Indirect Enzyme-linked Immunosorbent Assay Test Research Articles

Preliminary field evaluation of indirect ELISA test using the recombinant antigen rLicNTPDase-2 for serodiagnosis of canine leishmaniasis in Colombia

Leishmaniasis is a significant public health concern, with dogs as the primary reservoir in urban scenarios and facilitating transmission. Diagnosing infected dogs is a crucial step for public health interventions, and the development of new diagnostic platforms can significantly enhance efforts in various regions worldwide. Given the limited availability of diagnostic methods in Colombia, this study evaluates the effectiveness of an Indirect Enzyme-Linked Immunosorbent Assay (ELISA) based on the recombinant protein rLicNTPDase-2 to detect Leishmania in infected dogs. Serum samples were collected from dogs in both endemic and non-endemic areas and classified as natural standards based on prior parasitological diagnoses. The results revealed 24 true positives (TP) and 9 true negatives (TN). Subsequently, the test was then validated with samples from symptomatic and asymptomatic animals, alongside the standards, yielding a specificity of 96 %, a sensitivity of 81 %, efficiency of 90.6 %, a positive predictive value of 92.8 %, and a negative predictive value of 89.6 %. The positive likelihood ratio (RV+) was 20, while the negative likelihood ratio (RV-) was 0.19, indicating high relevance and a robust clinical utility. The area under the curve (AUC) was 1.00, suggesting that the test has excellent discriminatory ability, significantly deviating from the reference diagonal. This is further supported by the significant difference(p < 0.0001) between TN and TP results determined by Fisher's exact test. Involving 163 animals showed 47 % positive and 46 % negative results with a significant difference (p < 0.05) in the mean optical density (OD) values between positive and negative samples. These findings indicate that the ELISA test effectively differentiates between positive and negative samples based on OD values. This study suggests that ELISA based on the recombinant antigen rLicNTPDase-2 could serve as a viable alternative for the serodiagnosis of leishmaniasis in canines in Colombia.

Seroprevalence of infectious bursal disease in backyard chickens of selected districts of Buno Bedelle zone, Southwestern Ethiopia.

Infectious bursal disease (IBD) is a viral infectious disease that affects young chicks. A cross-sectional study was conducted from October 2020 to June 2021 to determine seroprevalence and associated risk factors of IBD on backyard chickens of purposively selected three districts of Buno Bedelle Zone, Southwestern Ethiopia. The study was carried out via a collection of serum samples, questionnaire surveys, and a total of 768 serum samples were collected from randomly selected backyard chickens of the study areas and examined for the presence of IBD virus. These serum samples were processed by using an indirect enzyme-linked immunosorbent assay test procedure in laboratories. SPSS version 20 was used for data analysis and descriptive statics techniques were used. Out of a total serum samples collected, 361 of them were found positive for the disease with an overall seroprevalence of 47%. Different seroprevalence rates of IBD with 55.9%, 43.3% and 41.2% were recorded in Didessa, Chora and Gechi districts, respectively. The result indicated that there was statistically significant difference (p<0.05) of the disease among the three districts. Among the nine Peasant Associations (PAs) observed for seroprevalence of IBD, highest (62.9%) and lowest (34.20%) seroprevalence of the disease was recorded in Yembero and Shengela PAs, respectively. However, IBD was statistically significant in only Shengela PA of Chora district (p=0.04, OR=1.93 and 95% CI 1.04-3.58) and Seko PA of Gechi district (p=0.05, OR=1.79 and 95% CI 1.01-3.16). Even though sex, breed, age, source and management-based seroprevalence was observed in the present study, the result indicated that there was only statistical significant difference (p=0.004, OR=0.62 and 95% CI 0.45-0.86) seen in which higher seroprevalence of IBD was determined in exotic (50.3%) than in local (38.6%) chickens breeds of the study areas. Generally, higher seroprevalence of IBD in the present study indicates a widely distribution of the disease and one of the potential threats for poultry production in the study areas. So, chicken vaccination and improved management system should be warranted in order to control the disease effectively.

