increase with extended incubation with patient sera. Yet, the negative readouts also show slower elevations, resulting in the highest P/N values at 1-h incubation for both genotype B and C detections. Together the serum incubation conditions have been determined as 1:1 dilution and 1 h at 37 °C.

The incubation time with the HRP-labeled detection mAbs was optimized for step iv at 37 °C, also in the range of 0.5 h to 2 h (Fig. 2i and j). Again, the positive readouts increase for longer incubation with detection mAbs. Yet, the slowly rising negative readouts make P/N values decline after 1-h incubation. Therefore, the optimal incubation with the detection mAbs has been chosen as 1 h at 37 °C.

The sensitivity, specificity and cut-off value of the ELISA kit

To calculate sensitivity and specificity, we first determined the HBV genotypes of more than 700 patient sera by PCR and DNA sequencing, among which 344 and 281 sera were identified as genotypes B and C respectively. Subsequently, these 625 sera were tested by both genotype B and C assays of our home-made ELISA kit (Fig. 3a and b). Based on the corresponding Receiver Operating Characteristic (ROC) analysis with GraphPad Prism (Fig. 3c and d), the cut-off values of optical density (OD) have been determined as 0.1516 and 0.1504 for genotype B and C detections respectively. Moreover, both assays exhibited good sensitivity and specificity. The sensitivity of genotype B detection is 95.35%, with a 95% confidence interval (CI95%) of [92.58–97.12%], while the sensitivity is 96.44% with a CI95% of [93.57–98.06%] for genotype C assay. The specificities of our ELISA kit are 95.37% (CI95% of [92.25–97.28%]) and 95.35% (CI95% of [92.58–97.12%]) for genotype B and C detections respectively. Meanwhile, at these cut-offs, the areas under the curve (AUC) are 0.9593 and 0.9739 for genotype B and C assays respectively, indicating an excellent overall performance for our ELISA kit. In our subsequent registration of the ELISA kit, the cutoffs for both genotype B and C assays are set at the same value of 0.15.

Fig. 3
figure 3

(a-b) Results of 344 genotype B sera and 281 genotype C sera in genotype B assay (a) and genotype C assay (b). (c-d). ROC curves of genotype B assay (c) and genotype C assay (d).

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Performance evaluation of the ELISA kit

We evaluated the performance of our ELISA kit on four aspects of repeatability, stability, anti-interference capability, and cross-reactivity. For repeatability, we tested the intra-assay and inter-assay repeatability with 2 sera of each genotype on the same day using three batches of ELISA kits manufactured at different time. The intra-assay and inter-assay coefficients of variation (CV) are summarized in Table 1. It is evident that the CVs for the positive controls (< 3%) are lower than that of the patient sera, probably because of their simpler composition. For the sera, the inter-assay CVs of the three batches for B assay ranged from 2.90 to 7.62%, while for C assay, the CVs varied between 1.87% and 5.90%. In terms of inter-assay, the CVs of C assay (1.73–4.94%) were slightly smaller than that of B assay (2.47–5.64%). Together, both intra-batch and inter-batch variations of our kit are below 8%, which is significantly lower than the standard of 15%, indicating satisfactory repeatability.

Table 1 Intra-assay and inter-assay CVs for the ELISA kit.
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The stability of our ELISA kit has been evaluated in four scenarios: accelerated stability, in-use stability, simulated transportation stability, and real-time storage stability. For each stability test, 6 genotype B sera and 6 genotype C sera were used in both B and C assays. Overall, no false positive cases were found and the OD values of all intended negative controls were well below the cut-off of 0.15. Hence, the stability evaluation was based on the positive results of each test.

For accelerated stability test, three batches of kits were kept at 37 °C for 10 days. The kits were tested on Day 0, 3, 7 and 10 (Fig. 4a and b). The OD values on Day 7 and 10 were normalized to the respective readouts on Day 0. It is found that on Day 7, the ratios of OD values ranged from 88.1 to 104.0% (B assay) and 83.50–99.6% (C assay), indicating an acceptable stability within one week at 37 °C. In contrast, a decline of the OD ratios was observed on Day 10, suggesting that the kit may have expired. Thus, we have estimated that our kit has a shelf life of at least one year at 4 °C, according to a previous report using the Arrhenius equation48.

For in-use stability test, the kits were kept at 4 °C and tests were performed on Day 3, 7, 10, 14, 18, 25, 35 and 40 after unpacking (Fig. 4c and d). Again, the OD values on Day 40 were normalized to the respective readouts on Day 0, which showed OD ratios within the ranges of 88.1-96.1% (B assay) and 84.5-99.1% (C assay) (Fig. 4c and d), exhibiting favorable in-use stability.

