Diagnostic Performance of Competitive ELISA and Western Blot Methods for the Detection of Antibodies against Theileria equi and Babesia caballi

Theileria equi (T. equi) and Babesia caballi (B. caballi) are the causative pathogens of Equine piroplasmosis (EP), a disease that has brought huge economic losses and great restrictions to the global equine industry. Rapid and accurate diagnostic methods are critical for the effective monitoring of the disease. In this study, we developed novel competitive ELISA methods and western blot assays based on the EMA1 or Bc48 proteins to detect antibodies against T. equi or B. caballi, respectively. In the novel cELISA, horseradish peroxidase (HRP)-labeled monoclonal antibodies are used in place of enzyme-conjugated secondary antibodies, in order to speed up the entire procedure. These methods have high sensitivity and no cross-reactivity with antibodies against other equine diseases. In the newly developed western blot assays, we optimized the dilution of T. equi or B. caballi positive serum samples to 1:200. Compared with the commercially available kit, both the novel cELISA assay and the western blot assay showed high coincidence rates in detecting antibodies against T. equi and B. caballi. Taken together, the novel cELISA and the western blot assays for detecting antibodies against T. equi or B. caballi have the potential to rapidly test for T. equi or B. caballi and to contribute to the surveillance and control of this disease.


Introduction
Equine piroplasmosis (EP) is an important protozoan infectious disease, caused by Theileria equi (T. equi) and Babesia caballi (B. caballi), and affects equids worldwide [1]. The clinical presentation is related to intravascular hemolysis and associated systemic illness, characterized by fever, anemia, loss of appetite, edema, jaundice, hepatomegaly, and splenomegaly [2][3][4]. The prevalence of this disease is related to many risk factors, including animal species, age, gender, breed, activities, and environmental factors [5]. EP has caused huge economic losses to the horse industry, including the cost of treatment and death. Moreover, its occurrence has caused restrictions in equine trading [6]. Because of the veterinary and economic impact of this disease, the World Organization for Animal Health (WOAH) has listed EP as a notifiable disease [7]. Many countries prevent the movement of T. equior B. caballi-positive animals into disease-free areas through strong restrictions [8]. This highlights the necessity of effective control measures in countries where the disease is endemic, and of enhancing the detection of EP in animals exposed to the parasites that cause it.
To date, the diagnostic methods used to detect T. equi and B. caballi infection include microscopic examination, the complement fixation technique (CFT), the indirect fluorescence antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA), and PCR [9][10][11][12]. Nevertheless, in both acute and early infections, serological testing becomes an important diagnostic method for confirming the involvement of T. equi or B. caballi, and ELISA has 2.4. Establishment of cELISA for Diagnosis of T. equi or B. caballi To optimize the amounts of coating antigen to be used, a checkerboard assay was conducted as follows. The coating protein EMA1 was diluted to five concentrations (0.1 µg/mL, 0.5 µg/mL, 0.75 µg/mL, 1 µg/mL, 1.5 µg/mL) and Bc48 was diluted to six concentrations (0.5 µg/mL, 0.75 µg/mL, 1 µg/mL, 1.25 µg/mL, 1.5 µg/mL, 2 µg/mL). To determine the optimal dilution of the sera to be tested, four positive and four negative sera were diluted to generate four serial twofold dilutions (1:1, 1:2, 1:4, and 1:8). 5% skimmed milk, 5% BSA and 5% PBS were used to determine the optimum block buffer, and the plate was stained with 3,3 ,5,5 -tetramethylbenzidine (TMB)-ELISA Substrate Solution (InnoReagents, Zhejiang, China) for different lengths of time (5 min, 10 min, 15 min, 20 min, and 25 min) to determine the optimum substrate reaction time. The optimum reaction conditions for use in this cELISA were selected based on the minimum ratio of OD 450nm values between positive and negative sera (P/N).
The standard cELISA procedure was as follows: a 96-well plate was coated with antigen (100 µL/well), which was in PBS buffer (0.1 M, PH 7.4) at optimum concentrations at 2-8 • C for 12-16 h. After washing three times with 200 µL PBST (PBS with 0.1% Tween), the plate was blocked with the optimum block buffer in incubator at 37 • C for two hours and washed three times with PBST again. Next, the tested sera at the optimal dilution were added to the wells (100 µL/well) and the plate was put into an incubator at 37 • C for thirty minutes. After washing again, 100 µL of diluted HRP-conjugated IgG was added to each well and mixed. Incubation of the plate was performed for 30 min at 37 • C and washed three times with 200 µL washing buffer. The plate was stained with TMB at 15-25 • C for the optimum reaction time and the reaction was stopped by the addition of 50 µL 2 M H 2 SO 4 . Finally, the OD 450nm value of the plate was measured using enzyme calibration (Biotek, Winooski, Vermont, USA) based on the values from the positive and negative sera.

