Immunochromatographic analysis of canine parvovirus (Parvoviridae: Protoparvovirus)

Full Text

Introduction

Canine parvovirus type 1 (CPV-1), belonging to the genus Parvovirus of the family Parvoviridae, was first identified in the 1960s as the causative agent of gastrointestinal and respiratory infections in dogs. Subsequently, mutations in CPV-1 led to the emergence of a new strain – canine parvovirus type 2 (CPV-2) – which was characterized by higher contagiousness and had spread worldwide by the 2000s [1, 2].

CPV-2 is a highly lethal and contagious virus that causes parvovirus enteritis, a condition characterized by fever, loss of appetite, lethargy, vomiting, and hemorrhagic gastroenteritis in dogs of all ages [2, 3]. However, the most severe clinical signs and high mortality rates (over 70%) are characteristic of puppies [2, 3].

The sources of infection are sick dogs and virus carriers, which shed the virus into the environment via feces; transmission occurs through the fecal-oral and airborne routes [4]. Canine parvovirus (CPV) exhibits a marked tropism for actively dividing cells. After infection, the virus initially replicates in the lymphoid tissue of the oropharynx and mesenteric lymph nodes, and then spreads hematogenously, reaching its highest concentration in the epithelium of the small intestinal crypts and lymphoid organs associated with the gastrointestinal tract [5]. As early as 4–7 days after infection, during the peak of clinical manifestations, the virus accumulates in large quantities in the intestines and feces of dogs, reaching up to 10⁹ TCID₅₀/g [4, 5]. It is during this period, especially in the early days of clinical manifestations, that the high concentration of the virus creates optimal conditions for early detection of the infection using immunochromatographic assay (ICA), which allows for the timely initiation of intensive therapy.

CPV is a non-enveloped virus consisting of a single DNA strand comprising 5,323 nucleotides and a capsid [6]. The genome contains two large open reading frames, one of which encodes non-structural proteins (NS1 and NS2), and the other encodes three structural proteins (VP1, VP2, and VP3) [6]. Among the virus's structural proteins, VP2 constitutes 90% of the capsid, which determines its leading role in the virus's interaction with the host [5], furthermore, it is the most immunogenic protein of the virus [7]. The unique amino acid sequence of VP2, which has no homologs among the dog's own proteins, makes this protein an optimal target for the early diagnosis of CPV infection [8].

Any clinical manifestations must be confirmed by laboratory diagnostic tests, as similar dyspeptic symptoms can be observed in many conditions. Currently, there are several main methods for detecting CPV infection, including loop-mediated isothermal amplification combined with enzyme-linked immunosorbent assay (ELISA) [9], polymerase chain reaction (PCR) [10, 11], ICA [11, 12] and the hemagglutination inhibition assay (HAI). Significant drawbacks of ELISA and PCR include the need for expensive equipment, qualified personnel, and a long time interval between sample collection and obtaining results. HAI, in turn, is rarely used today, as it is inferior to other more sensitive methods (PCR, ELISA, ICA) in terms of the clinical value of the result due to its lower informative value for early diagnosis. In this light, ICA appears to be the most optimal and inexpensive rapid test for obtaining results within a few minutes after receiving clinical samples.

These days, the market for ICA tests for CPV detection consists exclusively of imported diagnostic kits [12, 13], which leads to supply difficulties, increased diagnostic costs, and a shortage of consumables. These facts justify the relevance of developing domestic ICA tests aimed at detecting the VP2 protein in order to expand diagnostic capabilities for detecting parvovirus infection in dogs.

