Assessment of Functional Antibody Responses Induced by Tembusu Virus Vaccines Using a Blocking ELISA

Abstract

To establish a rapid, sensitive, and reproducible method for evaluating the immunogenic performance of Tembusu virus (TMUV) vaccines, we developed and optimized a blocking enzyme-linked immunosorbent assay (bELISA) using the TMUV envelope (E) protein as the coating antigen. By systematically screening the coating antigen concentration, mAb dilution, serum dilution, and chromogenic reaction time, we determined the optimal reaction conditions for this assay. The results showed that bELISA exhibited high specificity, yielding positive reactions only with TMUV-positive sera and no cross-reactivity with sera against other common duck viruses; the cutoff value for positivity was 48.89%, and the lowest detectable serum dilution was 1:10. Neutralization assays confirmed that the TMUV E-specific mAb significantly inhibited viral replication, supporting the functional relevance and reliability of the established bELISA. In a comparative investigation, this assay was used to assess five TMUV vaccines, including both inactivated and attenuated variants, in Cherry Valley ducks. The DF2 inactivated vaccine was found to elicit the highest antibody levels and blocking rates. This was followed by the WF100 attenuated vaccine, which also demonstrated a strong immune response. The TC2B inactivated vaccine, although effective, showed a comparatively lower response, whereas the FX2010-180P strain and mosquito cell-derived WF100 attenuated vaccine showed weaker immunogenicity. Neutralization assays further confirmed that the TMUV E-specific mAb significantly inhibited viral replication, supporting the functional relevance and reliability of the established bELISA. In summary, the bELISA described here demonstrates high specificity, sensitivity, and reproducibility and is suitable for evaluating the immune efficacy of different TMUV vaccines, providing a reliable technical platform for vaccine immunology studies and optimization of immunization strategies.

1. Introduction

Tembusu virus (TMUV), a member of the genus Orthoflavivirus in the family Flaviviridae, has been prevalent in waterfowl farms across numerous provinces in China since its initial outbreak in 2010. TMUV infection can significantly reduce egg production, accompanied by neurological manifestations, causing substantial economic losses in the waterfowl industry [1]. Currently, vaccination is the primary strategy for controlling and preventing TMUV infections [2]. Nevertheless, different vaccine types exhibit considerable variations in immunogenicity, spectrum of induced antibodies, and durability of protection. Consequently, the establishment of an efficient, sensitive, and easily standardized method for evaluating immune efficacy has emerged as a crucial issue in TMUV control.

Traditional virus neutralization tests (SNT) and plaque reduction neutralization tests (PRNT) are regarded as the “gold standard” for measuring neutralizing antibody levels and assessing vaccine protection [3]. However, both methods are labor-intensive and time-consuming, making them unsuitable for large-scale immunosurveillance and field diagnostics [4]. Consequently, there is an urgent need for an alternative assay for TMUV that preserves high specificity and sensitivity while being rapid, simple, and reproducible [5].

In recent years, the blocking enzyme-linked immunosorbent assay (blocking ELISA, bELISA) has been widely applied in serological studies of various viruses and has gradually become an effective alternative to traditional neutralization assays [6]. For example, in studies of SARS-CoV-2, bELISAs based on trimeric spike proteins and receptor-binding domains (RBD) have been successfully used to evaluate population-level IgG and immunoglobulin subclasses. Through the validation of precision, linearity, and specificity, these assays were harmonized against the WHO international standards, greatly enhancing standardization and inter-laboratory comparability [7]. In the field of animal infectious diseases, a nanobody-based liquid-phase bELISA has been employed to assess the immunogenic performance of Senecavirus A vaccines [8], with results showing a high correlation with SNT and effectively replacing challenge experiments. Similarly, for canine distemper virus, a bELISA constructed using monoclonal antibodies (mAbs) targeting neutralizing epitopes of the H protein enabled the rapid evaluation of neutralizing antibody levels [9].

Building on these advances, the potential application of bELISA in vaccine immunogenicity assessment has been broadly validated. In this study, we used the TMUV E protein as a coating antigen to develop and optimize a bELISA system. Through meticulous optimization of ELISA assay parameters, including the coating concentration of the antigen, dilution factor of mAb, serum dilution, concentration of enzyme-labeled secondary antibodies, and chromogenic conditions, we developed a highly sensitive and specific assay. We then applied this system to dynamically monitor blocking rates in the sera of ducks immunized with several commercial and laboratory-prepared TMUV vaccines and compared the levels and functional characteristics of the induced antibodies. This study aimed to establish a rigorous and reproducible technical framework and experimental basis for the objective evaluation of Tembusu virus (TMUV) vaccine efficacy in ducks.

