Lateral Flow Immunochromatographic Assay for Adenovirus Antibody Detection in Human Serum Using Gold Nanoparticles and Quantum Dots

1. Introduction

Adenoviral vectors, owing to their structural and functional characteristics, are widely used as delivery systems in vaccine production [1]. Despite improvements in vector design, first- and second-generation adenoviral vectors retain the ability to express certain adenoviral genes, which may induce undesirable immune responses in the hosts [2]. Clinical studies have demonstrated that the presence of pre-existing high titers of neutralizing antibodies against HAdV-C5 in vaccinated individuals can adversely affect immunization efficacy, leading to reduced seroconversion rates of neutralizing antibodies against SARS-CoV-2 [3]. Accordingly, the adenoviral serotype and pre-existing immune status should be carefully considered in the development of vector-based vaccines and gene therapy approaches.

The timely and effective implementation of antiviral measures requires diagnostic approaches that combine accessibility, ease of use, and rapid turnaround times. In modern laboratory practice, the most widely used methods for the diagnosis of viral infections are polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), both characterized by high sensitivity and specificity. Alternatively, the lateral flow immunochromatographic assay (LFIA) represents a single-step diagnostic approach that does not require complex equipment, additional reagent preparation, or prolonged sample processing [4]. The structural and technological features of the LFIA ensure rapid testing and straightforward interpretation of results, which are particularly advantageous when screening large populations at the point of care or in field settings [5]. In addition, immunochromatographic tests are characterized by cost-effectiveness, long-term storage stability, and minimal requirements for sample preparation [6]. The LFIA systems have considerable potential in environmental monitoring and food safety assessment [7]. Their reliability and effectiveness have been confirmed in the diagnosis of myocardial infarction through the detection of cardiac troponins [8] and human fatty acid-binding protein (H-FABP) [9]. Furthermore, LFIAs are widely applied in the diagnosis of bacterial, viral, and parasitic infections [4,5,6,10,11]. Overall, the LFIA is a promising and efficient tool for the rapid diagnosis of viral infections within the framework of epidemiological surveillance and mass preventive programs. Recent research has focused on improving the sensitivity of the LFIA and developing quantitative assay formats. For instance, studies have evaluated the use of novel labels, such as quantum dots (QDs) [12], which exhibit high stability, a large extinction coefficient, high quantum yield, and prolonged fluorescence lifetime. Accordingly, QDs are excellent labels that can be conjugated with antibodies to develop highly sensitive LFIA-based diagnostic systems [13,14].

While many LFIA platforms are designed for the direct detection of pathogens or antigens, serological immunochromatographic assays offer additional opportunities for assessing the host immune response. These assays enable evaluation of immune status following vaccination or after primary and secondary infections, thereby supporting epidemiological surveillance, retrospective diagnosis, and monitoring of population immunity. Based on these considerations, we hypothesized that an LFIA based on the recombinant HAdV hexon protein can enable reliable detection of HAdV antibodies using a serological method and that the use of quantum dots as signal-generating labels will increase the sensitivity of the assay compared to conventional gold nanoparticles (GNPs). In this study, we analyzed recombinant human adenovirus hexon protein, developed LFIAs based on GNPs and QDs, and evaluated the diagnostic performance of the proposed assays.

2. Materials and Methods

2.1. Bacterial Strains and Plasmids

Escherichia coli strains BL21 and DH5α, as well as the pET28 plasmid (Novagen, Cambridge, UK), were used in the study. Recombinant proteins were purified using Ni²⁺-NTA affinity chromatography columns (GE Healthcare, Uppsala, Sweden). For western blot analyses, mouse monoclonal antibodies against the His-tag (Thermo Fisher Scientific, Waltham, MA, USA) (RRID: AB_557403) and species-specific secondary antibodies conjugated with horseradish peroxidase (Sigma-Aldrich, St. Louis, MO, USA) were used. A Human Adenovirus IgG (ADV IgG) ELISA Kit (Abbexa Ltd., Cambridge, UK) was employed for serological analyses.

2.2. Expression of Recombinant HAdV Hexon Protein (rhHAdV)

Transformed E. coli BL21 cells were cultured in 5 mL of Luria–Bertani (LB) broth supplemented with kanamycin at 37 °C with shaking at 160 rpm. Upon reaching the logarithmic growth phase, isopropyl-β-d-1-thiogalactopyranoside (IPTG) was added to the culture at final concentrations of 0.2, 0.5, 1, and 5 mM to induce protein expression. The induced cells were further incubated at 37 °C with shaking at 160 rpm.