For simulated transportation stability test, kits of three batches were shaken on a rocker at 60 r/min for 7 days, and then fell from a height of 1.5 m for 3 times. The difference in OD values before and after the simulated transportation was minimal (Fig. 4e and f), indicating an excellent stability in transportation.

In order to test real-time storage stability, kits of three batches were kept at 4 °C for 1 year. The ELISA kits were tested at the ends of Months 3, 6, 9 and 12 (Fig. S6). The OD values slowly decline in 1 year as expected, but are well above the set threshold, which is 80% of the original OD values with freshly prepared kits. We then predicted the shelf life of the kit with Minitab Statistical Software, by projecting the OD values to the threshold line (lower specification of 2.4). It is found that the shelf lives of B and C assays are 26.0 and 23.5 months respectively (Fig. S6), which is in line with the shelf life of more than 1 year estimated by the accelerated stability test.

We have also investigated the stability of patient sera after repeated freeze-thaw cycles in order to provide a reference to the users of our kit. Patient sera before freeze-thaw cycling (Cycle 0) and after 1, 4 and 7 freeze-thaw cycles were tested with our home-made kit (Fig. S7). No significant difference was found between samples of Cycle 0 and Cycle 1. Yet, 7 out of 12 patient sera exhibited significant decline after 4 freeze-thaw cycles and all 12 sera showed significant differences after 7 cycles. Hence, to ensure the accuracy of the detection with our ELISA kit, it is advisable to minimize the freeze-thaw cycles of the sera.

Fig. 4
figure 4

(a-b) Accelerated stability tests of B assay (a) and C assay (b). (c-d) In-use stability tests of B assay (c) and C assay (d). (e-f) Simulated transportation stability tests for B assay (e) and C assay (f). (g-h) Anti-interference capability tests of B assay (g) and C assay (h). (i) Cross-reactivity tests of B assay and C assay.

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To evaluate the anti-interference capability of the ELISA kit, we assessed the impacts of 7 substances on the positive and negative detections, which include cholesterol (CHOL) (113.76 mmol/L), bilirubin (BIL) (6.84 mmol/L), triglycerides (TG) (156.48 mmol/L), hemoglobin (HGB) (4 mg/mL), albumin (ALB) (600 mg/mL), rheumatoid factor (RF) (174 IU/mL), and human anti-mouse antibody (HAMA) (26.8 ng/mL). These interferent solutions were serially diluted to obtain 4 dilutions (Fig. 4g and h). The undiluted and diluted interferent solutions were introduced into the patient sera and then detected with our ELISA kit. It is found that these interferents have no significant effect on negative readouts. For positive detections, a limit of 15% change was set as the threshold, which corresponded to the variation of OD readouts between 85% and 115% of the controls without interferents. The anti-interfering concentrations of these 7 substances have been determined by using this criterion. For example, when the concentration of CHOL is lower than 14.22 mmol/L (16x dilution), the variations of positive readouts of both B and C assays are within 85–115% of the OD values of non-interfered counterparts. When the concentration of CHOL is larger than 14.22 mmol/L, the OD values of some positive detections exceeds the set range. So, the anti-interfering concentration of CHOL was estimated as 14.22 mmol/L. Similarly, the anti-interfering concentrations of BIL, TG, HGB, ALB, RF and HAMA were determined as 3.42 mmol/L, 39.12 mmol/L, 2 mg/mL, 75 mg/mL, 43.5 IU/mL and 13.4 ng/mL respectively.

The cross-reactivity of our kit has been investigated with 7 other viruses including hepatitis A virus (HAV), hepatitis C virus (HCV), cytomegalovirus (CMV), treponemiapallidum (TP), human immunodeficiency virus (HIV), HBV genotype A (HBV-A) and HBV genotype D (HBV-D). Overall, the OD values are below the cut-off of 0.15 for all these tests (Fig. 4i), indicating low cross-reactivity of our kit with these examined viruses. It should be noted that HBV-A and HBV-D show larger OD values than the other five, which can be rationalized by their closer relationship with HBV genotypes B and C in evolution. In particular, the OD value of HBV-D in C assay is higher than that in B assay, presumably because the 6H3 mAb used in C assay cannot distinguish between genotypes C and D of HBV40.

Validation of ELISA-based genotyping with human sera

We have conducted two experiments with human sera to verify our ELISA kit for HBV genotyping. First, 307 HBV-negative sera from people without HBV infection in history were tested by the ELISA kit. None of them was tested positive in our assay (Fig. 5a), indicating a low false-positive rate for our kit.

Fig. 5
figure 5

(a) The detection of 307 HBV-negative sera from people without HBV infection in history using the ELISA kit (The average OD values are 0.0126 for B assay and 0.0314 for C assay. The standard deviations are 0.0168 for B assay and 0.0179 for C assay). (b) The genotyping results of 77 sera with HBV DNA ≥ 1000 IU/mL and HBsAg ≥ 250 IU/mL by three methods.