2. Materials and Methods

2.1. Experimental Animals

One-day-old healthy Cherry Valley breeding ducks were purchased from a commercial duck farm in Tai’an City, Shandong Province, and confirmed to be seronegative for Tembusu virus (TMUV) using serological testing (Figure S2).

2.2. Vaccines

A duck Tembusu virus (TMUV) live vaccine (strain WF100; Qilu Animal Health Products Co., Ltd, Jinan, China) was used in this study. The vaccine was administered at a dose of 0.5 mL, with a viral content of ≥10^4.5 TCID50 per dose. The TMUV live vaccine, strain FX2010-180P (Qingdao Yibang Bioengineering Co., Ltd, Qingdao, China), had a specification of one dose per 0.2 mL, with a viral content of ≥10^3.5 TCID50 per dose. The TMUV-inactivated vaccine (strain DF2) (Wuhan Keqian Biology Co., Ltd, Wuhan, China) was supplied at one dose per 0.5 mL, and the viral content before inactivation was 10^7.0 TCID50/mL. In addition, an inactivated vaccine was prepared in-house using the laboratory isolate TMUV-TC2B (GenBank: MH764605.1) with a pre-inactivation viral titer of 10^5.5 TCID50/mL. Concurrently, the WF100 strain was propagated in C6/36 cells to prepare an experimental attenuated vaccine, which was used in animal experiments.

2.3. Viruses, Cells, Sera, and Antibodies

The TMUV strain TC2B (GenBank: MH764605.1) was isolated, identified, and archived by the Avian Diseases Laboratory of the College of Veterinary Medicine, Shandong Agricultural University. The C6/36 cell line (Aedes albopictus clone C6/36, ATCC CRL-1660) was maintained in our laboratory, and duck embryo fibroblasts (DEF) were prepared from 10-day-old duck embryos. DuCV-positive serum, DHAV-3-positive serum, NDPV-positive-, NDRV-positive-, and GPV-positive sera were provided by our laboratory. A TMUV E-protein-specific monoclonal antibody (mAb) was generated and archived in our laboratory [10].

2.4. Indirect ELISA for Determination of mAb Titer

Monoclonal antibody titers were determined using indirect ELISA. Ninety-six-well plates were coated overnight at 4 °C with TMUV E protein (2 μg/mL, 100 μL per well). After washing three times with PBST, the plates were blocked with 5% (w/v) skim milk (200 μL per well) at 37 °C for 1 h. The TMUV E-specific monoclonal antibody was serially diluted (1:50–1:6400), added to the plates (100 μL per well), and incubated at 37 °C for 1 h. After three washes with PBST, HRP-conjugated goat anti-mouse IgG (1:10,000) was added (100 μL per well) and incubated at 37 °C for 1 h. After washing, TMB substrate (100 μL per well) was added and incubated at room temperature in the dark for 15 min. The reaction was stopped with 2 M H₂SO₄ (50 μL per well), and the absorbance at 450 nm was measured using a microplate reader.

2.5. Screening of Antigen and mAb Dilutions by Checkerboard Titration

The TMUV E protein was diluted to 8, 4, 2, 1, 0.5, 0.25, 0.125, and 0.0625 µg/mL with 0.05 mol/L carbonate buffer, 100 µL per well. Each concentration was set with three replicates, and the plates were incubated for 12 h at 4 °C. After washing with PBST, 5% skim milk blocking solution was added (200 µL per well). The plates were incubated at 37 °C for 1 h.

Subsequently, the TMUV E-specific mAb was diluted to 1:50, 1:100, 1:200, 1:400, 1:800, and 1:1600 with 5% skim milk, 100 µL per well, at 37 °C for 1 h. After washing the plates three times with PBST, an HRP-conjugated goat anti-mouse IgG antibody diluted 1:10,000 was added and incubated for 1 h at 37 °C. TMB substrate was added and incubated at room temperature for 20 min in the dark, the reaction was stopped with 50 µL of 2 mol/L H₂SO₄, and absorbance was measured at 450 nm.

The positive wells with an OD₄₅₀ value close to 1.0 were defined as the optimal reaction conditions for the antigen concentration and corresponding antibody dilution. Therefore, to determine the optimal coating time, the reaction conditions, such as 4 °C for 12 h, 37 °C for 1 h, and 37 °C for 2 h, were further compared.