Aliquots (100 µL) of the E. coli culture were collected every 2 h post-induction. Cells were harvested by centrifugation at 10,000 × g for 5 min at 4 °C. The resulting pellets were resuspended in 1 mL of buffer (20 mM HEPES, pH 7.5; 150 mM NaCl) and lysed using an OMNI Ruptor 4000 ultrasonic homogenizer (OMNI International, Kennesaw, GA, USA). Sonication was performed twice for 30 s at maximum power. The lysates were subsequently analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

The producer strain was cultured in 5 mL of LB broth supplemented with the appropriate antibiotic for 12 h at 37 °C with constant agitation. Subsequently, 50 µL of the overnight culture was inoculated in 50 mL of LB broth containing the antibiotic and incubated at 37 °C until the logarithmic growth phase was reached. Upon attaining the desired cell density, IPTG was added to a final concentration of 0.2 mM to induce protein expression. The induced cells were further cultured for 16 h at 37 °C. Cell biomass was harvested by centrifuging 50 mL of the culture at 3000 × g for 20 min. The cell pellet was resuspended in one-third of the volume of physiological saline and centrifuged again under the same conditions. The resulting pellet was then resuspended in 10 mL of TNE buffer (20 mM Tris, pH 7.5; 1 mM EDTA; 100 mM NaCl). The cells were disrupted by sonication (22 kHz, 4 cycles of 120 s each) and centrifuged at 17,000 × g for 60 min. The pellet was resuspended in 10 mL of buffer 1 (20 mM HEPES, 500 mM NaCl, 1 M urea) and incubated for 30 min on an orbital shaker at 25 °C, followed by centrifugation at 17,000 × g for 30 min. The resulting pellet was resuspended in metal-affinity chromatography binding buffer (20 mM HEPES, 500 mM NaCl, 20 mM imidazole, 8 M urea, 10 mM 2-mercaptoethanol) and subjected to sonication (22 kHz, 60 s). After sonication, the suspension was incubated for 30 min on an orbital shaker at 25 °C and then centrifuged at 3000 × g for 20 min.

Based on the optimization results, the optimized expression conditions were used for large-scale production of the recombinant hexon protein. Following induction and cultivation, the recombinant protein was purified from the bacterial lysate, subjected to refolding, and subsequently characterized using biochemical and proteomic approaches. The recombinant protein was purified from the supernatant using a Ni²⁺-NTA affinity column (GE Healthcare, Uppsala, Sweden). Elution was performed using the same buffer containing 8 M urea with a linear imidazole gradient (20–500 mM). Protein refolding was carried out by dialysis against a 300-fold excess volume of 25 mM sodium phosphate buffer (pH 7.25) containing 300 mM NaCl. The refolded protein was analyzed by denaturing electrophoresis in 15% SDS-PAGE gel. Western blot and LC–MS/MS were employed to evaluate the purified recombinant proteins.

2.3. Conjugation of GNPs with Protein G

Commercial colloidal GNPs with an average diameter of 20 nm (Sigma-Aldrich, Darmstadt, Germany) were used in this study. The nanoparticles were supplied as a standardized preparation and used according to the manufacturer's recommendations without additional physicochemical characterization. The obtained GNP solution was cooled and stored at 4 °C. Prior to conjugation, the pH of the GNP solution was adjusted to 8.5–9.0 using potassium carbonate. Protein G solution at various concentrations was then added to 10 mL of the pH-adjusted GNP suspension. The mixture was incubated for 60 min at 25 °C, after which 250 µL of 10% bovine serum albumin (BSA) was added. The suspension was further incubated for 10 min at 25 °C and subsequently centrifuged at 8000 × g for 30 min. The resulting pellet was resuspended in 1.5 mL of phosphate-buffered saline (PBS) containing 0.25% BSA.