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Next, 135 sera from patients with CHB were genotyped by three methods in parallel, including DNA sequencing, our ELISA-based kit, and a PCR-based kit commercially available from Shanghai ZJ Biotech. For DNA sequencing method, nested PCR with two pairs of primers was used to amplify the whole pre-S1 (2848–3204) and pre-S2 (3205–3215,1-154) gene of HBV and then the amplified fragments were sequenced. The obtained sequences were BLAST with the sequences in National Center for Biotechnology Information (NCBI) to identify the genotypes of HBV. The commercial PCR kit was based on real-time quantitative PCR (RT-qPCR) with primers specific for HBV genotypes B, C and D. The counts of successful cases by three methods were summarized in Table 2. Overall, more patients could be genotyped by our ELISA kit (114/135) than by the other two methods (87/135 and 82/135). Then, these 135 patient sera were divided into 6 sub-groups according to their HBV DNA and HBV surface antigen (HBsAg) levels. For the 39 sera with HBV DNA ≤ 50 IU/mL, no successful case was found for DNA sequencing and the commercial PCR kit. Yet, 17 out these 39 sera had HBsAg ≥ 250 IU/mL and 15 of these 17 could be successfully genotyped by our ELISA kit. For the 23 sera with HBV DNA in the range of 50-1000 IU/mL, the successful detection rates for DNA sequencing and the commercial PCR kit were still as low as 39.1% (9/23) and 26.1% (6/23) respectively. Interestingly, the majority of these sera (22/23) had high levels of HBsAg (≥ 250 IU/mL) and thus could be genotyped by our ELISA kit. Two DNA-based methods did yield high success rates for the 83 sera with HBV DNA ≥ 1000 IU/mL, which were 94.0% (78/83) for DNA sequencing and 91.6% (76/83) for the commercial PCR kit. However, our ELISA kit also exhibited a success rate of 91.6% (76/83) as good as the commercial PCR kit, since the majority of these sera (77/83) also had high levels of HBsAg and our kit had a very high success rate in these sera (76/77). Indeed, there were 116 sera with HBsAg level ≥ 250 IU/mL in total and our kit demonstrated a success rate of 97.4% (113/116) for them.

Table 2 Successful case counts of genotyping 135 patient sera by three methods.
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The detection limit claimed by the commercial PCR kit is at HBV DNA of 1000 IU/mL. Hence, we have chosen the 77 sera with HBV DNA ≥ 1000 IU/mL and HBsAg ≥ 250 IU/mL to compare the genotyping accuracies of these three approaches (Fig. 5b) The ELISA kit again demonstrated higher sensitivity, with a detection rate of 98.7% (76/77), which was higher than 93.5% (72/77) for DNA sequencing and 92.2% (71/77) for the commercial PCR kit. DNA sequencing failed to genotype 5 sera and yielded single genotype for 72 sera, including 26 genotype B and 46 genotype C sera. In contrast, mixed HBV infections by genotypes B and C were identified by both our ELISA kit (4 cases) and the commercial PCR kit (6 cases). Besides, 28 genotype B and 44 genotype C have been identified by the ELISA kit (only 1 negative case), whereas the commercial PCR kit has yielded 27 genotype B and 38 genotype C with 6 negative cases. Subsequently, the kappa statistical analysis was performed with these data at 95% confidence to investigate the correlation of these three methods. The kappa values are 0.836 (DNA sequencing vs. the ELISA kit), 0.692 (DNA sequencing vs. the commercial PCR kit), and 0.744 (the ELISA kit vs. the commercial PCR kit), indicating that our ELISA kit is more reliable than the commercial PCR kit.

We then compared the genotyping results of the 72 sera genotyped by DNA sequencing one by one and found that 58 sera were given consistent results by these three methods. Genotyping results of 14 sera showed discrepancies (Table 3). Among these 14 sera, 3 sera were identified as genotype B by both DNA sequencing and our ELISA kit, which yielded 2 negative results and 1 mixed infection in the test with the commercial PCR kit. It has also been noted that for the mixed infection with the commercial PCR kit, genotype B is more dominant than genotype C. Meanwhile, for the 11 sera reported as genotype C by DNA sequencing, 5 sera were also detected as genotype C by our ELISA kit. In addition, our ELISA kit showed 4 cases of mixed infection out of these 11 sera, which we considered as similar results. So, our ELISA kit actually only yielded two different results from DNA sequencing for these 11 sera: in one case, our ELISA kit identified genotype B, same as the commercial PCR kit; in the other case, our ELISA kit reported a negative result. In contrast, the commercial PCR kit only shared 2 same results (Sera 12 and 14) and 5 similar results (Sera 3 and 7–10) with DNA sequencing. Overall, the detailed comparison indicates that our ELISA kit has good consistency with the method of DNA sequencing.