Determination of the Cut-Off Value of the Novel cELISA
A total of 200 sera were collected from Inner Mongolia from 2019 to 2020 and kept in our laboratory. Of these, 47 samples were determined to be positive for T. equi and 25 samples were determined to be positive for B. caballi [21]. The serum samples were used to determine the cut-off value for the novel cELISA. The OD 450nm values of the samples were converted to a percent inhibition (PI) value using the following formula: PI (%) = (1 − (OD 450nm value of sample ÷ OD 450nm value of negative serum)) × 100%. In this study, we determine the baseline cut-off values by assessing the maximum consistency between the detection results of the novel cELISA methods and the identification of the serum samples.

Determination of the Analytical Specificity and Sensitivity of the Novel cELISA
To evaluate the analytical specificity of the novel cELISA, serum samples positive for T. equi and B. caballi, E. coli, S. abortus equi, S. equi, EHV-4, EAV, B. mallei, EIAV, and T. evansi, as well as serum samples negative for T. equi and B. caballi, were simultaneously tested. The analytical sensitivity of the novel cELISA was determined with a serial dilution of a separate serum sample positive for T. equi or B. caballi, which was diluted at ratios of 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, and 1:512 with PBS. These dilutions were also used to test the lowest dilution at which the novel cELISA could still detect the diseases.

Comparison of the Novel cELISA and Commercially Available cELISA Kit
The novel cELISA and the commercially available cELISA kit (VMRD, Pullman, WA, USA) were used in parallel to detect antibodies against T. equi and B. caballi. The 200 serum samples that were used in the cut-off determined section above were tested in this comparative analysis. These tests of the commercial kit were conducted following the manufacturer's instructions. We performed ROC curve analysis as a statistical tool for the diagnostic evaluation of the two cELISA assays in surveillance samples, where the value for the area under the curve (AUC) enabled a combined measure of diagnostic sensitivity and specificity (an AUC of 1 indicates perfect discriminatory value; an AUC of 0.5 or less indicates no discriminatory value).

Establishment of a Western Blot Assay That Detects T. equi and B. caballi Antibodies
SDS-PAGE Bis-Tris gels were prepared with PAGE Gel Fast Preparation Kit (Shanghai Epizyme Biomedical Technology Co., Ltd, Shanghai, China) for western blotting. The prepared protein EMA1 or Bc48 was separated by electrophoresis on SDS-PAGE Bis-Tris gel and run over two stages, including 80 V for 20 min and 120 V for 40 min. Next, the separated protein was transported onto nitrocellulose membranes and blocked with 5% skimmed milk (with NaN 3 ) for 2 h at room temperature. Membranes were incubated with test sera for 1.5 h and washed three times for 10 min with tris-buffered saline/Tween (TBST). Then, the membranes were incubated with DyLight 680-labeled goat anti-equine secondary antibodies (1:5000 in PBS) (NOVUS, Minneapolis, MN, USA) for 40 min and washed again. Finally, the target bands were detected using an Odyssey imaging system (LI-COR, Lincoln, NE, USA). To determine the optimal dilution of the tested sera, positive and negative sera were diluted to generate five serial dilutions (1:50, 1:100, 1:200, 1:400, and 1:800).