2.4. Conjugation of QDs with Protein G

The conjugation of Protein G with QDs with an excitation wavelength of 585 nm (Invitrogen, Carlsbad, CA, USA) was performed following a previously described method [15], with minor modifications. Briefly, 60 µL of the QD suspension was added to 840 µL of 0.05 M 2-(N-morpholino)ethanesulfonic acid buffer (MES) buffer (pH 5.8; Tocris Bioscience, Bristol, United Kingdom). To activate the carboxyl groups on the surface of the QDs, 50 µL of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC, HiMedia, Mumbai, India) and 50 µL of N-hydroxysulfosuccinimide sodium salt (NHS) (Glentham Life Sciences, Corsham SN13 9SW, United Kingdom), both at 10 mg/mL, were added to the mixture. The resulting suspension was incubated for 30 min at 25 °C. Subsequently, 400 µL of Protein G solution (1 mg/mL in PBS, pH 7.2) was added to the activated QDs, providing a molar ratio of 8:1. The reaction mixture was incubated for 2 h under continuous agitation using a PSU-10i orbital shaker (Biosan, Riga, Latvia) under light-protected conditions to prevent QD photodegradation and ensure efficient conjugation. Following incubation, the conjugate was purified to remove excess activating reagents and unbound Protein G. Purification was performed by concentration and dialysis against 10 mM borate buffer (pH 8.6) using Amicon Ultracel 30 K centrifugal filters (Millipore, Burlington, MA, USA) at 10,000 × g for 15 min. After purification, the conjugate was centrifuged at 15,000 × g for 20 min at 4 °C. The resulting pellet was resuspended in 400 µL of stabilizing buffer (20 mM Tris-HCl, pH 8.0; 0.5% BSA; 0.1% Tween 20; 5% sucrose; 0.05% NaN₃). The purified and concentrated conjugate was stored at 4 °C until further use.

2.5. Assembly of LFIA

GNP–Protein G (GNP-G) and QD–Protein G (QD-G) conjugates were applied onto separate PT-R5 membranes (Advanced Microdevices Pvt. Ltd., Haryana, India) at a volume of 11 µL per 1-cm strip using an AirJet Quanti 3000 dispensing system (XYZ3050 platform, BioDot, Irvine, CA, USA). Test and control lines were dispensed onto a laminated nitrocellulose membrane (CNPC-SS12-L2-H50, Advanced Microdevices Pvt., Ltd., Haryana, India) using a FrontLine 1000 dispensing system (XYZ3050 platform, BioDot). The test line consisted of purified rhHAdV at 0.5, 1, 1.5, 2, 3, and 4 mg/mL in PBS containing 10% glycerol. The control line was formed using mouse antibodies at 1 mg/mL in PBS containing 10% glycerol. All reagents were applied at a dispensing rate of 0.2 µL/mm.

The membranes with deposited reagents were dried at 23–25 °C for 20 h in a vacuum drying chamber. After drying, the membrane components were assembled into immunochromatographic composites and cut into 4-mm-wide strips using a CM4000 guillotine cutter module (BioDot). The membrane components were assembled at 23–25 °C in a controlled environment with a relative humidity of 25–30%.

2.6. Detection of Antibodies Against rhHAdV

The LFIA was performed at 25 °C. Briefly, 20 µL of the serum sample was mixed with 80 µL of buffer (Tris, pH 7.5; 150 mM NaCl; 0.1% Tween 20; 0.5% BSA) in an Eppendorf tube. A test strip was then vertically immersed into the mixture. The results were evaluated after 20 min. The test strips were analyzed visually.

2.7. Statistical Analysis

Receiver operating characteristic (ROC) curve analysis was performed using the online version of GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA). The diagnostic performance of the developed assays was evaluated by calculating sensitivity, specificity, and the area under the ROC curve (AUC).

3. Results

3.1. Expression and Purification of rhHAdV

Transformation of the pET28/hHAdV plasmid resulted in the generation of an E. coli BL21/pET28/hHAdV strain producing a recombinant fragment of the HAdV hexon protein spanning amino acid residues A120 to R316. The expression analyses demonstrated the production of a protein with an approximate molecular weight of 35 kDa. Optimization of expression and purification parameters enabled the isolation of purified rhHAdV protein. The recombinant antigen was purified using nickel-based metal-chelate affinity chromatography. Elution was performed using buffers containing varying concentrations of imidazole. SDS-PAGE revealed a single protein band in the purified preparation, indicating a high level of purity (Figure 1).

As shown in Figure 1a, the optimized expression and purification protocol enabled the production of rhHAdV preparations free of detectable impurities. Western blotting using anti-His-tag monoclonal antibodies revealed a protein band with a molecular weight of approximately 35 kDa, consistent with the expected size of the recombinant HAdV hexon (Figure 1b).

Further characterization of rhHAdV using LC–MS/MS analysis, including peptide identification (DITTTEGEEKPIYADK) with Mascot, yielded a maximum score of 1206, confirming that the antigen is a hexon protein of the genus Mastadenovirus within the family Adenoviridae (Figure 2).