Table 3 Genotyping discrepancies by these three methods.
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Last but not least, we have investigated the lowest levels of HBsAg and PreS1 for our ELISA kit to give positive genotyping results, given the fact that our ELISA kit shows a low success rate for patient sera with HBsAg < 250 IU/mL (1 out of 19 in Table 2). It is determined that the lowest level of HBsAg for our ELISA kit to yield a success rate higher than 90% is 90 IU/mL by the commercial quantitative kit for HBsAg from Abbott (Table S5). Meanwhile, the lowest level of PreS1 protein that can be genotyped in our ELISA assay is estimated as 400 ng/mL based on the assays with the positive control proteins of P-B and P-C (Fig. S8).

Discussion

HBV infection remains a serious global issue and has evolved to produce various HBV genotypes49,50. Although it is known that the infection of different HBV genotypes may vary in disease progression and treatment responsiveness51,52,53,54, HBV genotyping is rarely done in clinics. For one reason, it is because the gold standard of DNA sequencing is not convenient to be applied in clinical settings, which requires reliable primers for PCR, excellent PCR techniques, efficient DNA sequencing and manual sequence alignments. For another reason, genotype-specific treatment of HBV is still lacking. Hence, physicians are usually unwilling to go through so tedious a process to obtain a genotyping result that has little relevance in choosing the treatment for patients. Such a dilemma is indeed a “hen-and-egg” situation. Only with a quick and cheap genotyping method, can HBV genotyping be widely used in clinics, which can in turn generate enough data to formulate genotype-specific regimen for CHB patients and then promote broader use of HBV genotyping. The breakthrough of HBV genotyping may also set a good example for precision treatment of other diseases caused by virus infection.

To our delight, a company has taken the lead to develop an HBV genotyping kit based on RT-qPCR with genotype-specific primers. Such a DNA-based approach, together with DNA sequencing, will yield information on recombinant viruses, antiviral resistance and HBS antibody escape mutations. However, this genotyping kit shows low sensitivity and accuracy in our study, probably for two reasons. First, some patient sera have low HBV DNA levels, which makes PCR amplification difficult. In our 135 patient sera, HBV DNA levels of 52 sera are < 1000 IU/mL (Table 2) and the failure rate of the PCR-based genotyping is as high as 88.5% (46/52). Second, the PCR-based genotyping depends on genotype-specific primers. Yet, HBV is constantly evolving in patients and mutations could occur in the annealing sites of these primers, which may also cause failure of PCR amplification55.

We have intended to develop a protein-based HBV genotyping method by leveraging the widely available ELISA. HBV proteins like HBsAg (including HBS, MHBS and LHBS) are commonly more abundant in CHB patients and thus easier to detect. In particular, it is known that the immune control phase of HCC is characterized by low levels of HBV DNA (usually < 2000 IU/mL), while retaining high HBsAg levels56,57. Thus, protein-based HBV genotyping has better chance to work in this scenario, where DNA-based genotyping is difficult. In our work, the ELISA-based genotyping has demonstrated higher overall detection rate (114/135) than the other two DNA-based methods (87/135 and 82/135). More specifically, for the sera with HBV DNA < 1000 IU/mL, our ELISA kit has a success rate of 73.1% (38/52), whereas the DNA-based methods hardly work (< 20%).

Moreover, the actual virus population in one patient may have multiple variants. Yet, the standard method of DNA sequencing usually gives one genotype as the best hit of BLAST results. So, no mixed infection has been denoted by DNA sequencing. In contrast, both our ELISA kit and the commercial PCR kit have detected mixed infections by HBV genotypes B and C, which may be a better representation of the real-world situation. In Fig. 3a, there are 13 out of 281 genotype C sera exhibiting OD higher than 0.15 in genotype B assay, whereas in Fig. 3b, there are 17 out of 344 genotype B sera exhibiting OD higher than 0.15 in genotype C assay. We have identified these 30 sera and measured their HBsAg as listed in Table S6. When these sera are ranked by the HBsAg level, we can’t find a correlation between the HBsAg level and the cross-reactivity with the other genotype assay. In our opinion, the cross-reactivity of a serum in both genotype assays is likely due to two reasons. On one hand, HBV is constantly evolving and some HBV clones may carry a sequence that is between the ‘classic” genotype B and C sequences. For example, the PreS1 94–117 sequence of subgenotype C2 and isolate C3 (both from China) differs from P94C in 2 amino acids, but also differs from P94B in 2 amino acids (Table S7). In order to avoid that such a virus yields a positive signal in genotype B assay, we have also chosen genotype specific antibodies as capturing antibodies. The capturing 16D12 mAb is specific for genotype B40, while the capturing 6H3 mAb is specific for genotype C40. Although we believe such a double-check with genotype-specific capturing and detecting antibodies can give highly genotype-specific response, there is still a little chance for certain HBV clones that resemble both genotype B and C to cross-react in both genotype assays. On the other hand, the cross-reactivity in both genotype assays can partially be explained by the mixed infection of genotypes B and C virus. For the case of co-infections, PCR may amplify two genotypes or some unintended sequences and thus produce messy sequencing graphs (as illustrated in Fig. S9 for #9 of genotype C serum). But DNA sequencing can only yield the sequence of one genotype as the outcome and thus the co-infected serum is very likely to be mis-labeled with one genotype by DNA sequencing. It should also be noted that although clonal analysis would allow identification of mixed infections, it would not meet the need of quick and large-scale detection in clinics. Actually, it was also one reason for us to develop a genotyping kit based on ELISA, which may have a better chance to identify co-infections in the real world. To this end, we have chosen 6 sera of genotype B and 6 sera of genotype C representing low to high levels of PreS1 antigens (Table S8) and mixed them at 1:1 ratio in both normal order (forward mixed pair) and reverse order (reverse mixed pair). The test results of these 12 mixed sera with our ELISA kit indicate that our ELISA kit can indeed detect co-infections (Table S8).