2.6. Serum Dilution and Optimization of Competitive Reaction Conditions

The plates were coated with the optimized antigen concentration, and mAb at its optimal dilution was used as the blocking antibody. Duck positive and negative sera were diluted at 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, and 1:128, 100 µL per well was added and incubated at 37 °C for 1 h. The mAb was then added at the optimal dilution, and all subsequent steps remained unchanged. The dilution that yielded the highest PI value was defined as the optimal dilution. According to the determined optimal dilution ratio, the antibody-serum competition was subsequently performed at 37 °C for 0.5 h, 1 h, and 1.5 h to ascertain the most effective competition period.

2.7. Optimization of HRP-Conjugated Secondary Antibody Reaction Conditions

Under the established conditions, the HRP-conjugated secondary antibody was diluted in 5% skim milk to final concentrations of 1:4000, 1:7000, 1:10,000, 1:13,000, 1:16,000, and 1:20,000, whereas all other experimental parameters remained constant. The dilution yielding the highest percent inhibition (PI) value was identified as the optimal dilution for the assay. Based on this optimal dilution, the incubation duration at 37 °C was further evaluated at 0.5, 1, and 1.5 h to determine the most effective incubation time.

2.8. Optimization of TMB Substrate Color Development Conditions

The optimized conditions for TMB substrate incubation in the ELISA involved maintaining the reaction at 37 °C in the dark. Incubation times of 5, 10, 15, and 20 min were tested to determine the optimal color development period, defined as the time point yielding the highest percent inhibition (PI) value.

2.9. Establishment of the Cut-Off Criterion for Blocking Rate

Using the optimized bELISA system and reaction conditions, OD_450nm values were determined for 50 TMUV-negative duck sera, and the percent inhibition (PI) for each serum was calculated as PI = [(OD_{(negative control)} − OD_(sample))/OD_{(negative control)}] × 100% [11]. A test serum was interpreted as positive for specific antibodies when PI > x + 3SD; otherwise, it was interpreted as negative [12]. In parallel, SPSS (v29.0.2.0) software was used to compute the mean PI (x) and standard deviation (SD), and to assess the normality of the PI distribution among the 50 negative sera by calculating skewness, kurtosis, and their corresponding Z-scores.

2.10. Specificity Validation of the Blocking ELISA

Under the optimized blocking ELISA conditions, sera positive for duck Tembusu virus (TMUV), duck circovirus (DuCV), duck hepatitis A virus type 1 (DHAV-1), duck hepatitis A virus type 3 (DHAV-3), Muscovy duck parvovirus (MDPV), goose parvovirus (GPV), novel duck reovirus (NDRV), avian reovirus (ARV), and H9N2 subtype avian influenza virus were tested, with negative sera included as controls. TMUV-positive serum was used as a positive control to evaluate the specificity of the blocking ELISA. All serum samples were assayed in triplicate, and the results were expressed as percentage inhibition (PI).

2.11. Sensitivity Validation of the Blocking ELISA

Strongly positive, intermediate-positive, weakly positive, and negative sera against TMUV were serially diluted at ratios of 1:5, 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, and 1:640. All diluted serum samples were tested under the same optimized blocking ELISA conditions. Each sample was assayed in triplicate to systematically evaluate the sensitivity of the assay for detecting serum samples with different antibody levels.

2.12. Viral Titration and Neutralization Assay

TMUV-TC2B was titrated in C6/36 cells using the Reed–Muench method, and cytopathic effects were observed after serial dilution and incubation. The viral titer was expressed as TCID₅₀/mL. Six groups were established to evaluate the neutralizing activity of TMUV E protein-specific mAbs. In the experimental groups, the mAb was diluted at ratios of 1:2 and 1:10 and mixed with TMUV at a multiplicity of infection (MOI) of 1, followed by incubation at 37 °C for 1 h. The positive control group received an equal volume of virus suspension only, the negative control groups received mAb diluted at 1:2 or 1:10 only, and the blank control group received maintenance medium alone. Subsequently, the mixtures were inoculated into 96-well plates pre-seeded with monolayers of duck embryo fibroblast (DEF) cells at a density of 1 × 10⁵ cells/mL (100 µL per well), with three replicates per condition. After inoculation, the cells were incubated at 37 °C in a 5% CO₂ atmosphere. At 48 h post-infection, the cells were harvested, and intracellular viral RNA Ct values were determined using quantitative PCR (RT-qPCR). Viral RNA copy numbers were calculated by converting the Ct values using a pre-established standard curve (y = −0.3089x + 12.11, R² = 0.9969).