Serological detection of Newcastle disease virus in backyard poultry production system in Woliso District South West Shewa, Central Ethiopia.

Newcastle disease (ND)is one of the most important respiratory viral diseases. The disease is endemic in many parts of Ethiopia. However, there is no clear record about the introduction of the virus to the country (Ethiopia). Hence, detail about the ND is very important in its (ND) control and prevention. Despite these facts, there is no available research work done on ND in the current research area that would help either as references for researchers or that could help in the control and prevention of the disease. Therefore, the objective of this study was to detect the ND virus (NDV), using serological methods in from December 2018 to November 2019. A cross-sectional type of study was conducted to detect the NDV. The convenience sampling method was used for sample data collection. Before data collection, chicken with previous history of vaccination against the NDV was excluded from the sampling animals. Then, a total of 348 blood samples of 2 mLwere collected from the brachial vein in 3 mLdisposable syringes. The serum was collected in labeled 2 mL cryovial tubes. Indirect enzyme-linked immunosorbent assay(ELISA) tests were performed to detect antibodies against NDV and to determine its antibody titer. The test was performed using (ID.vetinnovative version 2) procedure. In the indirect ELISA test, 37.64% (131/348) were positive and antibody titer mean value of (1761.9088) was scored. The standard deviation of 2592.42160 and a percentage coefficient of variation of147% was scored. From the finding, we conclude that indirect ELISA test detected the presence of the NDV in the study area and the heterogeneousity of antibody titer in the study area. Therefore, further molecular characterization and epidemiological investigation should be carried and vaccination of animals is critical in the study area.

Seroprevalence of Toxoplasma gondii in Slaughtered Pigs in Kiambu, Kenya

Toxoplasma gondii is a ubiquitous protozoan parasite of public health concern, with severe health consequences among immunocompromised individuals and pregnant mothers. Pigs are intermediate hosts of this zoonotic parasite and consumption of undercooked pork is a risk factor for T. gondii infection. We investigated the seroprevalence and risk factors for T. gondii in pigs in Kenya. A cross-sectional study was conducted at a non-integrated abattoir in Kiambu, Kenya, where 446 blood samples were collected from slaughtered pigs between 5 January and 5 March 2021. For each pig sampled, additional data were collected on the county of origin, farm size, sex and live weight. Serum was harvested from each pig blood sample, and these were subjected to indirect enzyme-linked immunosorbent assay tests to detect the presence of immunoglobin G (IgG) for T. gondii infection. The overall seroprevalence for T. gondii was 34.53% (95% C.I. 30.16–39.17). Risk factors for T. gondii seropositivity in pigs included farm size (p &lt; 0.001) and the live weight of pigs (p = 0.044). The high seroprevalence of T. gondii indicates that consumers of raw and undercooked pork may be at a high risk of infection. It is therefore recommended that strategies for the mitigation of risk of exposure in populations should prioritize public health education for pig farmers, pork handlers and consumers on risk reduction measures along the pork value chains.

Advancements in Lily Viruses Management: Challenges and Solutions in Elimination and Detection

Lilies are important crops that are commonly used as cut flowers (Lilium spp.) and edible bulb crops (Lilium davidii var. unicolor). However, virus infections can significantly impact the quantity and quality of lily production. Various methods have been developed to eliminate viruses in lilies, including in vitro culture and virus detection techniques. Meristem culture is the most effective method, which can be combined with other techniques such as thermotherapy and chemotherapy. Nonetheless, virus elimination is affected by several factors, including cultivar, explants used, virus type, and duration of treatments. Efficient diagnostic methods, such as serological and molecular techniques, have been developed to detect viral infections in lilies, including enzyme-linked immunosorbent assay (ELISA) and real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR). However, cross-contamination and multiple-virus contamination can lead to unreliable results, and more sophisticated protocols and systems have been developed to address these issues. The objective of this review is to provide a comprehensive overview of the development of lily virus eradication, detection strategies, challenges, and solutions associated with these procedures, and how more sophisticated approaches such as multiplex RT-PCR, indirect ELISA (ID-ELISA), immunocapture RT-PCR (IC-RT-PCR), and immunochromatographic test strips (ICSs) can alleviate some of these setbacks.