Yet, there exist several limitations for our ELISA-based approach. First, although the ELISA-based approach has the advantages of higher success rate and easier procedure, the development of genotype-specific antibodies is time-consuming and the specificity of the antibodies may be impaired by recombinant viruses.

Second, our ELISA-based approach may fail to determine the genotype for HBV carrying mutations in the amino acid sequence of PreS1 94–117, given that our immunogens for genotype-specific antibodies were made based on these sequences. To reduce this risk, we retrieved 49 subgenotypes/isolates across 9 major HBV genotypes from GenBank and compared their sequences of PreS1 94–117 (Table S7) when designing the immunogens. The peptide we used to generate genotype B specific antibody belongs to subgenotype B2, which is the same as those of isolate B19-B21. Meanwhile, the peptide to generate genotype C specific antibody is of isolate C1, which has larger variation from B2 than the other C-isolates in Table S7. C1 differs from B2 for 4 amino acids: T97S, R103K, L108I and T115S. Nevertheless, it has been reported that the mutations in the PreS1 94–117 region are rare58. The alignments of our P94B and P94C peptides with the hits in GenBank (Fig. S10) also corroborate with this report.

Third, our ELISA kit is mainly intended for chronic hepatitis B cases with high levels of HBsAg. In theory, PreS1 should be present at a lower level than HBsAg. Hence, our ELISA kit shows a low success rate for patient sera with HBsAg < 250 IU/mL (1 out of 19 in Table 2). Yet, it should be noted that it is not unusual to find low HBV DNA levels with high HBsAg levels, especially in chronic hepatitis B59, where our ELISA kit may find better use than PCR-based approaches. Besides, our ELISA kit may also be used to genotype HBV in patients with low HBV DNA under nucleos(t)ide analog treatments, where PCR-sequencing method may not be applicable.

In summary, we have developed an ELISA-based method for HBV genotyping by using two pairs of genotype-specific antibodies for genotypes B and C of HBV, two major genotypes in China. Such a protein-based approach has demonstrated high successful detection rates independent of HBV DNA level. The genotyping results of our ELISA-based method are also in good agreement with the results by DNA sequencing. Hence, the protein-based approach can be used in clinics to complement or replace DNA-based genotyping, especially for patients with low HBV DNA level. Currently, World Health Organization (WHO) only recommends treatment for patients with HBV DNA exceeding 2000 IU/ml and HBV genotyping is not in the WHO guidelines for HBV diagnosis and treatment. Although our ELISA kit still requires extensive clinical evaluation, it is a situation of “hen and egg”. Indeed, we hope the broader use of this easier and cheaper method can generate enough clinical data for WHO to formulate genotype-specific treatment for hepatitis B patients.

Materials and methods

Preparation of mAbs

Balb/c mice were purchased from Vital River (Beijing, China) and immunized with P94B-KLH and P94C-KLH proteins in Abace Biotechnology (Beijing, China) to generate genotype-specific antibodies. All animals were housed and treated in accordance with protocols approved by Abace Biotechnology (Approval No: ABACE20151115-001. The original approval document in Chinese is available from the authors). Animal studies were conducted following relevant institutional and national guidelines and regulations, and this work adhered to ARRIVE guidelines.