Materials and methods

Reagents and materials

Recombinant canine parvovirus VP2 antigen was supplied by HyTest (Russia; Cat. No. 8CP2). Bovine serum albumin (BSA), complete and incomplete Freiden adjuvants, dimethyl sulfoxide (DMSO) solution, and culture media were ordered from PanEco (Russia). All inorganic reagents, 3,3',5,5'-tetramethylbenzidine (TMB), and sodium citrate (Na₃C₆H₅O₇) were purchased from the HimMed company (Russia). Biotin N-hydroxysuccinimide ester (Sigma, USA) was used. Protein G Sepharose 4 Fast Flow (Cytiva, USA) was used for affinity chromatography. The anti-mouse antibody conjugate with RAM Iss horseradish peroxidase (Imtek, Russia) was used for the control line of the ICA test. Costar Assay Plate 96-well high-binding flat-bottom plates (Corning Inc., USA) were used for the ELISA. The membrane composite was ordered from MDI (India).

Conjugation of the VP2 antigen with bovine serum albumin

Conjugation of the VP2 antigen with BSA was performed using glutaraldehyde. In 200 μL of dilution buffer (0.1 M Na₂HPO₄, pH 6.8), 100 μg of BSA was first dissolved, followed by 100 μg of VP2. A 25% glutaraldehyde solution was added to the reaction mixture to achieve a final concentration of 0.1%, and the mixture was incubated for 3 hours on a shaker in the dark. After that, a 10% glycine solution was added to the conjugate to a final concentration of 0.5% and incubated for 20 min on a shaker in the dark. After incubation, the conjugate was dialyzed against phosphate-buffered saline (PBS) (145 mM NaCl, 10 mM K₂HPO₄, pH 6.9) overnight at +4 °C. After dialysis, the conjugate was aliquoted into 100 μL tubes and stored at −20 °C.

Production of monoclonal antibodies

The production of monoclonal antibodies (mAbs) against VP2 CPV was based on hybridoma technology, with prior immunization of BALB/c mice.

For the immunization of mice, a solution of the VP2-BSA conjugate containing 100 μg of the target protein was mixed with an equal volume of complete (for the first stage) or incomplete (for the second stage) Freund's adjuvant until a homogeneous emulsion was obtained. Immunization was performed in the hind paw pads in two stages, 13 days apart.

Immunization was assessed using a solid-phase indirect ELISA with immune and non-immune sera collected from the tail vein of the corresponding BALB/c mice on day 3 after the second immunization. A graph showing the relationship between serum dilution and optical density was plotted using Microsoft Excel 2010.

Hybridoma producers were obtained according to standard procedures [14].

Ascites fluid was collected several days after the administration of hybridoma cells to BALB/c mice primed with mineral oil.

The collected ascites fluids were tested by solid-phase indirect ELISA for the presence of specific antibodies against the CPV VP2 protein, using serial dilutions of the ascites fluids ranging from 1 : 1,000 to 1 : 1,000,000.

Sub-isotyping of the mAbs present in the ascites fluids was performed using the Mouse Typer Sub-Isotyping Kit (Cat. No. 172-2055, BioRad, USA) according to the protocol.

Antibodies were purified from ascites fluid by affinity chromatography on a protein G-sepharose column (Cytiva, USA).

Characterization and selection of monoclonal antibody pairs using an enzyme-linked immunosorbent assay

Antibody concentration was measured using a FlexA-200 HT spectrophotometer at a wavelength of 280 nm; for this purpose, the sample was diluted 20-fold in PBS and placed in a quartz cuvette.

Protein concentration was determined using Equation 1:

$С\left(mAb\right)=\frac{O{D}_{280}x}{\epsilon }$, (1)

where C (mAb) – concentration of the mAb solution (mg/ml), OD₂₈₀ – optical density at a wavelength of 280 nm, x – dilution of the solution, ε – the molar extinction coefficient of 1.4 for immunoglobulins.

The purity of the mAb samples was determined by electrophoresis in a 12% polyacrylamide gel (SDS-PAGE) under reducing conditions using the Bio-Rad Mini-Protean 3-cell vertical electrophoresis system (Bio-Rad, USA).

The search for antibodies that recognize the VP2 antigen in solution and form pairs, for the further development of an ICA rapid test, was conducted using ELISA methods with prior biotinylation of the antibodies. To this end, 100 μg of each monoclonal antibody was mixed with a 10-fold excess of a 1% solution of biotin-N-hydroxysuccinimide in DMSO and incubated overnight at +4 °C. The conjugation reaction was stopped by adding 0.1% NaN₃.