3.2. Immunoreactivity of rhHAdV in ELISA with Human Positive Sera

The diagnostic performance of rhHAdV was evaluated using ELISA with sera from eight patients. Samples with antibody titers exceeding those of a standard weakly positive human serum were considered positive. Antibody titers against rhHAdV among patient sera varied. Specifically, six out of eight samples exhibited moderate antibody titers, one sample showed a high antibody level, and one sample was classified as negative (Figure 3).

Considering that weakly positive samples represent a major challenge in serological diagnostics, 33 serum samples with minimal antibody titers exceeding the cut-off threshold were selected for subsequent experiments. These weakly positive samples were used as control sera for comparisons of LFIAs based on GNPs and QDs.

3.3. Optimization of LFIA Components

GNP-G was prepared following the methods described by Sotnikov et al. [16]. To determine optimal conditions for immunochromatographic serodiagnosis of adenovirus infection, parameters for antigen immobilization on the working nitrocellulose membrane were optimized. The optimal concentration of rhHAdV for application on the CNPC-SS12-L2-H50 membrane was 1 mg/mL with a linear dispensing rate of 0.2 µL/mm. Further increases in antigen concentration resulted in nonspecific binding of the conjugate. The GNP-G conjugate was applied to the PT-R5 membrane at a volume of 2 µL/mm. Control sera were tested at a dilution of 1:20. The analytical sensitivity of the optimized parameters was evaluated using serial dilutions of a standard positive serum (1:20–1:1000). The limit of detection corresponded to a dilution of 1:300, at which a clearly distinguishable test line was observed (Figure 4).

The analytical performance of the LFIA based on QD-G was evaluated using the same concentrations of rhHAdV and identical serum dilutions as those applied for the GNP-based assay. Fluorescence of the test line was detectable up to a dilution of 1:1000 (Figure 5).

For subsequent experiments, a serum dilution of 1:20 was selected as it ensured clear and intense visualization of both test and control lines when analyzing positive samples, while only the control line was observed for negative sera. These findings indicated that the LFIAs based on GNP and QD conjugates possessed sufficient analytical sensitivity for the detection of adenovirus-specific antibodies.

3.4. Relative Specificity and Sensitivity of GNP- and QD-Based LFIAs

The performance of the LFIAs based on GNP and QD conjugates was evaluated using a panel of 90 serum samples. A commercial ELISA kit for the detection of human adenovirus IgG antibodies was used as the reference. Among 57 negative serum samples, both LFIAs yielded negative results in 56 cases, corresponding to a relative specificity of 98.2% (Table 1).

The relative sensitivity of the GNP-G assay was 96.9% (32/33; 95% CI: 84.7–99.5), whereas that of the QD-G assay was 100% (33/33; 95% CI: 89.6–100). The relative specificity of both LFIAs was 98.2% (56/57; 95% CI: 90.7–99.7).

Among the 33 positive samples, antibodies were detected in 32 samples using the GNP-G conjugate and in all 33 samples using the QD-G conjugate (Table 2). The relative sensitivities of the two LFIAs were 96.9% and 100%, respectively. These results demonstrated that fluorescence-based signal detection using QDs enhanced assay sensitivity.

The ROC curve analyses demonstrated high diagnostic performance for both LFIAs, with AUC values of 0.976 for the GNP-G–based assay and 0.991 for the QD-G–based assay. At comparable specificity (98.2%), the QD-G system exhibited superior sensitivity (100%). Cohen's kappa analysis demonstrated moderate agreement for the GNP-G–based assay (κ = 0.482) and substantial agreement for the QD-G–based assay (κ = 0.653) compared with the reference ELISA.

4. Discussion

In the present study, a recombinant human adenovirus hexon protein fragment (rhHAdV) was successfully expressed, purified, and evaluated as an antigen for serological detection of anti-adenovirus antibodies. Both gold nanoparticle- and quantum dot-based lateral flow immunochromatographic assays demonstrated high diagnostic performance, whereas the QD-based format showed superior analytical sensitivity. These findings support the potential application of rhHAdV and QD-based LFIA for rapid assessment of adenovirus-specific immunity.

Rapid and accurate diagnostics are instrumental in the effective control of viral diseases, as they enable early detection and timely isolation of infected individuals. Among available diagnostic methods, the LFIA is widely used owing to its rapidity, ease of use, affordability, and portability [17]. However, the LFIA has certain limitations, including insufficient sensitivity and specificity and substantial variation in performance with respect to the quality of the biological reagents employed, including antibodies, antigens, and conjugates [18].

