Development and application of an indirect ELISA for the serological detection of bovine viral diarrhea virus infection based on the protein E2 antigen.

BACKGROUND: Bovine viral diarrhea virus (BVDV) causes continuous economic losses to the livestock industry. Monitoring antibodies with enzyme-linked immunosorbent assay (ELISA) is a valuable tool to ensure the purification of BVDV in cattle. However, currently available ELISA kits based on the whole BVDV virion are both costly and time-consuming. The E2 protein has good immunogenicity, induces the secretion of neutralizing antibodies and is an essential immunogen for serological detection. METHODS AND RESULTS: We developed a novel recombinant E2 protein-based indirect ELISA (rE2-iELISA) and conducted a serological survey for BVDV antibodies in 2021-2022 in Beijing, China. The results showed that E2 protein was successfully expressed with high immunogenicity and the optimal rE2-iELISA displayed high sensitivity, reproducibility and specificity. Clinical testing of 566 serum specimens indicated that 318 BVDV positive samples and 194 BVDV negative samples were tested by rE2-iELISA and the IDEXX BVDV ELISA-Ab kit, with a positive coincidence rate of 93.3%, a negative coincidence rate of 86.3%, and an overall coincidence rate of 90.5%. CONCLUSION: This study established an rE2-iELISA method, which is a highly sensitive, specific and robust ELISA-test validated to detect anti-BVDV antibodies. These findings indicate that the newly developed rE2-iELISA method has the potential to be used as a rapid, reliable and cost-effective screening tool for BVDV infection and provides technical support for the evaluation of vaccine efficacy in cattle herds in the future.

Serological and molecular detection of Toxoplasma gondii and Neospora caninum in free-ranging rats from Nagpur, India.

Toxoplasma gondii and Neospora caninum are cyst-forming coccidian parasites that infect both wild and domestic non-felids as intermediate hosts, with rodents serving as important reservoir hosts during their life cycles. This study was aimed at investigating T. gondii and N. caninum infections and identifying factors favouring T. gondii infection in free-ranging rats from India. A total of 181 rodents were trap-captured, and blood and brain samples were subsequently collected for serological and molecular examination of T. gondii and N. caninum. Antibodies against T. gondii and N. caninum were detected by MAT/NAT and IFAT in 13.8% (25/181) and 1.65% (3/181) of rodents, respectively. All three N. caninum samples positive by NAT/IFAT were also positive for ELISA, while for T. gondii, 19 of 25 MAT/IFAT positive samples were also positive for ELISA. The antibody titers (MAT/NAT/IFAT) of rodents seropositive for T. gondii ranged from 25 to 400, while those of rats seropositive for N. caninum ranged from 25 to 100. Also, using PCR, DNA from T. gondii (B1 gene) and N. caninum (NC5 gene) was found in 2.76% (5/181) of brain samples and 0.55% (1/181) of brain samples. All PCR positive samples were also seropositive. No mixed infections were observed in the serological and molecular detections. A Chi-square analysis revealed that older rats and rats living in urban areas are significantly associated with T. gondii infection; however, rodent species, gender, location, habitat types, and seasonality were statistically nonsignificant. Overall, this study demonstrated that T. gondii was widely distributed while N. caninum was less prevalent among free-ranging rats in the studied area.

Identification of COVID-19 B-cell epitopes with phage-displayed peptide library.

BACKGROUND: Coronavirus disease 19 (COVID-19) first appeared in the city of Wuhan, in the Hubei province of China. Since its emergence, the COVID-19-causing virus, SARS-CoV-2, has been rapidly transmitted around the globe, overwhelming the medical care systems in many countries and leading to more than 3.3 million deaths. Identification of immunological epitopes on the virus would be highly useful for the development of diagnostic tools and vaccines that will be critical to limiting further spread of COVID-19. METHODS: To find disease-specific B-cell epitopes that correspond to or mimic natural epitopes, we used phage display technology to determine the targets of specific antibodies present in the sera of immune-responsive COVID-19 patients. Enzyme-linked immunosorbent assays were further applied to assess competitive antibody binding and serological detection. VaxiJen, BepiPred-2.0 and DiscoTope 2.0 were utilized for B-cell epitope prediction. PyMOL was used for protein structural analysis. RESULTS: 36 enriched peptides were identified by biopanning with antibodies from two COVID-19 patients; the peptides 4 motifs with consensus residues corresponding to two potential B-cell epitopes on SARS-CoV-2 viral proteins. The putative epitopes and hit peptides were then synthesized for validation by competitive antibody binding and serological detection. CONCLUSIONS: The identified B-cell epitopes on SARS-CoV-2 may aid investigations into COVID-19 pathogenesis and facilitate the development of epitope-based serological diagnostics and vaccines.