The capture mAb against the CPV VP2 protein was sorbed into the wells of the plate overnight at +4 °C. After washing, VP2 antigen was added and incubated for 1 hour at +37 °C. Biotinylated antibodies, also specific to VP2, were then added to form an immunocomplex consisting of capture mAb–antigen–detection mAb with biotin, and the mixture was incubated for 1 hour at +37 °C. For further visualization of the immunochemical interaction between antibodies and antigen, horseradish peroxidase-labeled streptavidin was used, which is capable of binding to biotin. The presence of the formed immune complexes with streptavidin was subsequently determined by adding TMB to visualize the reaction and detecting it on a FlexA-200 HT spectrophotometer (Allsheng, China) at a wavelength of 450 nm.

Synthesis of colloidal gold and conjugation with monoclonal antibodies

Colloidal gold (AuNPs) was prepared by the citrate method using gold chloride in accordance with the procedure described by N.V. Bogachev et al. [15].

Conjugation of monoclonal antibodies with colloidal gold

To determine the optimal conjugation conditions, 5 to 40 μg of antibodies per 1 mL of carrier were added to the carrier particles within a pH range of 5.5 to 9.0. The reaction was stopped by adding a stabilizing buffer. In parallel, a stability test was conducted by adding 1 M NaCl to samples of all samples with different pH values.

For the conjugation of mAbs with AuNPs, the detection antibodies were dialyzed against a 0.1 M Na₂CO₃ solution at pH 9.0 overnight at +4 °C. The precipitated antibodies were removed by centrifugation at 12,000 rpm for 5 min, after which the antibody concentration in the supernatant was measured using a FlexA-200 HT spectrophotometer at a wavelength of 280 nm. The required volume of the solution containing AuNPs was adjusted to pH 8.5 by adding 0.1 M Na₂CO₃ solution. MAbs were diluted in 0.1 M Na₂CO₃ solution to 40 μg/mL and added to the solution containing AuNPs in a 1 : 10 ratio. The reaction mixture was incubated at room temperature for 30 min. Afterward, 5% BSA and 50% sucrose solutions were added so that their final concentrations were 0.2% and 10%, respectively, and the mixture was incubated for 15 min. Next, the conjugate was centrifuged at 12,000 rpm at +4 °C for 5 min. Then, the maximum amount of supernatant was carefully removed, and PBS, a 0.2% BSA solution, and a 0.1% NaN₃ solution were added.

Production of experimental laboratory prototypes of the ICA test system

Using the Easy Printer Model LPM-02 (MDI, India) to apply reagents to an analytical membrane mounted on a substrate (Cat. No. 150CNPH-N-SS40-L2-P25, MDI, India), to apply polyclonal goat anti-mouse antibodies and mouse monoclonal VP2 G10(1) antibodies diluted in PBS, thereby forming control and test bands, respectively. The membranes were dried at room temperature in the dark overnight. Thereafter, using a programmable guillotine-type SS Automatic Strip Cutter Model M-100 (MDI, India), the analytical membrane with the applied antibodies was cut into 4-mm-wide strips.

7 μL of the VP2 E9 capture antibody conjugate (1) with AuNPs was applied to the conjugate membrane (Cat. No. 150CNPH-N-SS40-L2-P25) and left to dry at room temperature overnight in the dark.

To assemble the strips, an absorbent membrane (AP110, MDI, India) pre-cut into 4 mm wide strips and the conjugate-coated membrane were overlapped and attached to the substrate with the analytical membrane. A sample application membrane (Cat. No. GFB-R4(0.35), MDI, India), pre-cut into 4 mm wide strips, was adhered on top of the conjugate membrane.