2.13. Preparation of the Inactivated Vaccine

Medicinal white oil and Span-80 were mixed thoroughly at a ratio of 94:6 (v/v) and sterilized via autoclaving at 121 °C. The resulting mixture was used as the oil phase of the emulsion. For the aqueous phase, a fully inactivated TMUV suspension was combined with sterile Tween-80 at a 96:4 ratio and stirred until Tween-80 was completely dissolved. The oil and aqueous phases were then thoroughly emulsified following the method described by Huang, B. [13]. After emulsification, the vaccine was aliquoted, sealed, and stored as an emulsion-type inactivated formulation.

2.14. Propagation of the WF100 Attenuated Strain in Mosquito-Derived Cells

To verify the consistency and stability of the immunological evaluation system across different antigen source backgrounds, this study used the attenuated live Tembusu virus vaccine strain WF100 to generate antigen preparations with identical origins but distinct production backgrounds by propagating them in the C6/36 cell culture system. The WF100 virus suspension was filtered through a 0.22 µm membrane to remove bacteria and other impurities, ensuring sterility. The sterile filtrate was inoculated into C6/36 cells for continuous serial passaging. Viral supernatants were harvested at appropriate intervals and stored at −80 °C, resulting in the production of an attenuated vaccine.

2.15. Vaccine Immunization Experiment

A total of 120 one-day-old Cherry Valley ducks were maintained in the animal facility until they reached 7 days of age, at which point they were randomly assigned to six groups (

Table 1. Grouping and immunization protocols for vaccine immunogenicity testing.

Group	Age at Immunization (Days)	Route	Dose	No. of Birds
Negative control	7	Intramuscular	0.5 mL/bird	20
TC2B inactivated vaccine	7	Intramuscular	0.5 mL/bird	20
WF100 attenuated vaccine	7	Intramuscular	0.5 mL/bird	20
FX2010-180P attenuated vaccine	7	Intramuscular	0.2 mL/bird	20
DF2 inactivated vaccine	7	Intramuscular	0.5 mL/bird	20
Mosquito cell–derived WF100 attenuated vaccine	7	Intramuscular	0.5 mL/bird	20
Total				120

). Five groups were immunized with three commercial TMUV vaccines—the WF100 attenuated vaccine, FX2010-180P attenuated vaccine, and DF2 inactivated vaccine—and two in-house vaccines—the TC2B inactivated vaccine and the mosquito cell–derived WF100 attenuated vaccine—according to the immunization protocols outlined in Table 1. The remaining group served as a negative control and was injected with sterile saline. Blood samples were collected at 3-day intervals, beginning before immunization and continuing thereafter. Serum was separated and stored at −20 °C until further analysis.

2.16. Determination of Antibody Titers for Different Vaccines

Serum samples were serially diluted in 0.9% physiological saline at ratios of 1:25, 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12,800, and 1:25,600. A volume of 100 µL from each dilution was added to ELISA plates that had been pre-coated with TMUV E protein and blocked with 5% skim milk. Following incubation at 37 °C for 1 h, the plates were washed three times with PBST. Next, 100 µL per well of HRP-conjugated goat anti-duck secondary antibody, diluted 1:10,000 in 5% skim milk, was added and incubated at 37 °C for another hour. After three additional washes with PBST, TMB substrate was added for color development and incubated at room temperature in the dark for 15 min. The reaction was terminated with 50 µL of 2 M H₂SO₄, and the absorbance was immediately measured at 450 nm (OD₄₅₀) using a microplate reader.

2.17. Determination of Blocking Rates for Different Vaccines

Using the optimized blocking ELISA system and reaction conditions, serum samples from duck herds vaccinated with different TMUV vaccines were analyzed to assess antibody responses. Sera collected at various time points post-immunization were diluted 1:1 and added to 96-well plates that had been pre-coated with TMUV E protein and blocked. The plates were incubated at 37 °C for 1 h. Following the removal of the sera, the wells were washed three times with PBST, and TMUV E-specific mAb, diluted 1:400, was added and incubated at 37 °C for an additional hour. After a second washing step, HRP-conjugated goat anti-mouse IgG, diluted 1:10,000, was added and incubated at 37 °C for 1 h. The plates were then washed three more times, followed by the addition of TMB substrate and incubation at room temperature for 20 min in the dark. The reaction was terminated with 2 M H₂SO₄, and the absorbance was measured at 450 nm.