Enhanced Serologically Based Detection of Liberibacters Associated with Citrus Huanglongbing.

'Candidatus Liberibacter spp.' are associated with the most devastating disease of citrus Huanglongbing (HLB). In previous work, we established an in situ tissue print method for the detection of 'Ca. L. asiaticus' (CLas) in sweet orange. We optimized the protocol by preincubation of the anti-Omp antibody with 5% (w/v) extract of healthy rough lemon. This simple process eliminated cross reactions between citrus and the antibody. The optimized protocol enhanced the application of the polyclonal antibody, and we demonstrate detection of CLas from all parts of the world, including isolates from Japan, Thailand, Vietnam, Pakistan, Saudi Arabia, Brazil, the United States, and a selection of strains from China representative of the diversity extant there. The assay also was used to detect four isolates of 'Ca. L. africanus' (CLaf) representative of the diversity present in South Africa. The corresponding outer membrane genes of representative isolates were cloned and sequenced. The coding sequences were highly conserved, and isolates of CLas and CLaf shared 53.8 to 55.9% identity between species at the amino acid level. The optimized protocol is efficient for recognition of both CLas and CLaf in phloem cells of different citrus tissues regardless of geographic origin of the HLB samples. The method is simple and scales well to match the urgent need for accurate, sensitive, and high-throughput screening of HLB bacteria, and may play an important role especially for plant inspection and quarantine programs.

Evaluation of a conserved HA274-288 epitope to detect antibodies to highly pathogenic avian influenza virus H5N1 in Indonesian commercial poultry.

A peptide enzyme linked immunosorbent assay (ELISA) based on an epitope in the haemagglutinin (HA) of avian influenza virus H5N1, amino acid positions 274-288 (HA274-288) was evaluated for detection of H5N1-specific antibodies. An optimized ELISA based on the tetrameric form of the HA274-288 epitope designated MP15 gave low background with non-immune chicken sera and detected vaccinated and infected birds. The HA274-288 epitope was highly conserved in Indonesian H5N1 strains and antibody responses were detected in the majority of the vaccinated chickens regardless of the H5N1 strain used for vaccination. The HA274-288 epitope was also conserved in the majority of H5N1 strains from the neighbouring Asian region, and other H5 subtypes potentially allowing for a wider use of the MP15 ELISA in H5N1 vaccinated and infected flocks. The MP15 ELISA results correlated significantly with haemagglutination inhibition (HI) test results and test sensitivity and specificity were 87% and 92%, respectively. The MP15 ELISA titres were significantly higher than the HI titres in all immune sera allowing for sera to be tested at a single dilution of 1:400 which is of advantage in routine surveillance. The study indicated that the MP15 ELISA is potentially useful for serological detection of H5N1 vaccinated or infected poultry and to have some advantages over the standard HI test for routine monitoring of flocks' immunity after vaccination.

Development of a novel monoclonal antibody-based competitive ELISA for antibody detection against bovine leukemia virus.

Infection with bovine leukemia virus (BLV) leads to enzootic bovine leukosis, the most prevalent neoplastic disease in cattle. Due to the lack of commercially available vaccines, reliable eradication of the disease can be achieved through the testing and elimination of BLV antibody-positive animals. In this study, we developed a novel competitive ELISA (cELISA) to detect antibodies against BLV capsid protein p24. Recombinant p24 protein expressed by Escherichia coli, in combination with the monoclonal antibody 2G11 exhibiting exceptional performance, was used for the establishment of the cELISA. Receiver-operating characteristic curve analysis showed that the sensitivity and specificity of the assay were 98.85 % and 98.13 %, respectively. Furthermore, the established cELISA was specific for detecting BLV-specific antibodies, without cross-reactivity to antisera for six other bovine viruses. Significantly, experimental infection of cattle and sheep with BLV revealed that the cELISA accurately monitors seroconversion. In a performance evaluation, the established cELISA displayed a high agreement with Western blotting and the commercial BLV gp51 cELISA kit in the detection of 242 clinical samples, respectively. In conclusion, the novel p24 cELISA exhibited the potential to be a reliable and efficient diagnostic tool for BLV serological detection with a broad application prospect.