Immunochromatographic assay using VP2 protein and clinical samples from dogs

Serial dilutions of the recombinant VP2 CPV protein were prepared in the range of 1 to 200 ng/mL in phosphate-buffered saline with Tween (PBS-Tween) in 2-fold steps. Each dilution was applied to the prepared test strips in triplicate and incubated for 10 minutes at room temperature.

To prepare a positive control sample, recombinant VP2 CPV protein was added to a known negative clinical sample to a final concentration of 100 ng/mL.

Clinical samples and the model sample were diluted in PBS at a 1:2 ratio. The diluted samples were applied to the prepared test strips in a volume of 120 μL in triplicate. The exposure time was 10 minutes.

The test strips were photographed using a gel imaging system (Clinx Science, China). The intensity of the test zone staining was determined using Image Lab Software (Bio-Rad, USA) in relative units.

Interpretation of ICA Test Results

The graph showing the relationship between the concentration of the recombinant VP2 protein and the intensity of the test line was plotted using GraphPad Prism 8.0.1 with a 4PL fit. The observed limit of detection (vLOD) for the semi-quantitative analysis was determined according to M. Alhammadi et al. [16]. Standard deviations for tests using clinical samples were calculated using Microsoft Excel 2010.

Results

Production of immunoreagents

A VP2-BSA conjugate prepared using glutaraldehyde was used to immunize BALB/c mice.

Based on the results of immunization assessment using a solid-phase ELISA with unconjugated VP2 protein, it was found that serum from two immunizations with the VP2-BSA conjugate exhibits a specific immune response to pure, unconjugated VP2 antigen (Fig. 1).

 

Fig. 1. Testing of mouse sera after immunization with VP2-BSA.

VP2-BSA – the titration curve of immune serum; C – the titration curve of non-immune serum.

Рис. 1. Тестирование мышиных сывороток после иммунизации VP2-БСА.

VP2-БСА – кривая титрования иммунной сыворотки; К – кривая титрования неиммунной сыворотки.

 

The procedures for hybridizing immune lymphocytes with cells from the sp2/0 tumor line, cloning hybridoma clones, and collecting ascites fluid were performed according to standard protocols [14]. As a result, 10 stable clones were obtained, exhibiting high ascites-forming capacity and viability exceeding 90%: VP2 E9 (1), VP2 E9 (2), VP2 G2 (1), VP2 G2 (2), VP2 G3 (1), VP2 G3 (2), VP2 G10 (1), VP2 G10 (2), VP2 H5 (1), VP2 H5 (2).

Testing of ascites fluids and subsequent experiments with purified mAbs against VP2 showed that each hybridoma clone produces antibodies capable of recognizing both the solid-phase-immobilized and the solution-bound unconjugated VP2 protein.

The results of the immunochemical characterization of mAbs after their isolation from ascites fluids are presented in Table 1.

 

Table 1. Resulting data on the characterization of the obtained monoclonal antibodies against the VP2 protein of canine parvovirus

Таблица 1. Результирующие данные по характеризации полученных моноклональных антител против белка VP2 парвовируса собак

Clone name Название клона	Subclass Субкласс	Titer Титр	Volume, mL Объем, мл	Concentration, mg/mL Концентрация, мг/мл	Sample purity, % Чистота образца, %
VP2E9(1)	IgG1	1 : 1000	4.6	0.46	93.8
VP2E9(2)	IgG1	1 : 1000	9.3	1.89	95.5
VP2G2(1)	IgG2b	1 : 1000	9.2	2	94.7
VP2G2(2)	IgG2b	1 : 1000	7	0.64	97.8
VP2G3(1)	IgG1	1 : 1000	12.7	2.07	95.9
VP2G3(2)	IgG1	1 : 300	8.5	0.46	93.1
VP2G10(1)	IgG1	1 : 1000	8.1	0.6	93.9
VP2G10(2)	IgG1	1 : 1000	17.5	1.29	95
VP2H5(1)	IgG1	1 : 1000	5.8	1.9	98.1
VP2H5(2)	IgG1	1 : 1000	8.5	1.8	96.2

 

The antibody titer was determined as the endpoint of the titration curve, defined as a more than threefold difference in optical density, with each clone titrated starting from a concentration of 1 μg/mL. All clones were selected for further experiments to identify detection and capture antibodies.