P94B peptide (PASTNRQSGRQPTPLSPPLRDTHP) corresponding to the amino acids of 94–117 in the pre-S1 region of HBV genotype B (GenBank ID AAF87838.1) and P94C peptide (PASSNRQSGKQPTPISPPLRDSHP) corresponding to the amino acids of 94–117 in the pre-S1 region of HBV genotype C (GenBank ID AAL25952.1) were prepared by solid-phase peptide synthesis (Abace Biotechnology, Beijing, China). Then the P94B and P94C peptides were coupled to KLH using Imject™ Maleimide-Activated mcKLH kit (ThermoFisher, USA) and mixed with Complete Freund’s Adjuvant (MP Biomedicals) in equal volume. The obtained mixtures with adjuvants were immunized into 6 Balb/C mice (60 µg, 200 µL/mouse). 3 more doses of the mixtures (30 µg, 200 µL/mouse) were injected into the mice on Day 14, 30 and 55 to boost the immunizations. The respective antibody levels in the sera of immunized mice were titrated 7–10 days after each immunization until a high titer of the respective antibody was reached. Then one mouse immunized with P94B-KLH and one mouse immunized with P94C-KLH were intraperitoneally injected with 50 ng of respective protein for shock immunization. Mouse splenocytes were extracted three days after the shock immunization and fused with SP2/0 cells (Sigma-Aldrich, USA). Single cell clones were picked 12 days after fusion and cultured in 96-well plates. The antibodies in the supernatants of culture media were screened by indirect ELISA with plates coated with P94B and P94C peptides and then counter-screened with plates coated with P94C and P94B peptides respectively. 9 cell lines specific for P94B peptide and 6 cell lines specific for P94C peptide were identified. Then B19 and C04 cell lines were selected based on the assessments of specificity and sensitivity. The mAbs produced by these two cell lines were isolated and reduced by DTT (2 µg). The mAbs before and after DTT reduction were subjected to SDS-PAGE to verify the purified mAbs. The purified mAbs were sequenced by Chiyao Biotech (Beijing, China). The isotype of the obtained B19 and C04 mAbs were determined using goat anti-mouse isotype-specific antibodies (Southern Biotech, USA). Finally, the purified B19 and C04 mAbs were coupled to HRP as detection antibodies.

The specificity of B19 and C04 mAbs were verified with the control proteins, P-B and P-C, by indirect ELISA. P-B and P-C (5 µg/mL, 100 µL) were coated on the plate at 37 ℃ for 1 h. Then, HRP-conjugated B19 and C04 mAbs (4 µg/mL, 100 µL) were added and incubated at 37 ℃ for 1 h. Next, 100 µL 1-Step™ Ultra TMB-ELISA Substrate Solution (TMB) (ThermoFisher, USA) was added and incubated for 15 min. 50 µL 2 M H2SO4 (Sigma-Aldrich, USA) was added to stop the reaction. The plates were read with a plate reader (MOLECULAR DEVICES, SpectraMax Absorbance Reader, CMax Plus, BK-L96C) at the wavelengths (λ) of 450 nm and 630 nm. The OD values were determined as OD450 – OD630.

Preparation of control proteins

Recombinant P-B and P-C proteins representing the amino acids of 12–119 in the pre-S1 region, 1–55 in the pre-S2 region, and 1-186 in the S region of HBV genotypes B and C (349 amino acids in total) were cloned from patient sera and expressed in E. coli. First, genomic HBV DNA was extracted from patient sera with known genotypes by TaKaRa MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0 (TaKaRa, Japan) following the manufacturer’s protocol. PCR was conducted to amplify the targeted regions with genotype-specific primers. The primer sequences for P-B and P-C proteins are shown in Table S9. All primers used in this study were designed utilizing PRIMER (PREMIER Biosoft International, USA) and synthesized by Sangon Biotech (Beijing, China). The PCR amplification was performed by Sangon Biotech (Beijing, China). The PCR-amplified fragments and the pET-28a vector with His6-tag (Sigma-Aldrich, USA) were purified after overnight digestion with NdeI and HindIII restriction enzymes (ThermoFisher, USA), mixed at a ratio of 3:1 and then incubated with T4 DNA Ligase (New England Biolabs, USA) for overnight ligation at 16 °C to generate recombinant plasmids. Then, the pET-28a-P-B and pET-28a-P-C plasmids were separately transformed into E. coli DH5α (Tsingke Biotech, Beijing, China). Single cell clones were picked and inoculated into Luria-Bertani (LB) medium containing 50 mg/L kanamycin. The recombinant plasmids were extracted using Endo FREE Maxi Plasmid Kit (Tiangen Biotech, Beijing, China) and sequenced by Sangon Biotech (Beijing, China). The obtained sequences were aligned with the top 20 hits with known genotypes from the BLAST results. Next, the recombinant plasmids of correct sequences were transformed into E. coli BL21(DE3) (Tsingke Biotech, Beijing, China). Single cell clones were selected and inoculated into LB medium containing 50 mg/L kanamycin. Isopropyl β-D-1-thiogalactopyranoside (0.5 mM/L) (Sigma-Aldrich, USA) was added into the culture to enhance the expression of inserted gene. The His6-tagged P-B and P-C proteins were purified by Ni2+-NTA affinity chromatography and then verified by SDS-PAGE.