Serological detection of a novel ipomo-like virus infecting alfalfa in the U.S.

This study describes production of polyclonal antibodies against recently reported novel potyvirid infecting alfalfa (Medicago sativa L.). The virus was first found in alfalfa seed material and later identified in plant samples collected from commercial alfalfa fields in Arizona, USA. It was classified as a novel species related to the members of the genus Ipomovirus and potentially representing a new genus in the family Potyviridae (Nemchinov et al., 2023b). Polyclonal antibodies were produced against the predicted viral coat protein expressed in bacterial cells and used in different types of immunoassays for specific detection of this emerging virus. They could be helpful in plant virus certification programs, screening of alfalfa germplasm, research on pathogenicity, biology, and geographic distribution of this emerging virus.

Single Cell Expression Systems for the Production of Recombinant Proteins for Immunodiagnosis and Immunoprophylaxis of Toxoplasmosis.

Toxoplasmosis represents a significant public health and veterinary concern due to its widespread distribution, zoonotic transmission, and potential for severe health impacts in susceptible individuals and animal populations. The ability to design and produce recombinant proteins with precise antigenic properties is fundamental, as they serve as tools for accurate disease detection and effective immunization strategies, contributing to improved healthcare outcomes and disease control. Most commonly, a prokaryotic expression system is employed for the production of both single antigens and multi-epitope chimeric proteins; however, the cloning strategies, bacterial strain, vector, and expression conditions vary. Moreover, literature reports show the use of alternative microbial systems such as yeast or Leishmania tarentolae. This review provides an overview of the methods and strategies employed for the production of recombinant Toxoplasma gondii antigenic proteins for the serological detection of T. gondii infection and vaccine development.

Prevalence of Toxoplasma gondii Antibodies and Risk Factors in Two Sympatric Invasive Carnivores (Procyon lotor and Nyctereutes procyonoides) from Zgorzelec County, Poland.

The intracellular protozoan Toxoplasma gondii is distributed worldwide and infects many species of warm-blooded animals. Most mammals, including humans, can serve as intermediate hosts. This pathogen, with its zoonotic potential, causes toxoplasmosis, a condition that can range from subclinical to fatal in humans. It is therefore important to assess the occurrence of the pathogen, even if only indirectly through the detection of antibodies. Epidemiological data on the seroprevalence in wild animals, including invasive species, are rare in Poland. Therefore, we tested 197 wild raccoons (Procyon lotor) and 89 raccoon dogs (Nyctereutes procyonoides) from Zgorzelec County, southwestern Poland, for the presence of antibodies. Samples were collected between January 2019 and December 2020 and analysed using a commercial indirect modified agglutination test (MAT, cut-off 1:25). The statistical analysis revealed significant differences in seroprevalence between the two predatory species. Of the 197 surveyed raccoons, 96 (48.73%; 95% confidence interval (CI): 41.73-55.73%) tested positive, while 25 of the 89 raccoon dogs (28.09%; 95% CI: 18.70-37.48%) were positive. Regarding risk factors, body weight and sex influenced the presence of T. gondii antibodies in both the species, with a higher likelihood of seropositivity among heavier animals and females, respectively. For raccoon dogs, juveniles were more likely to be seropositive than adults at a given weight. Our results suggest that T. gondii infection is widespread in the regional raccoon and raccoon dog populations, indicating a high level of parasite circulation in the environment.

Dot Immunobinding Assay for the Rapid Serodetection of Scedosporium/Lomentospora in Cystic Fibrosis Patients.