Comparative Analysis of the Clinical Samples
A total of 208 clinical serum samples were collected from Inner Mongolia, China in 2022 and stored in our laboratory. In order to perform comparative analysis of the clinical samples, the novel cELISA, the commercial cELISA kit (VMRD, Pullman, WA, USA) and the western blot were used simultaneously to detect the antibodies against T. equi and B. caballi. The Venn diagram shows the comparative results of the novel cELISA method, the western blotting method and the commercial cELISA method for the diagnosis of clinical samples of T. equi and B. caballi. Venn plot map analyses were performed using the Jvenn tool, a free online platform (http://www.bioinformatics.com.cn (accessed on 28 October 2022)) for data analysis [22].

Preparation of the Proteins EMA1 and Bc48 and the Corresponding HRP Labeled mAbs
SDS-PAGE verification showed that the EMA1 and Bc48 proteins were successfully expressed using the aforementioned prokaryotic expression system for the production of proteins of the expected sizes of 42 kDa and 40 kDa, respectively [21]. Finally, 20 mg (1 mg/mL) of purified recombinant EMA1 protein and 20 mg (1 mg/mL) of purified recombinant Bc48 protein were obtained, respectively. The correctly identified EMA1 and Bc48 proteins were stored at −80 • C. Next, the purified proteins were used as antigens for the immunization of mice to generate mAbs for use in cELISA development. The specificity of 1A12 (EMA1) and 1A3 (Bc48) were further confirmed by results of competitive binding assays, designed to detect the capability of immune sera of known specificity to inhibit the binding of each mAb to its respective antigens. Hybridoma cells that could secrete mAbs against EMA1 or BC48 were collected and mAbs clearly reacted with the recombinant EMA1 and Bc48 proteins. After identification by using an antibody subtype kit, mAbs 1A12 and 1A3 were both determined to be IgG1 subtypes. Then, 15 mg (1 mg/mL) of the purified mAb 1A12 and 15 mg (1 mg/mL) of the purified mAb 1A3 were obtained. Both mAbs 1A12 and 1A3 were then labeled with HRP using the HRP-labeling kit, and 5 mg of each HRP-labeled mAb was stored in −80 • C refrigerator with 50% glycerol.

Establishment of the cELISA Method
The checkerboard titration method was used to optimize our cELISA diagnostic method for T. equi or B. caballi, separately [23]. The results showed that the optimal EMA1 protein coating concentration for the detection of T. equi was l µg/mL ( Figure 1A), and the most suitable blocking buffer was 5% BSA ( Figure 1B). The optimal dilution of the equine serum sample to be tested was 1:1 ( Figure 1C). The optimal colorimetric reaction time was 15 min since the P/N value at this time was the minimum ( Figure 1D). Two hundred equine serum samples were assessed using our novel cELISA to deter- The optimal coating concentration of Bc48 protein for the detection of B. caballi was 0.5 µg/mL ( Figure 1E) and the most suitable blocking buffer was 5% BSA ( Figure 1F). The optimal dilution of the equine serum sample to be tested was found to be 1:2 ( Figure 1G). The optimal colorimetric reaction time was 10 min since the P/N value at this time was the minimum ( Figure 1H).

Determination of the cELISA Cut-Off Value (PI)
Two hundred equine serum samples were assessed using our novel cELISA to determine the cut-off value of the cELISA. The samples were collected from Inner Mongolia from 2019 to 2020 and were kept in our laboratory. Of these samples, 47 were positive for T. equi and 153 were negative for T. equi, and 25 samples were positive for B. caballi and 175 were negative for B. caballi. Our results suggested that when the cut-off value was set to 40%, the consistency and identification results of the serum samples by the novel cELISA methods were at their maximum, so the cut-off value for both novel cELISA was 40.0% (Figure 2A,B). When the PI value of the tested serum sample was equal to or greater than 40%, it was determined to be antibody-positive and the sample was regarded to be antibody-negative at a PI < 40%.

Determination of the cELISA Cut-off Value (PI)
Two hundred equine serum samples were assessed using our novel cELISA to determine the cut-off value of the cELISA. The samples were collected from Inner Mongolia from 2019 to 2020 and were kept in our laboratory. Of these samples, 47 were positive for T. equi and 153 were negative for T. equi, and 25 samples were positive for B. caballi and 175 were negative for B. caballi. Our results suggested that when the cut-off value was set to 40%, the consistency and identification results of the serum samples by the novel cELISA methods were at their maximum, so the cut-off value for both novel cELISA was 40.0% (Figure 2A,B). When the PI value of the tested serum sample was equal to or greater than 40%, it was determined to be antibody-positive and the sample was regarded to be antibody-negative at a PI < 40%. Figure 2. Determination of the cut-off value of the novel cELISA assays. Two hundred equine serum samples were assessed using the novel cELISA to determine the cut-off value. When the consistency and identification results of the serum samples by the novel cELISA methods were at their maximum, the cut-off values of both novel cELISA assays were found to be 40% (A,B).