Based on the OD₄₅₀ values obtained, the percent inhibition (PI) for each serum sample was calculated as PI (%) = [(OD_{(negative control)} − OD_(sample))/OD_{(negative control)}] × 100% [11].

2.18. Statistical Analysis

Statistical differences among experimental groups were analyzed using Student’s t-test with GraphPad Prism software (version 8.0; GraphPad Software Inc, San Diego, CA, USA). Statistical significance was set at p < 0.05 and p < 0.01.

3. Results

3.1. Determination of the Optimal Antigen Coating Concentration and Monoclonal Antibody Dilution

The TMUV-E mAb prepared in this study reached a titer of 1:12,800 (Figure 1A). When the E protein was diluted to 2 μg/mL and the mAb to a dilution of 1:400, the OD₄₅₀ value of the positive serum approached 1.0, and the P/N ratio reached its highest level under the optimized ELISA conditions (

Table 2. Determination of the optimal dilutions of the antigen and monoclonal antibody.

Antigen Dilution Concentration	Monoclonal Antibody Dilution
	1:50	1:100	1:200	1:400	1:800	1:1600
8 μg/mL	3.807	3.26	2.497	1.592	0.866	0.466
4 μg/mL	3.761	3.252	2.503	1.595	0.916	0.492
2 μg/mL	3.215	2.376	1.583	0.98	0.498	0.281
1 μg/mL	1.749	1.042	0.654	0.366	0.199	0.111
0.5 μg/mL	0.806	0.477	0.316	0.17	0.094	0.058
0.25 μg/mL	0.451	0.26	0.176	0.098	0.067	0.048
0.125 μg/mL	0.204	0.121	0.137	0.06	0.054	0.05
0.0625 μg/mL	0.099	0.062	0.06	0.055	0.055	0.053

). The ideal coating conditions for the E protein involved incubation at 4 °C for 12 h using the 2 μg/mL antigen concentration (Figure 1B).

3.2. Determination of the Optimal Serum Dilution

Gradient dilution of both TMUV-positive and negative duck sera revealed that the percent inhibition (PI) reached its highest value at a 1:1 dilution (

Table 3. Determination of optimal dilutions of positive and negative sera.

Serum Dilution	1:1	1:2	1:4	1:8	1:16	1:32	1:64	1:128
Positive serum	0.403	0.527	0.667	0.766	0.816	0.994	1.097	1.153
Negative serum	1.123	1.193	1.252	1.265	1.196	1.240	1.2873	1.2713

and Figure 2A), with the ideal incubation condition being blocking at 37 °C for 1 h (Figure 2B).

3.3. Optimal Dilution Determination for HRP-Conjugated Secondary Antibodies

Following gradient dilution of the enzyme-labeled secondary antibody, the percent inhibition (PI) reached its maximum at a dilution of 1:10,000 (Table S1 and Figure 2C). The ideal incubation condition was 1 h at 37 °C (Figure 2D).

3.4. Determination of the Optimal Reaction Time for the TMB Substrate

The substrate was incubated at 37 °C in the dark for 5, 10, 15, and 20 min (Table S2). The percent inhibition (PI) value peaked at 20 min, indicating that this was the optimal incubation time (Figure 2E).

3.5. Determination of the Cut-Off Value

The mean PI value of 50 TMUV-antibody-negative duck sera was determined to be 27.97%, with a standard deviation (SD) of 6.97%. Therefore, when the PI of a test serum exceeds 48.89%, it is judged as TMUV antibody-positive; otherwise, it is negative (Figure 3A). Further analysis of the normality of PI values for the 50 samples using SPSS software revealed skewness = 0.185, standard error of skewness = 0.337, kurtosis = −0.351, standard error of kurtosis = 0.662, skewness Z-score = skewness value/standard error of skewness ≈ 0.55, and the kurtosis Z-score = kurtosis value/kurtosis standard error ≈ −0.53. Both Z-scores fell within ±1.96, indicating that the data followed a normal distribution (Figure 3B).