The detection of Scedosporium/Lomentospora is still based on non-standardized low-sensitivity culture procedures. This fact is particularly worrying in patients with cystic fibrosis (CF), where these fungi are the second most common filamentous fungi isolated, because a poor and delayed diagnosis can worsen the prognosis of the disease. To contribute to the discovery of new diagnostic strategies, a rapid serological dot immunobinding assay (DIA) that allows the detection of serum IgG against Scedosporium/Lomentospora in less than 15 min was developed. A crude protein extract from the conidia and hyphae of Scedosporium boydii was employed as a fungal antigen. The DIA was evaluated using 303 CF serum samples (162 patients) grouped according to the detection of Scedosporium/Lomentospora in the respiratory sample by culture, obtaining a sensitivity and specificity of 90.48% and 79.30%, respectively; positive and negative predictive values of 54.81% and 96.77%, and an efficiency of 81.72%. The clinical factors associated with the results were also studied using a univariate and a multivariate analysis, which showed that Scedosporium/Lomentospora positive sputum, elevated anti-Aspergillus serum IgG and chronic Pseudomonas aeruginosa infection were significantly associated with a positive result in DIA, while Staphylococcus aureus positive sputum showed a negative association. In conclusion, the test developed can offer a complementary, rapid, simple and sensitive method to contribute to the diagnosis of Scedosporium/Lomentospora in patients with CF.

Detection of Toxoplasma gondii Infection in Small Ruminants: Old Problems, and Current Solutions.

Toxoplasmosis is a parasitic zoonosis of veterinary importance, with implications for public health. Toxoplasma gondii infection causes abortion or congenital disease in small ruminants. Moreover, the consumption of infected meat, cured meat products, or unpasteurized milk and dairy products can facilitate zoonotic transmission. Serological studies conducted in various European countries have shown the high seroprevalence of specific anti-T. gondii antibodies in sheep and goats related to the presence of oocysts in the environment, as well as climatic conditions. This article presents the current status of the detection possibilities for T. gondii infection in small ruminants and their milk. Serological testing is considered the most practical method for diagnosing toxoplasmosis; therefore, many studies have shown that recombinant antigens as single proteins, mixtures of various antigens, or chimeric proteins can be successfully used as an alternative to Toxoplasma lysate antigens (TLA). Several assays based on DNA amplification have been developed as alternative diagnostic methods, which are especially useful when serodiagnosis is not possible, e.g., the detection of intrauterine T. gondii infection when the fetus is not immunocompetent. These techniques employ multicopy sequences highly conserved among different strains of T. gondii in conventional, nested, competitive, and quantitative reverse transcriptase-PCR.

Comparison of Antibody Isotype Response to Angiostrongylus cantonensis in Experimentally Infected Rats (Rattus norvegicus) Using Hawai'i 31 kDa Antigen in an Indirect ELISA.

Neuroangiostrongyliasis (NAS) is an emerging tropical disease in humans and some animals which is caused by infection with the parasitic nematode Angiostrongylus cantonensis. It is the leading cause of eosinophilic meningitis worldwide. Diagnoses in humans and susceptible animals are generally presumptive and easily confused with other central nervous system disorders. The 31 kDa antigen is currently the only NAS immunodiagnostic assay that has achieved 100% sensitivity. However, little is known about the humoral immune response against the 31 kDa antigen in NAS infections, which would be critical for widespread adoption of this assay. We used the Hawai'i 31 kDa isolate in an indirect ELISA assay to confirm the presence of immunoglobulin IgG, IgM, IgA, and IgE isotypes in six-week post-infection plasma from lab-reared rats infected with 50 live, third-stage, A. cantonensis larvae isolated from a wild Parmarion martensi semi-slug. Our results confirmed the presence of all four isotypes against the Hawaii 31 kDa isolate, with sensitivity ranging from 22-100%. The IgG isotype showed 100% sensitivity in detecting A. cantonensis infection, which validates the use of IgG indirect ELISA with 31 kDa antigen as an effective immunodiagnostic assay for rats six weeks post-infection. Given each isotype may be present at different times during NAS infections, our data provides preliminary information on the humoral immune response to A. cantonensis infection in lab-reared rats and serves as a baseline for future studies.

Study on antigenic protein Omp2b in combination with Omp31 and BP26 for serological detection of human brucellosis.