Analysis of the Specificity of the Novel cELISA
To determine the analytical specificity of detection of T. equi antibodies, sera positive for other common equine diseases, including EIAV, EAV, EHV-4, EIV, B. caballi, B. mallei, T. evansi, E. coli, S. equi, and S. abortus equi, were assessed using the cELISA to determine cross-reactivity. As shown in Figure 3A, the resulting PI values for T. equi antibody- Figure 2. Determination of the cut-off value of the novel cELISA assays. Two hundred equine serum samples were assessed using the novel cELISA to determine the cut-off value. When the consistency and identification results of the serum samples by the novel cELISA methods were at their maximum, the cut-off values of both novel cELISA assays were found to be 40% (A,B).

Analysis of the Specificity of the Novel cELISA
To determine the analytical specificity of detection of T. equi antibodies, sera positive for other common equine diseases, including EIAV, EAV, EHV-4, EIV, B. caballi, B. mallei, T. evansi, E. coli, S. equi, and S. abortus equi, were assessed using the cELISA to determine cross-reactivity. As shown in Figure 3A, the resulting PI values for T. equi antibody-positive sera were higher than 40%, while the PI values of sera positive for other equine diseases were all under 40%, and were therefore assessed as negative. Similarly, when the cELISA was tested for specificity for antibodies against B. caballi as well as other common equine diseases (EIAV, EAV, EHV-4, EIV, T. equi, B. mallei, T. evansi, E. coli, S. equi, and S. abortus equi), the PI values of the serum samples positive for other common equine diseases were all lower than 40%, and therefore tested as negative, while those positive for B. caballi were above 40% and therefore considered to be positive ( Figure 3B). These results indicated that the novel cELISA assays do not detect antibodies against pathogens except for T. equi or B. caballi in equine sera, and the tests, therefore, have high specificity. equine diseases (EIAV, EAV, EHV-4, EIV, T. equi, B. mallei, T. evansi, E. coli, S. equi, and S abortus equi), the PI values of the serum samples positive for other common equine dis eases were all lower than 40%, and therefore tested as negative, while those positive fo B. caballi were above 40% and therefore considered to be positive ( Figure 3B). These result indicated that the novel cELISA assays do not detect antibodies against pathogens excep for T. equi or B. caballi in equine sera, and the tests, therefore, have high specificity.

Analysis of the Sensitivity of the Novel cELISA
Different dilutions of T. equi-positive serums were used to determine the analytica sensitivity of the novel cELISA. The results showed that the PI was greater than 40% whe the T. equi-positive serum was diluted 1:128 and was less than 40% when the T. equi-pos tive serum was diluted 1:256 ( Figure 4A). Similarly, the analytical sensitivity of the nove cELISA for B. caballi was determined using different dilutions of B. caballi-positive serum The results showed that the PI was greater than 40% when the B. caballi-positive serum was diluted 1:64 and was less than 40% when the B. caballi-positive serum was dilute 1:128 ( Figure 4B). So, the data indicated the analytical sensitivity of the novel cELISA fo T. equi and B. caballi were 1:128 and 1:64, respectively. Overall, the developed nove cELISA methods exhibit good sensitivity and have promise as candidates for a comme cial kit.