Comparison of Three Serologic Tests for the Detection of Anti-Coxiella burnetii Antibodies in Patients with Q Fever.

The performance of a commercial immunofluorescence assay (IFA commercial), an in-house immunofluorescence assay (IFA in-house) and an indirect enzyme-linked immunosorbent assay (ELISA) were evaluated in the detection of antibodies anti-C. burnetii in the serum of Q fever patients and persons without the disease. For the study, seropositive and seronegative samples for Q fever (n = 200) from a serum bank of the Instituto Adolfo Lutz in Brazil were used. Commercial IFA was considered in this study as the gold standard for diagnosing Q fever. The in-house IFA demonstrated good agreement with the commercial test, showing high sensitivity (91%) and specificity (97%) compared to the gold standard, with a Kappa coefficient of 0.8954. The indirect ELISA test showed lower agreement with the gold standard, showing low sensitivity (67%), although the specificity of the technique was high (97%) and the Kappa coefficient was moderate (0.6631). In-house IFA is an excellent alternative for diagnosing Q fever.

Comparison of Commercial Enzyme-linked Immunosorbent Assays for Diagnosis of Contagious Agalactia Caused By Mycoplasma Agalactiae.

IntroductionContagious agalactia (CA) is a disease affecting small ruminants with worldwide distribution and caused by several mycoplasmas, especially M. agalactiae. The main option for systematic diagnosis under monitoring control programmes is the enzyme-linked immunosorbent assay (ELISA) test.Material and MethodsThis study was designed to appraise the performance of two commercial indirect ELISA tests using M. agalactiae p48 protein and one using total protein, for antibody detection in small ruminants after natural infection with different M. agalactiae strains. We carried out the test evaluation using sera of confirmed M. agalactiae-positive goats with clinical signs. In addition, test agreement was assessed by kappa between the three commercial ELISA tests.ResultsAll three ELISA tests showed high validity scores (Youden’s J: 72.9–84%). The sensitivity values for the P48 protein-based tests were 76.9% and 84.6%, and was 79% for the total protein-based test. The specificity of all tests was 100%. In addition, between the total protein-based ELISA test and the other two ELISA tests based on the P48 protein, the agreement was substantial (kappa: 0.762–0.763) and the agreement between the latter two tests was almost perfect (kappa: 0.93).ConclusionThe validity parameters for all tests allowed their application for diagnostic purposes in lactating goats excreting M. agalactiae in milk and presenting clinical signs. The agreements show that any of these ELISA tests could be equally well used for diagnosis in programmes against CA.

Development and evaluation of indirect enzyme-linked immunosorbent assay using recombinant dense granule antigen 7 protein for the detection of Toxoplasma gondii infection in cats in Thailand.