BACKGROUND: Brucellosis is a very common zoonosis in certain localized areas worldwide, with a high prevalence in most developing countries. The detection of brucellosis still faces many challenges such as the need for more sensitive and specific diagnostic antigens. METHODS: To evaluate the efficacy of Brucella outer membrane proteins (Omps) Omp2b in combination with omp31 and BP26 as diagnostic antigens for the serological detection of human brucellosis, these proteins were prepared by a prokaryotic expression system. Human brucellosis-positive and-negative sera were collected, and the detection effects of the diagnostic antigens were evaluated using an established indirect ELISA (iELISA) method. Receiver operating characteristic (ROC) curves and the area under the ROC curve (AUC), true positives, true negatives, false positives, false negatives, accuracy, positive predictive value, negative predictive value, analytical specificity, and sensitivity were obtained to evaluate the effectiveness of Omp2b and antigen combinations. RESULTS: The iELISA results showed that the AUC of the antigenic proteins was 0.9100, 0.9387, 0.9343, and 0.9448, respectively, and that the combination of Omp31 and BP26 improved the accuracy and was superior to that of Omp2b alone. Analysis at the determined cut-off values showed that the analytical sensitivity of the assay was 0.8739 (95% CI:0.7974-0.9293) and the analytical specificity was 0.8539 (95% CI:0.7632-0.9199) when using Omp2b alone and 0.8649 when using the combination of Omp2b + BP26 (95% CI:0.7869-0.9223) with an analytical specificity of 0.9213 (95% CI:0.8446-0.9678) and 0.8468 (95% CI:0.7662-0.9082) and an analytical sensitivity of 0.9101 (95% CI:0.8305-0.9604). When Omp2b + Omp31 + BP26 was combined, the analytical sensitivity and specificity were 0.8559 (95% CI:0.7765-0.9153) and 0.9326 (95% CI:0.8590-0.9749), respectively. Protein antigens, including antigen combinations, did not cross-react with Yersinia enterocolitica O9 and E. coli O157: H7, indicating that their specificity was better than that of lipopolysaccharide (LPS). CONCLUSIONS: Compared with individual Omp2b, antigen combinations improved the effectiveness in detecting brucellosis, but were still not as effective as LPS antigen. Omp2b, combined with Omp31 and BP26 as diagnostic antigens, can be used to detect human brucellosis.

Epidemiological evidence of acute transmission of Zika virus infection in dengue suspected patients in Sri-Lanka.

BACKGROUND: Zika Virus (ZIKV) is a re-emerging, arthropod-borne flavivirus transmitted by Aedes mosquitoes (Ae. aegypti and Ae. albopictus). The coexistence of dengue virus (DENV) and ZIKV concurrently has been associated with a wide array of neurological complications, which may influence the clinical outcomes of infections. Sri Lanka witnessed a severe dengue epidemic in 2017, characterized by extraordinary and severe disease manifestations with considerable morbidity. Therefore, this study assessed the potential occurrence of ZIKV infection during DENV outbreak in Sri Lanka from 2017 to 2019, which could bear substantial implications for public health. METHODS: Five hundred ninety-five serum samples were procured from individuals suspected of dengue and admitted to Kandy National Hospital between 2017 and 2018 and the Negombo District General Hospital between 2018 and 2019. These samples underwent quantitative real-time RT-PCR (qRT-PCR) to identify the presence of the ZIKV gene, while enzyme-linked immunosorbent assay was employed to detect ZIKV-specific IgM and IgG antibodies. Focus reduction neutralization tests were subsequently conducted to confirm ZIKV infection. RESULTS: Among the 595 serum samples, 6 (1.0%) tested positive for ZIKV using qRT-PCR. Anti-ZIKV IgM and IgG were identified in 18.0% and 38.6% patients. Sixty-six (11.0%) samples demonstrated the presence of anti-ZIKV IgM and IgG. Within ZIKV IgM-positive samples, 2.2% exhibited neutralizing antibodies against ZIKV. Through the implementation of qRT-PCR, ZIKV IgM detection, and neutralization testing, 2% and 3.7% cases of ZIKV infections were confirmed in the Kandy and Negombo regions, respectively. CONCLUSION: This study is the inaugural endeavor to substantiate the existence of ZIKV infection in Sri Lanka utilizing molecular and serological analysis. The findings of this investigation imply that ZIKV was circulating throughout the 2017-2019 DENV outbreak. These results underscore the necessity for improved preparedness for future outbreaks, fortifying governmental policies on public health, and establishing effective early warning systems regarding the emergence of these viruses.