In the present study, we developed and evaluated two LFIAs for the serological detection of antibodies against human adenovirus using rhHAdV as the target antigen. Modern serological assays for infectious disease diagnosis are predominantly based on recombinant antigens, which provide high specificity, reproducibility, and ease of standardization [19,20]. The recombinant fragment corresponding to amino acid residues A120–R316 of the hexon protein was successfully expressed in a prokaryotic system and purified by metal-chelate affinity chromatography. The A120–R316 fragment includes a substantial portion of the exposed hexon domain and retains hypervariable regions that are major targets of antibody responses during adenoviral infection. The rhHAdV antigen exhibited high purity and strong immunoreactivity, as confirmed using western blotting, LC–MS/MS, and ELISA. Comparative evaluation of the LFIAs revealed that the QD-based assay exhibited higher analytical sensitivity than the GNP-based assay while maintaining high diagnostic specificity (98.2%).

Despite the promising diagnostic performance of the rhHAdV- and QD-based test systems, several limitations should be acknowledged. First, the study included a relatively small number of clinical samples, which may restrict the generalizability of the obtained diagnostic characteristics. Second, serum samples representing different stages of adenovirus infection were not comprehensively evaluated. Third, the potential cross-reactivity with antibodies against other adenovirus serotypes was not extensively evaluated. In addition, the repeatability and reproducibility of the developed LFIA were not assessed through intra-assay and inter-assay precision studies. Further validation using quantitative signal registration is required to determine assay robustness. Furthermore, although the QD-based LFIA demonstrated superior analytical sensitivity, fluorescence detection may require additional instrumentation compared with conventional GNP-based assays that can be interpreted visually. These limitations should be addressed in future studies aimed at large-scale validation and practical implementation of the developed diagnostic platform.

Truncated adenoviral hexon proteins containing hypervariable regions have previously been used in immunization and serological studies, including neutralization assays and antibody detection systems [21,22]. Consistent with the findings of these previous studies, the rhHAdV used in the present study retained strong immunoreactivity and effectively detected specific antibodies in patient sera.

Fluorescent nanoparticles, including QDs, can enhance LFIA sensitivity owing to their high fluorescence quantum yield and resistance to photobleaching [10,23]. Our findings are consistent with these reports, as the QD-based LFIA enabled antibody detection at higher serum dilutions than the GNP-based format. The diagnostic sensitivity achieved by the QD-based assay (100%) further supports growing evidence that fluorescence-assisted LFIA platforms can outperform conventional colorimetric systems, particularly when antibody concentrations are low.

The improved performance of the developed assays is likely attributable to both the structural properties of the selected antigen and the physicochemical characteristics of the applied labels. The presence of two hypervariable regions within the selected fragment may have contributed to the strong immunoreactivity observed in both ELISA and LFIA. The high analytical sensitivity of the QD-based LFIA can be attributed to the unique optical properties of QDs, including high fluorescence intensity, excellent photostability, and low background signal. These characteristics facilitate signal detection even at low antibody concentrations, which is particularly important for weakly positive samples within the diagnostic “gray zone.” The variability observed in ELISA antibody titers among patient sera most likely reflects the heterogeneity of humoral immune responses associated with adenoviral infections.

The results of this study demonstrate that recombinant human adenovirus hexon protein is a promising antigen for serological diagnostic applications. Incorporation of QDs into the LFIA improved assay sensitivity without a substantial loss of specificity, suggesting that fluorescence-based LFIA platforms may be particularly valuable for early antibody detection and identification of low-titer antibody responses. However, additional validation studies are required before routine diagnostic implementation can be recommended. Future investigations should include larger and geographically diverse patient cohorts, comprehensive evaluation of cross-reactivity with other adenovirus serotypes, and assessment of assay performance across different stages of infection. Further optimization of fluorescence detection systems may also improve the practicality and accessibility of QD-based LFIA for both clinical diagnostics and point-of-care applications.

5. Conclusions

While the LFIA offers rapidity, simplicity, and accessibility, it has certain limitations relating to the stability of its components. The developed QD-based LFIA represents a promising rapid serological platform for assessment of pre-existing immunity to adenoviral vectors and may support large-scale screening programs. The sensitivity of the fluorescence-based assay (100%) was found to exceed that of the GNP-based assay (96.9%). These findings confirm the potential of combining rhHAdV with QD-based detection for efficient serological diagnostics.