Screening of a panel of monoclonal antibodies against canine parvovirus VP2

A biotin-based sandwich ELISA was used to select a pair of antibodies, with one acting as the capture antibody and the other as the detection antibody.

Each of the 10 clones obtained was conjugated with N-hydroxysuccinimide biotin (biotin). After testing the monoclonal antibody (mAb) conjugates with biotin in an indirect ELISA, an optimal concentration of 1 μg/mL was selected, which was used in the sandwich ELISA for all biotin-conjugated mAbs. Each of the 10 monoclonal antibodies was evaluated both as a detection antibody and as a detection antibody in all possible combinations. Based on the experimental results, mAbs VP2 E9 (1), VP2 G2 (1), and VP2 G10 (1) were selected for further studies as the most promising.

Preparation and characterization of colloidal gold and conjugates with monoclonal antibodies

Colloidal gold was selected as the detection label due to its properties, which include ease of synthesis, high biocompatibility with antibodies–owing to the ability of AuNPs particles to bind non-covalently to immunoglobulins–as well as the rapid and straightforward interpretation of the ICA test results [17]. Following the method of N.V. Bogachev et al. [15], AuNPs were synthesized using the citrate method, and their physicochemical properties were determined using a Malvern ZetaSizer Nano ZS (UK). The synthesis resulted in a AuNP solution with particles 59 nm in size, a zeta potential of −21.1 ± 3.38 mV, and a polydispersity index (PDI) of 0.603 ± 0.043. The measurement results are presented in Figs. 2 and 3. Despite the relatively high PDI value, according to the literature, particles with a size of approximately 40–60 nm significantly increase the test sensitivity compared to smaller particles [18, 19]. Furthermore, the obtained zeta potential value of the particles indicates their high stability in the colloidal solution [20].

 

Fig. 2. Particle size distribution graph of colloidal gold (nm) relative to the scattered light intensity.

Рис. 2. График распределения частиц коллоидного золота по размеру (нм) относительно интенсивности рассеянного света.

 

Fig. 3. Zeta potential distribution graph of colloidal gold particles (mV) relative to the total number of events.

Рис. 3. График распределения дзета-потенциала частиц коллоидного золота (мВ) относительно общего количества событий.

 

Prior to conjugating mAbs with AuNPs, pH values and mAb concentrations were selected at which AuNP particle aggregation did not occur. As both the pH and the antibody concentration increased, it was found that the AuNP solution retained its original color, indicating that no particle aggregation had occurred. The experimental results revealed that the optimal mAb concentration for conjugation was 40 μg/mL, with a solution pH of 8.5.

After conjugation of the antibody clones selected by sandwich-ELISA with AuNPs, several tests were performed on ICA strips, where each of the selected antibodies served as both a capture antibody and a detection antibody.

Based on the results of the experiments, the pair of mAbs VP2 E9 (1) and VP2 G10 (1) showed the best result with no background on the test line, where the first antibody acted as the detection antibody in the conjugate with the antigen, and the second as the capture antibody.

The spectra of the AuNP solution and the AuNP conjugate with VP2 E9 antibodies (1) were recorded in the ultraviolet (UV)-visible range of 400–700 nm using an Allsheng FlexA-200HT spectrophotometer. The red shift of the conjugate solution spectrum, with a maximum optical density at 529 nm, compared to the AuNP solution spectrum, with a maximum optical density at 534 nm, indicates the transition of AuNPs from the native state to the antibody-conjugated state [18, 21]. The UV spectra plots, generated in Microsoft Excel 2010, are shown in Fig. 4.

 

Fig. 4. UV spectra graphs.

E9-AuNPs – the graph of the spectrum of the solution of the conjugate AuNPs and mAbs VP2 E9 (1), AuNPs – the graph of the spectrum of the AuNPs solution.