Serological evidence of SARS-CoV-2 infection in pets naturally exposed during the COVID-19 outbreak in Argentina.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of coronavirus disease 2019 (COVID-19), has rapidly spread worldwide. The monitoring of animals has shown that certain species may be susceptible to be infected with the virus. The present study aimed to evaluate the presence of SARS-CoV-2 antibodies by ELISA and virus neutralization (VN) in pets from owners previously confirmed as COVID-19-positive in Argentina. Serum samples of 38 pets (seven cats and 31 dogs) were obtained for SARS-CoV-2 antibody detection. Three out of the seven cats and 14 out of the 31 dogs were positive for SARS-CoV-2 by ELISA, and one cat and six dogs showed the presence of neutralizing antibodies in which the cat and two of the six dogs showed high titers. Another dog from which three serum samples had been obtained within eight months from the diagnosis of its owner showed the presence of antibodies at different times by both ELISA and VN. However, the results showed that the antibodies decreased slightly from the first to the third sample. Our results provide evidence that SARS-CoV-2 infection in pets living with COVID-19-positive humans from Argentina during the outbreak of SARS-CoV-2 can be detected by serology assay.

Detection of Tomato Spotted Wilt Virus (TSWV) Infection in Plants Using DAS-ELISA and Dot-ELISA.

Plant viruses cause severe damages to crop productions each year worldwide. To prevent the losses caused by plant viruses, it is necessary to develop specific and efficient diagnostic tools to detect viruses. Among the current virus detection techniques, serological detection methods are considered to be rapid, simple, sensitive, and high throughput. Therefore, serological detection methods such as double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), triple antibody sandwich ELISA (TAS-ELISA), antigen coated plate-ELISA (ACP-ELISA), Dot-ELISA and tissue print-ELISA as well as colloidal gold immunochromatographic strip are now wildly used to detect viruses in plants. In this chapter, we describe the DAS-ELISA and Dot-ELISA methods, and their applications in the detection of Tomato spotted wilt virus (TSWV) infection in plants. These two methods can be easily adapted for diagnosis of other plant viruses.

Research progress in laboratory detection of SARS-CoV-2.

BACKGROUND: Nucleic acid testing is a reliable method for diagnosing viral infection in clinical samples. However, when the number of cases is huge and there are individual differences in the virus itself, the probability of false-negative results increases. With the advancement in research on the new coronavirus, new detection technologies that use serum-specific antibodies as detection targets have been developed. These detection technologies have high efficiency and shorter turnaround time, which ultimately shortens the time required for diagnosis. This article summarizes the methods that have been reported to date for the detection of the new coronavirus and discusses their principles and technical characteristics. AIMS: Compare the advantages and disadvantages of various SARS-CoV-2 detection methods and analyze their principles. METHODS: Searched reports on SARS-CoV-2 detection methods published so far, extracted the data and analyzed them. Use the primer blast function of NCBI to analyze the primers used in qRT-PCR detection. RESULTS: The detection sensitivity was the highest when nucleocapsid protein gene was used as the target, reaching 96.6%. The detection efficiency of the remaining targets ranged from 66.7% to 96.0%. Various new detection methods, like Serum specific antibody detection, can speed up the test time. However, due to the complexity of the method and higher testing requirements, it seems that it cannot be used as a complete replacement for qRT-PRC testing. CONCLUSIONS: With the advancement of technology and the improvement of methods, the detection methods of SARSCoV-2 have become more mature. These advances provided great help to the detection of SARS-CoV-2.

An Efficient and Rapid Assay for Detecting Neutralizing Antibodies Against Serotype 4 Fowl Adenovirus.

Currently, the outbreak of serotype 4 fowl adenovirus (FAdV-4) has spread worldwide and caused tremendous economic loss to the poultry industry. Although inactivated vaccines have been licensed against FAdV-4 in China, a rapid and efficient serological method for measuring the titer of neutralizing antibodies (NAbs) specific for FAdV-4 post-infection or vaccination is rarely reported. Classical virus neutralization test (VNT) is superior in sensitivity and specificity for detecting NAbs but is either time-consuming or laborious. In this study, a recombinant virus FA4-EGFP expressing EGFP-fiber-2 fusion protein, rather than wild type (WT) FAdV-4 was used to develop a novel VNT for detecting FAdV-4 NAbs. Specificity analysis showed that the approach only reacted with the sera against FAdV-4, not with the sera against other avian pathogens tested. The novel VNT was effective in the detection of NAbs against FAdV-4 in sera from both experimentally infected and clinically vaccinated chickens, and had good linear correlation with the classical VNT. Moreover, the novel VNT not only significantly simplifies the procedure for detection of NAbs, but also shortens the timeline to 24 h in comparison with the classical VNT with 3-4 d. All these data demonstrate that the FA4-EGFP based VNT developed here provides an efficient diagnostic method for monitoring the immunological state of the vaccination or diagnosing the clinical infection of FAdV-4 in a quick and funding-saving manner.