3.6. Specificity Test

Under the optimized reaction conditions of the blocking ELISA, sera positive for TMUV, DuCV, DHAV-1, DHAV-3, MDPV, GPV, NDRV, ARV, and H9N2, as well as negative sera, were tested. As shown in the results, only TMUV-positive sera exhibited inhibition values exceeding the predefined cut-off (48.89) and were therefore classified as positive, whereas all other virus-positive and negative sera displayed inhibition values below the cut-off and were classified as negative. These results indicate that the blocking ELISA showed no cross-reactivity with sera against other common waterfowl viruses and demonstrated high assay specificity (Figure 4A).

3.7. Sensitivity Test

Strongly positive, intermediate-positive, and weakly positive TMUV sera were serially diluted and tested under the optimized blocking ELISA conditions to evaluate assay sensitivity, with negative sera included as controls. When serum dilutions were below 1:10, the percent inhibition values of strong-positive and intermediate-positive sera were above the predefined cut-off value (48.89%), while weak-positive sera approached or exceeded the cut-off at low dilution levels. As the dilution factor increased, the inhibition rates of all positive sera decreased progressively and fell below the cut-off at dilutions of 1:20 or higher, whereas negative sera consistently exhibited inhibition values below the cut-off across all dilution points (Figure 4B). These results indicate that the blocking ELISA reliably discriminated sera with different antibody levels, with a sensitivity detection dilution of 1:10.

3.8. Validation of the Neutralizing Activity of the Monoclonal Antibody

The RT-qPCR results were analyzed by comparing the viral replication levels in each group relative to the virus-only control, which was used as a positive reference for maximal viral copy numbers. The positive control group exhibited the highest viral RNA copy numbers, whereas no viral signal was detected in either the blank or negative control groups. In the experimental groups, both 2-fold and 10-fold dilutions of the monoclonal antibody markedly reduced intracellular viral RNA levels compared to the positive control, indicating effective inhibition of viral replication. The comparable degree of viral suppression observed at the two antibody dilutions suggests that the monoclonal antibody (mAb) maintained stable neutralizing activity within this concentration range (Figure 5).

3.9. Changes in Antibody Levels After Immunization

Indirect ELISA results demonstrated that all five vaccines elicited detectable levels of TMUV-specific antibodies following immunization; however, notable differences were observed among the vaccine groups with respect to the rate of antibody increase, peak titers, and duration of antibody persistence (Figure 6A). Prior to immunization, the antibody titers in all groups were negative and exhibited a gradual decline over the 7-day period, with consistent trends across groups. Following immunization on day 7, antibody titers in all five vaccinated groups increased significantly and remained at relatively high levels throughout the duration of the experiment, whereas the blank control group consistently maintained the baseline levels.

Distinct immunogenic profiles were observed in the vaccine groups. The DF2 inactivated vaccine group exhibited the most rapid immune response, with a minor early peak detected at 16 days post-immunization, followed by a continuous rise, culminating in the highest endpoint titer. The mosquito cell-derived WF100 attenuated vaccine group also displayed a small peak at day 16; however, the antibody level was lower than that of the DF2 group and showed greater fluctuations, ultimately resulting in the lowest endpoint titer. The TC2B inactivated vaccine group demonstrated a rapid post-immunization rise and maintained a relatively high and stable antibody titer throughout the study. The WF100 and FX2010-180P attenuated vaccine groups exhibited intermediate responses, with WF100 outperforming FX2010-180P, which showed the lowest peak antibody titer among the five vaccine groups.

3.10. Blocking Rates of Different Vaccines

Blocking ELISA results demonstrated that all five vaccines induced the production of blocking antibodies following immunization; however, the antibody responses exhibited distinct dynamic profiles among the groups (Figure 6B). The DF2 inactivated vaccine group exhibited relatively pronounced fluctuations in the blocking rate throughout the experimental period but achieved the highest peak value and maintained this superior level until the end of the trial, indicating a strong and sustained immune response. The WF100 vaccine group consistently showed a positive blocking rate beginning at 7 days post-immunization, with the smallest overall fluctuation amplitude observed. It ranked second in peak value and remained relatively high at the conclusion of the study. The TC2B inactivated vaccine group displayed a steadily increasing blocking rate after immunization, characterized by minimal fluctuations and high stability. Although its peak and final blocking rates were slightly lower than those of the top two groups, it maintained a strong position in third place. The FX2010-180P attenuated vaccine group followed a trend similar to that of the TC2B group but at a consistently lower level, with both its peak and final blocking rates ranking fourth. In contrast, the mosquito cell-derived WF100 attenuated vaccine group exhibited greater variability and the lowest peak blocking rate, suggesting that it induced comparatively lower levels of functional antibodies.