Background and Aim:Toxoplasma gondii is recognized as a zoonosis causing toxoplasmosis in animals globally. Cat is a definitive host of T. gondii and sheds oocyst through feces, which can infect human beings and animals through contaminated food ingestion. A precise diagnostic test is essential to prevent T. gondii infection in both humans and animals. This study aimed to develop and evaluate the pETite-dense granule antigen 7(GRA7)-based indirect enzyme-linked immunosorbent assay (ELISA) to detect T. gondii infection in cats.Materials and Methods:T. gondii-GRA7 was cloned and expressed in the Expresso®small ubiquitin-related modifier (SUMO) T7 Cloning and Expression System. The recombinant pETite-GRA7 was purified using HisTrap affinity chromatography and confirmed using Western blot analysis. The recombinant protein was used to develop and evaluate the indirect ELISA for T. gondii infection detection. In total, 200 cat sera were tested using pETite-GRA7-based indirect ELISA and indirect fluorescent antibody test (IFAT). The statistical analysis based on Kappa value, sensitivity, specificity, positive predictive value, negative predictive value, χ2 test, and receiver operating characteristic (ROC) curve was used to evaluate the performance of the test.Results:A 606 bp GRA7 polymerase chain reaction (PCR) product was obtained from T. gondii RH strain genomic DNA. The gene was cloned into the pETite™ vector and transformed to HI-Control Escherichia coli BL21 (DE3) for protein expression. Approximately 35 kDa of recombinant pETite-GRA7 was observed and Western blot analysis showed positive bands against anti-6-His antibody and positive-T. gondii cat serum. A sample of 0.5 μg/mL of pETite-GRA7 was subjected to indirect ELISA to detect T. gondii infection in the cat sera. The results showed sensitivity and specificity of pETite-GRA7-based indirect ELISA at 72% and 96%, respectively. An acceptable diagnostic performance was characterized by high concordant results (94%) and substantial agreement (Kappa value=0.65) with IFAT. The seroprevalence levels of ELISA and IFAT were 10% and 9%, respectively, and were not significantly (p>0.05) different. The expected performance of ELISA at different cutoff points using the ROC curve analysis revealed 89% sensitivity and 92% specificity at the cutoff value of 0.146, with a high overall assay accuracy (area under the curve=0.94).Conclusion:In this study, the pETite™ vector, N-terminal 6xHis SUMO fusion tag, was used to improve the solubility and expression level of GRA7. The recombinant pETite-GRA7 showed enhanced protein solubility and purification without special condition requirements. This pETite-GRA7-based indirect ELISA showed high concordant results and substantial agreement with IFAT. ELISA revealed an acceptable sensitivity and specificity. These initial data obtained from cats’ sera demonstrated that pETite-GRA7-based indirect ELISA could be a useful method for local serological diagnosis of T. gondii infection in cats in Thailand.

Serological responses to a soluble recombinant circumsporozoite protein-VK210 of Plasmodium vivax (rPvCSP-VK210) among Iranian malaria patients

BackgroundCircumsporozoite protein (CSP) has a central immune domain that includes short regions of repeating amino acid sequences. This immunodynamic region is an epitope of B cells that can elicit an immune response in human and laboratory animals. The aim of the present study was to express the recombinant PvCSP-VK210 antigen and evaluate it for assaying antibodies obtained during human P. vivax infection by Western blotting and indirect ELISA (enzyme-linked immunosorbent assay).MethodGenomic DNA of P. vivax was isolated from a blood sample of an Iranian person with vivax malaria, and by PCR, the fragment of the PvCSP-VK210 gene was amplified. The gene fragment was cut after gel purification by BamHI and HindIII enzymes and then cloned into pET28a expression vector. Finally, the recombinant pET28a was transformed into the E.coli BL21 (DE3) as the expression host. In order to produce His-tagged protein, the expression host was cultured in LB medium. The protein was purified by Ni–NTA columns and immobilized metal affinity chromatography, and after confirmation by Western blotting technique, was used as the antigen in the indirect ELISA test.ResultsThe recombinant protein was expressed and purified as a 32-kDa protein. The sensitivity and specificity of the indirect ELISA test with the recombinant PvCSP-VK210 antigen were 61.42% and 97.14%, respectively, based on OD = 0.313. Between the results of the microscopic test and the indirect ELISA test with the recombinant PvCSP-VK210 antigen there was a Kappa coefficient of 0.586. The positive and negative predictive value and validity of the ELISA test with the recombinant PvCSP-VK210 antigen were 95.55%, 71.57%, 79.28%, respectively.ConclusionThe sensitivity of the indirect ELISA method with the recombinant PvCSP-VK210 antigen was 61.42%, which is the first report from Iran.