High-specificity targets in SARS-CoV-2 N protein for serological detection and distinction from SARS-CoV.

Numerous serological detection kits are being rapidly developed and approved for screening and diagnosing suspected coronavirus disease 2019 (COVID-19) cases. However, cross-reactivity between pre-existing antibodies against other coronaviruses and the captured antigens in these kits can affect detection accuracy, emphasizing the necessity for identifying highly specific antigen fragments for antibody detection. Thus, we performed a conservation and specificity analysis of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein. We also integrated various B-cell epitope prediction methods to obtain possible dominant epitope regions for the N protein, analyzed the differences in serological antibody levels for different epitopes using ELISA, and identified N protein epitopes for IgG and IgM with high-specificity. The SARS-CoV-2 N protein showed low mutation rates and shared the highest amino acid similarity with SARS-CoV; however, it differed substantially from other coronaviruses. Tests targeting the SARS-CoV-2 N protein produce strong positive results in patients recovering from SARS-CoV. The N18-39 and N183-197 epitopes for IgG and IgM detection, respectively, can effectively overcome cross-reactivity, and even exhibit good specificity between SARS-CoV-2 and SARS-CoV. The antibody levels detected with these were consistent with those detected using the complete N protein. These findings provide a basis for serological diagnosis and determining the kinetics of SARS-CoV-2 antibody detection in patients.

Characterization and evaluation of a recombinant multiepitope peptide antigen MAG in the serological diagnosis of Toxoplasma gondii infection in pigs.

BACKGROUND: Toxoplasmosis caused by Toxoplasma gondii is a serious disease threatening human and animal health. People can be infected with T. gondii by ingesting raw pork contaminated with cysts or oocysts. Serological test is a sensitive and specific method usually used for large-scale diagnosis of T. gondii infection in humans and animals (such as pigs). Commercial pig Toxoplasma antibody ELISA diagnostic kits are expensive, which limits their use; moreover, the wide antigen composition used in these diagnostic kits is still unclear and difficult to standardize. The multiepitope peptide antigen is a novel diagnostic marker, and it has potential to be developed into more accurate and inexpensive diagnostic kits. METHODS: The synthetic multiepitope antigen (MAG) cDNA encoding a protein with epitopes from five T. gondii-dominant antigens (SAG1, GRA1, ROP2, GRA4, and MIC3) was designed, synthesized, and expressed in Escherichia coli BL21 (DE3) strain. The recombinant protein was detected through western blot with pig anti-T. gondii-positive and -negative serum, and then IgG enzyme-linked immunosorbent assay (ELISA) named MAG-ELISA was designed. The MAG-ELISA was evaluated in terms of specificity, sensitivity, and stability. The MAG-ELISA was also compared with a commercial PrioCHECK® Toxoplasma Ab porcine ELISA (PrioCHECK ELISA). Finally, the trend of pig anti-T. gondii IgG levels after artificial infection with RH tachyzoites was evaluated using MAG-ELISA and two other ELISA methods (rMIC3-ELISA and PrioCHECK ELISA). RESULTS: MAG antigen could be specifically recognized by pig anti-T. gondii-positive but not -negative serum. MAG-ELISA showed high diagnostic performance in terms of specificity (88.6%) and sensitivity (79.1%). MAG-ELISA could be used for detecting anti-T. gondii IgG in the early stage of T. gondii infection in pigs (at least 7 days after artificial infection). CONCLUSIONS: Our results suggest that MAG antigen can be applied to specifically recognize anti-T. gondii IgG in pig, and MAG-ELISA has the potential for large-scale screening tests of T. gondii infection in pig farms and intensive industries.