4. Discussion

Duck Tembusu virus (TMUV) emerged in 2010 and has since become a persistent and significant pathogen in China’s waterfowl industry, causing substantial declines in egg production and overall farm productivity [14]. Vaccination remains the primary and most effective strategy to control TMUV [2]. In this study, we compared the immune responses induced by five different TMUV vaccines using a traditional indirect ELISA alongside a newly developed blocking ELISA. To ensure the serological results reflected actual protective immunity, we also performed virus neutralization assays, which confirmed the blocking ELISA’s reliability. This approach is similar to that of Mu et al. [8], who also used a blocking ELISA to assess vaccine-induced protection in a similar context.

Indirect ELISA results showed that all five vaccines induced detectable TMUV-specific antibodies, but there were marked differences in antibody magnitude and kinetics among the vaccines. The DF2 inactivated vaccine elicited a rapid antibody response with a high peak titer that remained relatively stable, indicating favorable immunological performance, likely reflecting good antigen stability and formulation [15]. The self-prepared TC2B inactivated vaccine also induced a stable antibody response, although at lower overall levels, suggesting its immunogenicity could be improved [16]. The two commercial live attenuated vaccines produced intermediate responses: the WF100 strain induced higher antibody levels than the FX2010-180P strain, reflecting differences in immune responsiveness between these attenuated strains [17,18]. By contrast, the mosquito cell-derived WF100 attenuated vaccine showed pronounced fluctuations in antibody levels and the lowest final titers. This finding suggests that without further optimization, the heterologous cell-based propagation may compromise antigen effectiveness and limit the immune response [19].

Before vaccination, ducklings had low levels of TMUV-specific antibodies (baseline titer ~1:256), presumably due to maternally derived immunity. These passive antibodies declined over the first week of life, consistent with the natural waning of yolk-transferred maternal IgY [20]. After vaccination at 7 days of age, all five groups showed clear seroconversion by indirect ELISA, although antibody kinetics differed markedly among vaccines. Notably, the antibody titer curves were often “jagged” rather than smooth: some groups had an early modest peak (~16 days post-immunization) followed by a continued rise to a high final titer. This biphasic dynamic likely reflects waning maternal antibodies overlapping with the developing active immune response [21], as well as the normal maturation of a primary antibody response.

Conventional indirect ELISA measures overall TMUV-specific antibody levels in serum but does not provide information on the proportion or functional relevance of antibodies targeting key viral epitopes. Therefore, total antibody levels alone are insufficient to fully reflect vaccine-induced functional humoral responses [22]. To more accurately assess functional antibody responses, we established a blocking ELISA to detect serum antibodies that compete for critical functional epitopes on the TMUV E protein and interfere with virus–host interactions. This assay uses a competitive binding principle between a functional monoclonal antibody and immune serum, allowing sensitive detection of antibodies targeting key viral epitopes and their levels [23]. Following systematic optimization of key parameters, including antigen coating conditions, dilution ratios of the monoclonal antibody and serum samples, and overall reaction conditions, the assay demonstrated favorable overall technical performance. Specificity validation showed that detectable signals were exclusively derived from TMUV-positive sera, with no cross-reactivity observed with sera positive for multiple common waterfowl viruses, indicating high analytical specificity. Sensitivity evaluation further demonstrated that the assay effectively discriminated serum samples with different antibody levels, allowing reliable differentiation among strongly positive, moderately positive, and weakly positive sera. In addition, repeated measurements yielded consistent results, indicating good assay stability and reliability. Overall, the blocking ELISA was systematically validated with respect to specificity, sensitivity, and reproducibility, and it provides a stable and controllable technical approach for monitoring TMUV-associated antibody levels and supporting subsequent immunological studies.

The envelope (E) protein of duck Tembusu virus (TMUV) exhibits a high degree of sequence conservation among different vaccine strains, and its major neutralizing epitopes are distributed across domains I, II, and III, which are relatively conserved in both structure and function [24,25,26]. Therefore, a blocking ELISA employing homologous TMUV E protein as the coating antigen is not readily affected by minor sequence differences among vaccine strains.

We then used the blocking ELISA to compare the functional antibody levels induced by each vaccine in ducklings. DF2 (inactivated) induced the highest blocking antibody levels, which remained positive throughout the observation period. This suggests that DF2 elicited a high proportion of functional antibodies capable of targeting key viral epitopes and blocking virus–host binding, thereby providing stable antiviral activity [27]. By comparison, the WF100 live attenuated vaccine induced blocking levels that fluctuated little and stayed relatively high by the end of the period. This indicates that WF100 stably activated responses against critical viral epitopes and induced sustained functional antibodies [28,29]. Such robust, durable responses suggest that the WF100 vaccine could provide long-term population-level protection and help control TMUV transmission in duck flocks.