Comparative Study of Indirect Fluorescent Antibody, ELISA, and Immunochromatography Tests for Serological Diagnosis of Bovine Babesiosis Caused by Babesia bovis.

Simple SummaryCurrently serological diagnosis of bovine babesiosis is based on the detection of Babesia-specific antibodies (immunoglobulin-G). Antibody detection is commonly used in seroepidemiological studies or in the assessment of antibabesial antibody titers after cattle vaccination. The indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA) are the most widely used diagnostic tests, although there their implementation has some drawbacks, principally due to the requirements for trained personnel, specific materials, and special laboratory equipment. This study compared a newly designed rapid immunochromatography test (ICT), which has been reported recently and used for Babesia bovis-specific antibody detection with promising results, with an in-house ELISA for the serological diagnosis of cattle exposed to B. bovis (Babesia bovis) in Mexico. Higher sensitivity and specificity values were found by ICT, proving its effectiveness over ELISA. ICT also had better concordance than ELISA when IFAT was used as the “gold standard”. The rapid ICT was shown to have diagnostic utility for the detection of antibodies against B. bovis and could be used as a field test in Mexico due to its practicality, as it does not need laboratory equipment for implementation and interpretation of results.The indirect fluorescent antibody test (IFAT) is the most frequently used test to conduct seroepidemiological studies so far, and it is regarded as the "gold standard" test for the serological diagnosis of bovine babesiosis. The aim of the present study was to compare the enzyme-linked immunosorbent assay (ELISA) and the rapid immunochromatography test (ICT) for use in the serological diagnosis of cattle exposed to B. bovis in Mexico. The evaluation of test performance was carried out with 30 positive and 30 negative reference sera. A total of 72 bovine sera samples collected from cattle in a region with endemic bovine babesiosis were analyzed by ELISA and ICT, and the results were compared with those of IFAT. Kappa value (k) was also calculated to determine the agreement between tests. The sensitivity and specificity of ELISA for detecting antibodies against B. bovis were 87% (26/30) and 80% (24/30), respectively. The sensitivity and specificity of ICT for detecting antibodies against B. bovis were 90% (27/30) and 83.3% (25/30), respectively. The overall concordance determined for ELISA and ICT was 94.4% (68/72) and 98.6% (71/72), respectively, when the results were compared with those of IFAT. ICT was more sensitive and specific in this comparative study, showing good strength of agreement (k = 0.79) with respect to IFAT. ICT combines a strip-based assay system that is fast, practical, and sensitive for detection of antibodies to B. bovis, which suggests that it could be applied in the field without requiring any laboratory equipment for its use and interpretation of test results.

Seroprevalence and risk factors for brucellosis in small ruminant flocks in Karnataka in the Southern Province of India.

Background and Aim:Brucellosis is a zoonotic disease of high economic and public health importance in large and small ruminant populations worldwide. A cross-sectional study was conducted to determine the seroprevalence and risk factors of brucellosis in small ruminants in organized farms in the southern region of India.Materials and Methods:Farms exclusively rearing sheep and goats were selected based on the number of animals (small, medium, or large) and the location of the farm (urban, periurban, or rural). A total of 1499 serum samples; 1001 from sheeps and 498 from goats were sourced from six sheep and four goat farms and tested using Rose Bengal Plate and indirect Enzyme-Linked Immunosorbent Assay tests.Results:The apparent prevalence of brucellosis was higher in sheep (8.29%, 95% CI 6.7-10.1) than goats (5.82%, 95% CI 4.0-8.2). The true adjusted population level seroprevalence was also higher in sheep, at 7.7% (95% CI 6.0-9.6) than in goats, at 5.1% (95% CI 3.2-7.6). According to bivariate categorical analysis, six highly significant (p<0.001) animal- and farm-level risk factors for sheep were age, breed, number of lambings, history of abortion, rural farms, and presence of dogs on the farm. In goats, five significant risk factors were found: History of abortion, separate sheds, dogs on the farm, weekly veterinary consultation, and lack of brucellosis awareness. In a logistic regression model, abortion (OR adjusted 10.8, 95% CI 1.2-96.12), rural farms (OR adjusted 8.5, 95% CI 3.6-20.0), and absence of separate sheds on the farms (OR 1.9, 95% CI 1.1-3.5) were found to be significant risk factors for ovine brucellosis.Conclusion:The use of complementary measures to tackle the multiple animal- and farm-level risk factors may help to reduce the disease burden in the absence of a vaccination policy for small ruminants in India.