Serum collection

Sera were collected from CHB patients at the First Hospital of Jilin University (Changchun, China). This study was approved by the ethical commission at the First Hospital of Jilin University (Approval No: 19K111-001. The original approval document in Chinese is available from the authors) and complied with the principles of the Declaration of Helsinki. All participants signed informed consent forms. HBV DNA levels of patient sera were measured with RT-qPCR kit (Roche, USA) by following manufacturer’s protocol at the First Hospital of Jilin University (Changchun, China). HBV HBsAg levels of patient sera were measured with HBsAg Quantitative kit (Abbott, USA) by following manufacturer’s protocol.

Genotyping by DNA sequencing

HBV DNA was extracted from patient sera by using TaKaRa MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0 (TaKaRa, Japan). The extracted viral DNA was amplified by nested PCR. The sequences of the two pairs of primers for nested PCR are shown in Table S9. The first pair of primers (P-1-Foward and P-1-Reverse) was only used in amplification and the second pair of primers (P-2-Foward and P-2-Reverse) served in both amplification and sequencing. The nested PCR and sequencing of the PCR-amplified fragments were performed by Sangon Biotech (Beijing, China). The obtained sequences were BLAST in NCBI to identify HBV genotypes.

Genotyping by PCR-based method

The PCR-based method was conducted with a commercial PCR-genotyping kit for HBV (Shanghai ZJ Biotech, Shanghai, China) by following manufacturer’s protocol. Briefly, HBV DNA was extracted from patient sera with the extraction buffer of the kit. Then a pre-mixed solution containing fluorescent-labeled primers was added to the extracted DNA and RT-qPCR was performed on an ETC811 Thermal Cycler (Ultrassay BioTech, China). HBV genotyping results were read by QuantStudio™ Design & Analysis Software (ThermoFisher, USA).

Optimization of the sandwiched ELISA

To determine the optimal concentrations of capture antibodies and detection antibodies, various antibody combinations of 16D12 with B19 and 6H3 with C04 at different concentrations were used to detect HBV in patient sera with known genotypes by checkerboard titration. The patient sera identified as genotype B by DNA sequencing were used as the positive samples for genotype B assay but negative samples for genotype C assay, and vice versa. Two commercial antibodies, 16D12 and 6H3 mAbs (Cnpair Biotech, Hangzhou, China), were coated on the ELISA plate at 0.5, 1, 2, 3, 4, 5 and 6 µg/mL (100 µL/well), and two home-made genotype-specific antibodies B19 and C04 were used at 1, 2, 3 and 4 µg/mL in detection (100 µL/well) respectively. The highest P/N values were used to determine the optimal concentrations of capture antibodies and detection antibodies.

To optimize the coating condition, 16D12 (4 µg/mL) and 6H3 (3 µg/mL) mAbs were coated on the ELISA plate at 4 °C for 16 h and 37 °C for 1 h, 2 h, 3 h and 4 h. 13 positive sera and 2 negative sera were chosen for each of B and C assays. The incubation with sera (30 µL/well without dilution) and detection antibodies (3 µg/mL for B19 and 2 µg/mL for C04) were conducted at 37 °C for 1 h. The highest positive readouts were used to determine the optimal coating condition.

To optimize the serum dilution, pooled patient sera of genotypes B and C were diluted in PBS with 8% horse serum and 0.2% BSA at ratios varying from 1:9 to 9:1. ELISA plates were coated with 16D12 (4 µg/mL) and 6H3 (3 µg/mL) at 4 °C for 16 h and then 100 µL of diluted patient sera was added to each well and incubated at 37 °C for 1 h. The incubation with detection antibodies (3 µg/mL for B19 and 2 µg/mL for C04) was also conducted at 37 °C for 1 h. The highest P/N values were used to determine the optimal serum dilution.

Similarly, the incubation times with the sera and HRP-labeled detection mAbs were optimized based on the above optimal conditions. Briefly, patient sera of 1:1 dilution were added to coated ELISA plate (100 µL/well) and incubated at 37 °C for 0.5 h, 1 h, 1.5 h and 2 h. For detection antibody incubation, HRP-labeled B19 (3 µg/mL) and HRP-C04 (2 µg/mL) were added (100 µL/well) and incubated at 37 °C for 0.5 h, 1 h, 1.5 h and 2 h. The highest P/N values were used to determine the optimal incubation times with the sera and detection antibodies.