For the TC2B inactivated vaccine, the blocking ELISA pattern generally mirrored the total antibody trend. Its peak blocking rate was slightly lower than DF2’s, but the overall response was stable. This suggests that TC2B’s antigen closely matches the key viral epitopes of the wild-type virus, so that increases in total antibodies were accompanied by proportional increases in functional antibodies [30]. Thus, the TC2B vaccine demonstrated reliable immunogenicity in inducing stable functional antibody levels. In contrast, the FX2010-180P attenuated vaccine induced relatively low blocking rates, consistent with its use primarily in older meat or laying ducks. The immature immune system of ducklings likely limited their response to this attenuated strain, resulting in poor induction of high-level functional antibodies [31]. Accordingly, the FX2010-180P group showed weak functional antibody responses—despite measurable total antibody titers, the proportion of functional antibodies remained low. These observations highlight the importance of considering host immune maturity when evaluating vaccines in young animals. The mosquito cell-derived WF100 vaccine induced the lowest peak blocking levels, and its functional antibody titers were not sustained, indicating a failure to establish a stable functional response [16]. This suggests that the antigenic stimulus of this strain was insufficient to induce strong functional antibodies [32]. These findings indicate that this strain’s attenuation strategy and stability require further optimization. On the other hand, the poor performance of this vaccine underscored the high sensitivity and discriminatory power of our blocking ELISA, which clearly distinguished the functional responses among the different vaccines.

We further validated the blocking ELISA’s relevance with an in vitro virus neutralization assay [33]. A TMUV E protein-specific monoclonal antibody significantly inhibited viral replication, confirming that it targets a critical viral epitope and effectively blocks TMUV infection [6,34]. This provides functional evidence that the blocking ELISA truly reflects protective antibody activity [35]. Similarly, previous studies have shown a strong correlation between blocking ELISA results and protective immunity, supporting its use as an effective alternative or complement to classical neutralization tests [36]. In this study, the neutralization assay was performed as a proof-of-concept experiment to verify the functional relevance of the monoclonal antibody used in the blocking ELISA. The analysis focused on demonstrating antibody-mediated inhibition of viral replication, rather than on comprehensive serum neutralization or titer determination. Collectively, these findings demonstrate that a monoclonal antibody-based blocking ELISA is a rapid and reliable tool for evaluating functional antibody responses induced by different TMUV vaccines.

All five vaccine strains evaluated in the present study belong to TMUV Cluster 2, which has been the predominant lineage in duck flocks in recent years. However, Cluster 3 TMUV has emerged in poultry populations in Asia, and experimental serology has demonstrated significant antigenic variation between a Cluster 3 chicken-origin isolate and a representative Cluster 2.2 strain based on cross-neutralization testing [37]. Therefore, although the E protein is generally conserved among TMUV lineages, the applicability of the current E-based bELISA to Cluster 3–induced antibodies cannot be assumed and should be validated using Cluster 3 reference sera and isolates in future work. In this context, the established bELISA is supported for comparative evaluation of Cluster 2–derived vaccines in this study, while its performance against Cluster 3 requires additional verification.

In summary, live attenuated vaccines are characterized by a rapid onset of immunity and a relatively high proportion of functional antibodies, making them suitable for primary vaccination or emergency immunization [16]. In contrast, inactivated vaccines offer advantages in safety and durability of immune responses and are therefore more appropriate for baseline immunization and booster vaccination [38]. A rational combination of these two vaccine types may achieve a complementary immunization strategy that provides rapid protection followed by long-term immune maintenance [39]. In addition, the comparative results obtained for the mosquito cell-derived WF100 live attenuated vaccine and the TC2B inactivated vaccine indicate that vaccine strain adaptability and manufacturing processes are critical determinants of immunogenicity [40]. Overall, although blocking rates generally follow trends observed for total antibody levels, they are able to reveal functional differences that are not captured by antibody titers alone in certain vaccines. Therefore, the blocking ELISA established in this study serves as a valuable complementary tool for assessing vaccine-induced protective immune efficacy among different TMUV vaccines, and it provides useful reference information for comparative evaluation of vaccine performance and the optimization of immunization strategies.