Analysis of the Sensitivity of the Novel cELISA
Different dilutions of T. equi-positive serums were used to determine the analytical sensitivity of the novel cELISA. The results showed that the PI was greater than 40% when the T. equi-positive serum was diluted 1:128 and was less than 40% when the T. equi-positive serum was diluted 1:256 ( Figure 4A). Similarly, the analytical sensitivity of the novel cELISA for B. caballi was determined using different dilutions of B. caballi-positive serums. The results showed that the PI was greater than 40% when the B. caballi-positive serum was diluted 1:64 and was less than 40% when the B. caballi-positive serum was diluted 1:128 ( Figure 4B). So, the data indicated the analytical sensitivity of the novel cELISA for T. equi and B. caballi were 1:128 and 1:64, respectively. Overall, the developed novel cELISA methods exhibit good sensitivity and have promise as candidates for a commercial kit.
indicated that the novel cELISA assays do not detect antibodies against pathogens except for T. equi or B. caballi in equine sera, and the tests, therefore, have high specificity.

Analysis of the Sensitivity of the Novel cELISA
Different dilutions of T. equi-positive serums were used to determine the analytical sensitivity of the novel cELISA. The results showed that the PI was greater than 40% when the T. equi-positive serum was diluted 1:128 and was less than 40% when the T. equi-positive serum was diluted 1:256 ( Figure 4A). Similarly, the analytical sensitivity of the novel cELISA for B. caballi was determined using different dilutions of B. caballi-positive serums. The results showed that the PI was greater than 40% when the B. caballi-positive serum was diluted 1:64 and was less than 40% when the B. caballi-positive serum was diluted 1:128 ( Figure 4B). So, the data indicated the analytical sensitivity of the novel cELISA for T. equi and B. caballi were 1:128 and 1:64, respectively. Overall, the developed novel cELISA methods exhibit good sensitivity and have promise as candidates for a commercial kit.

Comparison of the Novel cELISA and the Commercially Available cELISA Kit
To evaluate the performance of this novel cELISA assay, a total of 200 serum samples were tested using the novel cELISA and a commercially available cELISA kit. All samples were tested with the novel cELISA, as well as the commercially available cELISA kit, and the PI value of each sample was determined. We performed receiver operating characteristic (ROC) curve analysis as a statistical tool for the diagnostic evaluation of the novel cELISA in surveillance samples, where the area under the ROC curve (AUC) was employed to assess the accuracy of the novel cELISA assays (an AUC of 1 indicates perfect discriminatory value; an AUC of 0.5 or less indicates no discriminatory value). Compared with the commercial cELISA method, the AUCs of the novel cELISA assay were 0.979 for T. equi and 0.999 for B. caballi (p < 0.001) ( Figure 5A,B), and the diagnostic sensitivity and specificity of our novel cELISA assay were 91.49% (43/47) and 99.35% (152/153) for T. equi, and 88% (22/25) and 99.43% (174/175) for B. caballi ( Figure 5A,B). The agreement among the assays was calculated as previously reported [24], and the coincidence rate between the novel cELISA and the commercial cELISA assays was 97.5% for T. equi and 98% for B. caballi.

Comparison of the Novel cELISA and the Commercially Available cELISA Kit
To evaluate the performance of this novel cELISA assay, a total of 200 serum samples were tested using the novel cELISA and a commercially available cELISA kit. All samples were tested with the novel cELISA, as well as the commercially available cELISA kit, and the PI value of each sample was determined. We performed receiver operating characteristic (ROC) curve analysis as a statistical tool for the diagnostic evaluation of the novel cELISA in surveillance samples, where the area under the ROC curve (AUC) was employed to assess the accuracy of the novel cELISA assays (an AUC of 1 indicates perfect discriminatory value; an AUC of 0.5 or less indicates no discriminatory value). Compared with the commercial cELISA method, the AUCs of the novel cELISA assay were 0.979 for T. equi and 0.999 for B. caballi (p < 0.001) ( Figure 5A,B), and the diagnostic sensitivity and specificity of our novel cELISA assay were 91.49% (43/47) and 99.35% (152/153) for T. equi, and 88% (22/25) and 99.43% (174/175) for B. caballi ( Figure 5A,B). The agreement among the assays was calculated as previously reported [24], and the coincidence rate between the novel cELISA and the commercial cELISA assays was 97.5% for T. equi and 98% for B. caballi.

Establishment of the Western Blot Immunoassay
To determine the optimum dilution of the test sera for the novel western blot immunoassay, we assessed five serial dilutions of the serum samples either negative or positive for T. equi or B. caballi using the established western blot assay. The results showed that the optimum serum dilution in the western blot assay of antibodies against T. equi or B. caballi was determined to be 1: 200 ( Figure 6A,B). Under these conditions, T. equi or B. caballi-positive sera showed the target band, while the sera negative for these diseases did not have the target band.