Seroprevalence of Crimean Congo Hemorrhagic Fever in cows by ELISA in Mosul city

Crimean-Congo hemorrhagic fever (CCHF) is described as a tick-borne viral zoonosis highly prevalent in Africa, Asia, Russia and the Balkans within the distribution range of ticks that belong to the genus Hyalomma. This research aimed to verify the seroprevalence of CCHF in cows employing Indirect enzyme-linked immunosorbent assay (I-ELISA) in Mosul city / Iraq, to ​​examine some epidemiological risk factors related to the incidence of CCHF. From October 2019 - September 2020, one hundred eighty-four blood samples were taken from 3-8-year-old cattle of both sexes, from several management systems and origins, from various parts in Mosul city, 10 ml of blood was taken from the jugular vein in test tubes without anticoagulant to obtain the sera for I-ELISA test, while epidemiological data were obtained by interviewing the farm-owners. I-ELISA was applied to detect the antibodies of CCHF in the serum. The overall seroprevalence of CCHF in cows was 40/184 (21.7%). As for the risk factors associated with increased seroprevalence of the disease, an increase in seroprevalence rates was observed at ages 6, 7 and 8 years 30.5%, among females 26.3% and imported animals 27.5%. which showed that the serological prevalence in indoor feeding and outdoor feeding, was not significantly different (p<0.05). This study concluded that there is a higher seroprevalence of CCHF in Mosul city along with many risk factors related to its incidence.

Detection of Toxoplasma gondii-specific immunoglobulin (IgG) antibodies in meat juice of beef

Toxoplasmosis is an important worldwide foodborne zoonotic disease. Infected cattle meats is considered a serous cause of human toxoplasmosis. Here, this study assesses the infection with Toxoplasma gonddi (T. gondii) in cattle using meat juice samples from diaphragmatic muscles collected at the slaughter. An in house indirect enzyme linked immunosorbent assay (ELISA), and commercial latex agglutination test (LAT) followed by immunoblotting were developed on the meat juice (fluids) using tachyzoites of locally isolated T. gondii strain. The comparative analysis of the results of the tested juice samples showed an excellent agreement between the in-house indirect ELISA and LAT test in the positive and negative of meat juice. Relative sensitivity was higher for ELISA on diaphragms fluids random samples 80.39%, for the LAT test was 68.6%. Immune-reactive bands of T. gondii local strain Ag with naturally infected meat juice were 116, 83, 65, 30 and 23 KDa. The obtained results concluded that the development of an effective ELISA test to be used in for detection of toxoplasmosis infection of slaughtered cattle in large-scale would be exactly valuable, since the important role that beef plays in epidemiology of T. gondii, in particular the hazard of transmission to human and food safety.

Development of an indirect ELISA based on soluble antigen produced from virus-infected cells for detection of porcine hemagglutinating encephalomyelitis virus