Рис. 4. Графики ультрафиолетовых спектров.

E9-КЗ – график спектра раствора конъюгата коллоидного золота (КЗ) и моноклональных антител VP2 E9 (1); КЗ – график спектра раствора КЗ.

 

Selection of a membrane composite and optimization of ICA test parameters

The membrane composite was selected from among the membranes manufactured by MDI (India). For an analytical membrane, pore size is a critical factor, as it determines the capillary flow rate as well as the efficiency of labeled antibody accumulation in the control and test line zones [22]. For the selection process, membranes with different pore sizes (8, 10, 12, 15, 90, 150, 200 μm), and among them, a membrane with a pore size of 150 μm was selected, as it exhibited the most intense signal and ensured optimal analyte flow rate with minimal background. Of the two conjugate membranes, PT-R5 and PT-R7, the latter was selected due to its uniform and complete release of the conjugate. The absorbent membrane must ensure optimal capillary flow across the entire strip and possess sufficient absorbency [23]. Based on these requirements, the AP110 was preferred over the AP080 among the absorbent membranes.

To optimize the conditions of the ICA test system, the optimal amounts of antibodies were selected for application to the test and control bands. To optimize the control zone, goat anti-mouse polyclonal antibodies and rabbit anti-mouse antibodies (RAM Iss, Imtek) were applied at concentrations of 0.5, 1.0, and 2.0 μg per strip, and to optimize the test zone, capture antibodies were applied at concentrations of 1.0, 1.5, 2.0, and 4.0 μg per strip. At the same time, drying conditions for the test strips were also selected: 1 hour in a thermostat at +37 °C, overnight at room temperature, or overnight in a thermostat at +37 °C. As a result, it was concluded that 2.0 μg of antibodies per strip was the most optimal amount for both the control strip and the test strip. Drying the test strips overnight at room temperature yielded the best results compared to drying in a thermostat, due to a higher signal intensity and lower variability between replicates.

Immunochromatographic assay using the VP2 protein

According to M. Alhammadi et al. [16], the vLOD was defined as the minimum concentration of VP2 protein that produces the faintest color on the test line but is still distinguishable from the negative control. Fig. 5 shows that a test line is still visible on test strip number 8, from which it was concluded that the observed limit of detection was 1.56 ng/mL. At the same time, the graph of test line intensity versus VP2 concentration exhibits linearity in the target protein concentration range from 12.5 to 200 ng/mL, which can be used in future studies as a quantitative assessment of protein content in clinical samples (Fig. 6).

 

Fig. 5. Determination of the apparent limit of detection (vLOD) of the ICA test system for detecting VP2 protein in serial dilutions.

VP2 protein solutions with different concentrations were applied to test strips 1–9: 1 – 200 ng/mL, 2 – 100 ng/mL, 3 – 50 ng/mL, 4 – 25 ng/mL, 5 – 12.5 ng/mL, 6 – 6.25 ng/mL, 7 – 3.125 ng/mL, 8 – 1.56 ng/mL, 9 – 0 ng/mL.

Рис. 5. Определение наблюдаемого предела обнаружения (vLOD) ИХА-тест-системы при детектировании белка VP2 в серийных разведениях.

На тест-полоски 1–9 наносили растворы белка VP2 с разными концентрациями: 1 – 200 нг/мл, 2 – 100 нг/мл, 3 – 50 нг/мл, 4 – 25 нг/мл, 5 – 12,5 нг/мл, 6 – 6,25 нг/мл, 7 – 3,125 нг/мл, 8 – 1,56 нг/мл, 9 – 0 нг/мл.

 

Fig. 6. Graph of the dependence of the concentration of recombinant VP2 protein on the intensity of staining of the test line.

The y-axis shows the relative units of band staining intensity; the x-axis shows the concentration of recombinant vp2 protein (ng/ml) (n = 3).

Рис. 6. График зависимости концентрации рекомбинантного белка VP2 от интенсивности окрашивания тестовой линии.