The protocol for the home-made ELISA kit

After optimizing various conditions, the protocol for the home-made ELISA kit was established as followings. 16D12 mAb (4 µg/mL, 100 µL/well) specific for HBV genotype B and 6H3 mAb (3 µg/ mL, 100 µL/well) specific for HBV genotype C diluted in PBS (1x, pH = 7.4) were alternatively coated onto 96-well ELISA plates and incubated at 4 °C for 16 h. The wells were washed 4 times with 1x PBST (300 µL/well) containing 0.05% Tween-20 (Sinopharm Chemical Reagent, Beijing, China). Then blocking buffer (1x PBS) with 0.2% BSA (Bovogen Biologicals, Australia), 0.1% Tween-20, 8% horse serum (Biological Industries, Israel), 4% sucrose (Sinopharm Chemical Reagent, Beijing, China), and 0.1% ProClin300 (Sinopharm Chemical Reagent, Beijing, China) was added (180 µL/well) and incubated at 37 °C for 2 h. Next, the wells were washed 4 times with 1x PBST (300 µL/well) and the ELISA plates were air-dried at 25 °C for 4 h (relative humidity < 30%). The air-dried plates were packaged with vacuum sealing machine. Other reagents (sample diluent, positive controls, B19 mAb, and C04 mAb) were also packaged together. The sample diluent is 1x PBS containing 0.2% BSA, 0.1% Tween-20, 4% horse serum, 0.3% NaCl (Sinopharm Chemical Reagent, Beijing, China), 0.1% ProClin300 and 0.02‰ phenol red (Sigma-Aldrich, USA). The concentrations of positive controls, P-B and P-C, are both 5 µg/mL in sample diluent. The concentrations of HRP-B19 and HRP-C04 mAbs are 3 µg/mL and 2 µg/mL respectively, diluted in 1x PBS containing 0.2% BSA, 0.1% Tween-20, 8% horse serum, 0.3% NaCl, and 0.1% ProClin300. The assembled ELISA kits were stored at 4 °C. Before sample testing, the ELISA kits were kept at room temperature for 30 min. Then patient sera were added (50 µL/well) with 50 µL of sample diluent (1:1 dilution) to both genotype B assay and C assay wells, and incubated at 37 °C for 1 h. Subsequently, the wells were washed 4 times with 1x PBST (300 µL/well). The B assay and C assay wells were incubated with HRP-B19 (3 µg/mL, 100 µL/well) and HRP-C04 (2 µg/mL, 100 µL/well) at 37 °C for 1 h respectively. The wells were washed 4 times with 1x PBST (300 µL/well). Next, TMB was added (100 µL/well) and incubated at 37 °C for 10 min. The reaction was stopped with 2 M H2SO4 and the plates were read with a plate reader at the wavelengths (λ) of 450 nm and 630 nm. The OD values were determined as OD450 – OD630.

Performance evaluation of the ELISA kit

To calculate the intra-assay and inter-assay coefficients of variation (CVs), 2 positive sera of each genotype were tested (16 duplicate wells/serum for the evaluation of intra-assay variation) on the same day with three batches of ELISA kits manufactured at different time (for the evaluation of inter-assay variation). For accelerated stability, in-use stability, simulated transportation stability, and real-time storage stability, 6 genotype B sera and 6 genotype C sera were tested in triplicates in both B and C assays. The threshold for stability tests was set as 80–120% of the original OD values with freshly prepared kits. To evaluate the stability of patient sera after repeated freeze-thaw cycles, the collected sera were frozen at -20 ℃, thawed at 4 ℃, and immediately tested after indicated cycle. For anti-interference capability tests, CHOL, BIL, TG, HGB, ALB were purchased from Sigma-Aldrich, USA, whereas RF and HAMA were purchased from Meridian, USA. These interferent were added into the sera at indicated concentrations with or without designated dilutions. For cross-reactivity tests, HAV, HCV, CMV, TP, HIV, HBV-A and HBV-D were obtained from the positive controls of respective commercial kits (GBI Biotech, Beijing, China). These positive controls were 1:1 diluted (same as the patient sera) and then detected by the ELISA kit in triplicates following the same protocol for the patient sera. The sera with viral infection that were serendipitously identified from supposedly “healthy” people were also tested in triplicates in the cross-reactivity tests, including 4 samples for HAV, 8 samples for HCV, 2 samples for CMV, 3 samples for TP, 3 samples HIV, and 4 samples HBV-A. All tests were conducted according to the established protocol of the home-made ELISA kit.

Statistical analysis and software

Data are presented as mean ± standard deviation (SD) where applicable. Significance for the differences were determined by One-way ANOVA. Statistical significance is denoted as *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001.

ROC curves were plotted and analyzed by GraphPad Prism (Version 8.3.0) to determine the cut-off value, sensitivity, specificity, and AUC. Kappa statistics analysis was performed with IBM SPSS Statistics (Version 26) to evaluate the correlation among three different genotyping methods. ESPript (Version 3.0)60 was used for protein sequence alignments. Minitab Statistical Software (Version 2021) was adopted in real-time stability analysis and shelf-life prediction. QuantStudio™ Design & Analysis Software was used to interpret the genotyping results of the commercial PCR-based kit.