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a member of the genus Betacoronavirus and is the etiologic agent of encephalomyelitis or vomiting and wasting disease in neonatal pigs. Although there are only a few epidemiological studies that document the seroprevalence of PHEV infection, there are reports of sporadic outbreaks, including recent documentation of an influenza-like respiratory disease associated with PHEV in the United States. To address this issue, we have developed a new indirect enzyme linked immunosorbent assay (ELISA) for use in sero-epidemiological research of PHEV infection. One hundred and fifty porcine serum samples that were determined as antibody-positive or antibody-negative in virus neutralization (VN) tests were used in conjunction with PHEV-specific antigen extracted from virus-infected FS-L3 cells using RBS buffer containing 0.2 % NP-40 to develop this assay. The ELISA showed a high sensitivity (95.35 %) and specificity (96.88 %) by receiver operating characteristic (ROC) analysis, with an area under the curve (AUC) of 0.996 attesting to its accuracy. Our results revealed a strong correlation between the results of the indirect ELISA and VN test (R = 0.850, P < 0.05), with near-perfect agreement (kappa value = 0.932). These results indicate that this new indirect ELISA might be useful for diagnosis and sero-epidemiological tracking of PHEV infection.

Comparison of Cattle Serum Antibody Responses to Five Different Mycobacterial Antigens in an ELISA System.

The presence of common zoonosis diseases caused byMycobacterium tuberculosiscomplex (MTBC) and nontuberculous mycobacteria (NTM), such as Johne's and Crohn's diseases, poses a public health threat and economic losses to Iranian livestock. Therefore, the early detection of mycobacteria is of paramount importance. In this regard, enzyme-linked immunosorbent assay (ELISA) is a new, simple to use, rapid, and useful diagnostic tool. This study was performed to evaluate different crude antigens obtained from Mycobacterium species using an indirect ELISA test to identify the mycobacterial infection in infected livestock. Five different strains of Mycobacteria including M. tuberculosis,M. phlei,M. bovis,M. avium subspecies paratuberculosis, andM. bovisAN5 were cultured. The crude antigens in the samples were precipitated with trichloroacetic acid 4%. Thesodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude antigens isolated from different Mycobacterium species was reported. The total level of protein was determined by the Lowry protein assay. After the crude antigen preparation, the ELISA test was performed and the results were compared with the purified protein derivative skin test. Data analysis was performed using SPSS software version 25. All five strains were detected in more than 92% of healthy animals. The highest sensitivity of ELISA tests was inM. bovisAN5 antigen which was greater than 83%. The highest diagnostic specificity and efficiency of assays were in M.aviumsubspecies paratuberculosiswhich was 95.83% and over 83%, respectively. Regarding the results,M. aviumsubspeciesparatuberculosisandM. bovisAN5 antigens were promising candidates for the design of diagnostic ELISA due to their sensitivity, specificity, and efficiency.

Relationship between natural and infection-induced antibodies in systemic autoimmune diseases (SAD): SLE, SSc and RA.

Infection or vaccine-induced T cell-dependent immune response and the subsequent high-affinity neutralizing antibody production have been extensively studied, while the connection between natural autoantibodies (nAAbs) and disease-specific antibodies has not been thoroughly investigated. Our goal was to find the relationship between immunoglobulin (Ig)M and IgG isotype nAAbs and infection or vaccine-induced and disease-related autoantibody levels in systemic autoimmune diseases (SAD). A previously described indirect enzyme-linked immunosorbent assay (ELISA) test was used for detection of IgM/IgG nAAbs against citrate synthase (anti-CS) and F4 fragment (anti-F4) of DNA topoisomerase I in 374 SAD samples, with a special focus on systemic lupus erythematosus (SLE) (n=92), rheumatoid arthritis (n=73) and systemic sclerosis (n=157) disease groups. Anti-measles IgG and anti-dsDNA IgG/IgM autoantibodies were measured using commercial and in-house indirect ELISA tests. In all SAD groups the anti-measles IgG-seropositive cases showed significantly higher anti-CS IgG titers (P=0·011). In anti-dsDNA IgG-positive SLE patients, we detected significantly higher levels of anti-CS and anti-F4 IgG nAAbs (P=0·001 and<0·001, respectively). Additionally, we found increased levels of IgM isotypes of anti-CS and anti-F4 nAAbs in anti-dsDNA IgM-positive SLE patients (P=0·002 and 0·016, respectively). The association between IgG isotypes of pathogen- or autoimmune disease-related antibodies and the IgG nAAbs may underscore the immune response-inducible nature of the diseases investigated. The relationship between protective anti-dsDNA IgM and the IgM isotype of anti-F4 and anti-CS may provide immunoserological evidence for the beneficial roles of nAAbs in SLE patients.