Establishment of the Western Blot Immunoassay
To determine the optimum dilution of the test sera for the novel western blot immunoassay, we assessed five serial dilutions of the serum samples either negative or positive for T. equi or B. caballi using the established western blot assay. The results showed that the optimum serum dilution in the western blot assay of antibodies against T. equi or B. caballi was determined to be 1: 200 ( Figure 6A,B). Under these conditions, T. equi or B. caballi-positive sera showed the target band, while the sera negative for these diseases did not have the target band.

Clinical Performance
For evaluating whether the novel cELISA and the western blot assay can be used to accurately detect an instance of disease in clinical samples, a total of 208 serum samples To determine the optimum dilution of the test sera for the novel western blot immunoassay, we assessed five serial dilutions of the serum samples either negative or positive for T. equi or B. caballi using the established western blot assay. The results showed that the optimum serum dilution in the western blot assay of antibodies against T. equi or B. caballi was determined to be 1: 200 ( Figure 6A,B). Under these conditions, T. equi or B. caballi-positive sera showed the target band, while the sera negative for these diseases did not have the target band.

Clinical Performance
For evaluating whether the novel cELISA and the western blot assay can be used to accurately detect an instance of disease in clinical samples, a total of 208 serum samples collected in Inner Mongolia, China in 2022 were assessed using the novel cELISA, the commercially available cELISA kit, and the western blot assays. Venn diagrams were used to compare the performance of the different assays in detecting antibodies against T. equi or B. caballi.
The three methods of detection agreed in 177 out of the total 208 serum samples tested for the presence of T. equi antibodies ( Figure 7A). Across all 208 samples analyzed for the presence of T. equi antibodies, coincidence rates of the novel cELISA with the western blot assay and commercial cELISA assay were 93.27% (194/208) and 87.98% (183/208), respectively. tion of the test sera for the western blot assay for T. equi detection. (B) The determination optimum dilution of the test sera for the western blot assay for B. caballi detection.

Clinical Performance
For evaluating whether the novel cELISA and the western blot assay can be u accurately detect an instance of disease in clinical samples, a total of 208 serum sa collected in Inner Mongolia, China in 2022 were assessed using the novel cELIS commercially available cELISA kit, and the western blot assays. Venn diagrams wer to compare the performance of the different assays in detecting antibodies against or B. caballi.
The three methods of detection agreed in 177 out of the total 208 serum sa tested for the presence of T. equi antibodies ( Figure 7A). Across all 208 samples an for the presence of T. equi antibodies, coincidence rates of the novel cELISA with the ern blot assay and commercial cELISA assay were 93.27% (194/208) and 87.98% (18 respectively. The three methods of detection agreed in 167 out of the total 208 serum sa tested for the presence of B. caballi antibodies. The coincidence rate between the cELISA and the commercial cELISA was 91.83% (191/208), and that between the w blot assay and the commercial cELISA was 84.13% (175/208). Collectively, these suggest that the novel cELISA and the western blot assay have a high agreement w commercial kit, and they are promising candidates for further clinical testing.  The three methods of detection agreed in 167 out of the total 208 serum samples tested for the presence of B. caballi antibodies. The coincidence rate between the novel cELISA and the commercial cELISA was 91.83% (191/208), and that between the western blot assay and the commercial cELISA was 84.13% (175/208). Collectively, these results suggest that the novel cELISA and the western blot assay have a high agreement with the commercial kit, and they are promising candidates for further clinical testing.