По оси Y – относительные единицы интенсивности окрашивания полосы, определенных в программе Image Lab Software; по оси X – концентрация рекомбинантного белка VP2 (нг/мл) (n = 3).

 

ICA testing using clinical samples from dogs

To evaluate the performance of the developed test system, clinical samples with positive and negative VP2 values–confirmed by a commercial test (QBQVET, Russia)—were used, as well as a positive control sample with a known VP2 concentration of 100 ng/mL (Fig. 7).

 

Fig. 7. Determination of VP2 in clinical samples.

Test strips 1, 2, 3 are positive material in three replicates, test strips 4, 5, 6 are negative material in three replicates, test strips 7, 8, 9 are a model positive sample with added recombinant VP2 protein at a concentration of 100 ng/mL in three replicates.

Рис. 7. Определение VP2 в клинических образцах.

Тест-полоски 1, 2, 3 – положительный материал в трех повторах; тест-полоски 4, 5, 6 – отрицательный материал в трех повторах; тест-полоски 7, 8, 9 – модельный положительный образец с добавленным рекомбинантным белком VP2 в концентрации 100 нг/мл в трех повторах.

 

The intensity of the test line on the strips containing the positive control (69 ± 10.4 relative units) is consistent with the intensity of the test line on the strips to which a solution of recombinant VP2 protein was applied in the PBST (67.3 ± 4.6 relative units), indicating the high accuracy and specificity of the developed test system.

Discussion

In this study, an ICA test system was developed for the detection of VP2 in CPV. As the major capsid protein, VP2 is highly immunogenic and is therefore widely used in the diagnosis of parvovirus infection [9–12, 24, 25]. This is precisely why this protein was selected as a target for the production of mAbs and the subsequent development of an ICA test system based on them.

A review of the literature revealed that researchers employ various formats of ICA rapid tests to detect VP2 CPV in biological samples. For example, in the study by C. Sharma et al., polyclonal antibodies are used as capture antibodies instead of mAbs against VP2 CPV, while in the study by E. Salmanli et al., a test system is described that is designed to detect antibodies against VP2 rather than the protein itself [12, 26]. The ICA test system described in this study is designed to detect the viral VP2 protein. This allows for the confirmation of the virus's presence in dogs, which is important for early diagnosis when the CPV antibody titer has not yet peaked. Furthermore, the test system is based on two different monoclonal antibodies that do not compete with each other for binding, which ensures high specificity and reduces the likelihood of cross-reactions compared to polyclonal.

It should be noted, however, that the sensitivity of these tests varies across studies and is somewhat lower than that of other methods for detecting VP2. For example, in a study by S.S. Tinky et al., where the PCR test was used as the reference, ICA demonstrated sensitivity and specificity values of 72.2% and 92.8%, respectively [11]. Another study aimed at comparing tests for detecting CPV showed that ICA had the lowest sensitivity among all the tests considered, with a value of 56.1% [27]. In turn, a study by K. Felix et al. demonstrated that a commercial ICA kit had a relative sensitivity of 95.4% and a specificity of 71.4%, with PCR used as the reference method [28]. Despite the differing values, the authors agree that ICA tests are indispensable in the rapid diagnosis of CPV infection, since performing this analysis requires neither specialized training nor expensive equipment.

In this study, diagnostic sensitivity and specificity were not calculated due to the small number of clinical samples (n = 2). Instead, a comparative evaluation was conducted of the ICA test results using a model positive sample obtained by adding recombinant VP2 protein to a validated negative sample, against the ICA test results on the recombinant protein. Experiments showed comparable intensity of the test line on the model sample and on pure recombinant protein at the same concentration, indicating that sample components do not affect the test result. However, it should be noted that model samples do not always reflect the interactions of the native antigen with biomaterial components. The lack of a large number of clinical samples did not allow us to identify diagnostic criteria such as sensitivity and specificity in this study; therefore, further research on a larger sample is required to determine these parameters.

The ICA test system developed is of great significance for veterinary diagnostics in our country, as the main costly components, such as mAbs and AuNPs, were produced at the N.N. Blokhin National Medical Research Center for Oncology, a federal state budgetary institution under the Russian Ministry of Health. The use of domestically produced reagents reduces dependence on imported supplies, ensures high reproducibility of results, and opens up opportunities for scaling up and producing rapid tests.

Conclusion

In this study, an ICA test system was developed for the detection of the VP2 protein of CPV. A panel of mAbs against VP2 was created, and binding and detection antibodies were identified. Colloidal gold was synthesized, and mAb conjugates were prepared. To improve the assay's performance, optimal conditions for assembling the test strips and conducting the assay were selected. The observed lower limit of detection (vLOD) for the system was 1.56 ng/mL. The test system showed no signal on a confirmed negative clinical sample, as well as consistent staining intensity on a model positive sample and on pure recombinant VP2 protein, indicating the accuracy and specificity of the system. Thus, the resulting test system can be applied for the diagnosis of CPV.

About the authors

Nikolay E. Varlamov

N.N. Blokhin National Medical Research Center of Oncology

Email: n.varlamov@ronc.ru
ORCID iD: 0000-0002-8602-6599

Researcher, Laboratory of Biocollection and Antibody-Based Preparations

Russian Federation, 115478, Moscow

Mikhail A. Ryzhikov

N.N. Blokhin National Medical Research Center of Oncology; National Research University Higher School of Economics

Author for correspondence.
Email: m.ryzhikov@ronc.ru
ORCID iD: 0009-0000-2292-8537

Laborant Researcher, Laboratory of Biocollection and Antibody-Based Preparations; Postgraduate Student

Russian Federation, 115478, Moscow; 101000, Moscow

Natalya Y. Serebryakova

N.N. Blokhin National Medical Research Center of Oncology

Email: n.serebrjakova@ronc.ru
ORCID iD: 0009-0003-7210-2882

Researcher, Laboratory of Biocollection and Antibody-Based Preparations

Russian Federation, 115478, Moscow

Dmitriy V. Gusev

N.N. Blokhin National Medical Research Center of Oncology

Email: d.gusev@ronc.ru
ORCID iD: 0000-0003-0218-8265

Cand. Sci. (Chem.), Head of the Laboratory of Chemical Synthesis

Russian Federation, 115478, Moscow

Yana O. Sadovskaya

N.N. Blokhin National Medical Research Center of Oncology

Email: ja.sadovskaja@ronc.ru
ORCID iD: 0009-0009-7115-7797

Junior Researcher, Laboratory of Biocollection and Antibody-Based Preparations

Russian Federation, 115478, Moscow

Margarita G. Khotuleva

N.N. Blokhin National Medical Research Center of Oncology

Email: m.khotuleva@ronc.ru
ORCID iD: 0009-0008-6104-5233

Laborant Researcher, Laboratory of Biocollection and Antibody-Based Preparations

Russian Federation, 115478, Moscow

Anastasia O. Karimova

N.N. Blokhin National Medical Research Center of Oncology; National Research University Higher School of Economics

Email: a.karimova@ronc.ru
ORCID iD: 0009-0000-0317-9948

Junior Researcher, Laboratory of Biocollection and Antibody-Based Preparations; Postgraduate Student

Russian Federation, 115478, Moscow; 101000, Moscow

Olga N. Solopova

N.N. Blokhin National Medical Research Center of Oncology

Email: o.solopova@ronc.ru
ORCID iD: 0000-0002-5465-6094

Cand. Sci. (Biol.), Head of the Laboratory of Biocollection and Antibody-Based Preparations

Russian Federation, 115478, Moscow

Pavel K. Ivanov

N.N. Blokhin National Medical Research Center of Oncology

Email: p.ivanov@ronc.ru
ORCID iD: 0000-0003-1403-8520

D. Sci. (Med.), Leading Researcher, Laboratory of Biocollection and Antibody-Based Preparations

Russian Federation, 115478, Moscow

References

Supplementary files