Discussion
EP has emerged as an important protozoan infection and is caused by T. equi and B. caballi [25]. A successful vaccine against EP has yet to be developed, and the significant genetic diversity within T. equi and B. caballi clades complicates vaccine development efforts. The economic impact of this disease and the restrictions on the trade of infected animals have led the WOAH Animal Health Code to categorize EP as a notifiable disease [26]. This highlights the need for robust and effective measures of control against the disease in countries where it is endemic, and for combined high-sensitivity serological techniques to enhance the detection of the disease in animals exposed to the parasites that cause EP. The current techniques for diagnosing T. equi and B. caballi infections include microscopic examination, CFT, IFAT, cELISA, and PCR [27]. Currently, the WOAH considers cELISA to be the preferred test for EP in the international horse trade [7]. The cELISA method overcomes problems of antigen purity since the specificity of the cELISA depends solely on the mAb used. For this reason, the cELISA method is ideal for detection with recombinant antigens. The use of recombinant protein in diagnostic assays also precludes the need to infect animals or cells with the pathogen for antigen production [28]. To prevent the further spread of the infection, some countries (the USA, Canada, Australia, and Japan) require verification of the seronegativity of imported horses [29]. The commercial cELISA kit that allows testing for EP is relatively time-consuming and requires complex procedures. However, there is no developed commercial test kit for EP in China. This makes the control of this disease in China greatly challenging. This study aimed to establish cELISA methods for the detection of EP. In order to improve the accuracy of EP diagnosis, the presence of cross-reactions and potential performance issues of the several assays available should always be considered when interpreting laboratory results. Therefore, a western blotting method was established as a supplementary method for the diagnosis of T. equi and B. caballi.
Rapid and sensitive detection methods are critical for performing epidemiological investigations and for disease control and prevention. ELISA is therefore a good choice in the large-scale monitoring of disease outbreaks. Due to their low cost and high throughput, ELISA assays have been widely used for the serological diagnosis of T. equi and B. caballi [30]. However, the design of some commercial cELISA kits relies on species-specific HRP-labeled secondary antibodies [18]. The difference between the novel cELISA and the commercial cELISA is that the novel cELISA directly uses HRP-labeled mAbs as the detection antibody, while the commercial cELISA uses HRP-labeled anti-horse IgG secondary antibody as the detection antibody. The advantages of using competitive mAbs for detecting antibodies against T. equi and B. caballi include the reduced number of operation steps, a reduction in the testing time, and unlimited provision of a standardized reagent. In addition, the detection results were made more specific by reducing the operational step of one-step cascade amplification. The proteins EMA1 and Bc48 are commonly used as diagnostic antigens against T. equi and B. caballi, respectively. In this study, HRP-labeled 1A12 and 1A3 mAbs were used as detection antibodies to establish the cELISA assay. The results showed that the novel cELISA had high sensitivity and specificity (Figures 3 and 4), and had a high coincidence rate with the commercial cELISA assay ( Figure 5).
Western blotting is used as a supplementary diagnostic test for important human and veterinary diseases, including human immunodeficiency virus (HIV), transmissible spongiform encephalopathy (TSE), and equine infectious anemia virus (EIAV) [31][32][33]. In this study, the newly established western blotting detection method showed high consistency with the results of the commercial cELISA kit from VMRD ( Figure 7A,B). A potential explanation for this discrepancy observed in some clinical samples is that the native structure of EMA1 and Bc48 proteins and recombinant EMA1 and Bc48 proteins were different. In conclusion, we have defined the most important quality parameters and have found that the performance of our western blot assay is sufficient to make it a reliable diagnostic tool to be used as a follow-up test after a doubtful ELISA test result. This tool is particularly relevant because it represents a new method to confirm discordant or inconclusive test results from cELISA. Following more extensive validation, the use of this assay in screening horses prior to inter-country movement may help with the continued control of EP on a global scale.
Overall, this study has established novel cELISA and western blotting methods for the detection and diagnosis of EP. The novel cELISA and western blotting techniques showed comparable sensitivity and specificity to the commercially available cELISA. We suggest the use of western blot assay as a complementary test, available for the confirmation of conflicting results, that will contribute to resolving legal and/or sanitary situations that could lead to the spread of EP in China.
Author Contributions: Conceptualization, Writing-review and editing, X.W. and C.D.; Methodology, Validation, Data curation, B.Z. and K.C.; Formal analysis, resources, Z.H. and W.G.; Methodology, Validation, Data curation, Writing-original draft, G.Y. All authors have read and agreed to the published version of the manuscript.
Funding: This study was supported by grants of the National Key Research and Development Program of China (grants number 2022YFD1800200, 2020YFE0203400 and 2021YFD1800500).
Data Availability Statement: Not applicable.