A novel anti-NS2BNS3pro antibody-based indirect ELISA test for the diagnosis of dengue virus infections.

Dengue virus reportedly circulates as four genetically distinct serotypes for which there is no widely accepted vaccine or drug at present. Morbidity and mortality caused by this virus are alarming for the possible increased threat to human health. A suitable diagnostic test is the prerequisite for designing and developing control measures. But, the tests being employed at present possess one or the other drawback for this disease diagnosis. During the dengue virus infections, NS2B is essential for the stability and catalytic activity of the NS3 protease. N-terminal 185 amino acids of NS3 protease domain along with hydrophilic portion of NS2B (NS2BNS3pro) is being used to screen dengue inhibitors but not for diagnosis until now. In the present study, we have used purified NS2BNS3pro as an antigen to trap anti-NS2BNS3pro antibodies of the clinical samples. Antibodies were detected successfully in both Western blot analysis and enzyme-linked immunosorbent assay (ELISA) tests. In ELISA, antibodies were detected in both primary and secondary infections of all serotypes. Interestingly, 17 samples declared as other febrile infections by NS1 and IgM/IgG tests were found to be positive in present test, which were further confirmed by reverse-transcription polymerase chain reaction. In silico studies suggested the absence of conserved epitopes between NS2BNS3pro and the counterpart in JEV, Zika, and CHIKV, indicating less possibility of crossreaction, which was in turn confirmed by using synthetic peptides representing the above epitopes. Statistical analysis with 76% specificity, 87% sensitivity, and 95% concordance also supported the present test as a suitable test for large scale diagnosis of dengue virus infections.

Enzyme-Linked Immunosorbent Assay for the Detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) IgM/IgA and IgG Antibodies Among Healthcare Workers.

BackgroundThe outbreak of the novel coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been spreading rapidly across the world. A nucleic acid real-time quantitative polymerase chain reaction (RQ-PCR) test of nasopharyngeal samples is the standard method for the diagnosis of an active SARS-CoV-2 infection. However, many limitations of the RQ-PCR tests make them unsuitable for the simple and rapid diagnosis of COVID-19 patients. Moreover, some individuals with COVID-19 present an asymptomatic infection. Thus, assessing the asymptomatic transmission of COVID-19, especially in healthcare workers (HCWs), is crucial for evaluating the efficiency of the current preventive measures. Serological tests such as enzyme-linked immunosorbent assay (ELISA) are needed to quickly identify a large number of asymptomatic carriers to prevent the further spread of the virus and assess level of possible serological immunity in a community.MethodBetween April 18 and June 17, 2020, 330 HCWs from five Madinah region-affiliated hospitals underwent a seroprevalence screening for anti-SARS-CoV-2 antibodies (immunoglobulin [Ig]M/IgA and IgG) using indirect ELISA testing.ResultAmong the 330 samples, 80 (24.24%) were positive for SARS-CoV-2 IgM/IgA and/or IgG antibodies. There were no significant differences observed in the seroprevalence among the different occupations of the HCWs (excluding the pharmacists) with respect to the percentage of their seropositive samples.ConclusionThe current study presented the seroprevalence of anti-SARS-CoV-2 IgM/IgA and IgG antibodies in HCWs. The regular screening of HCWs for these antibodies is necessary; subsequently, a molecular test is recommended for those with seropositive (IgM, IgA, and IgG) samples to assess their viral load and potential shedding.

Evaluation of an in-house indirect enzyme-linked immunosorbent assay of feline panleukopenia VP2 subunit antigen in comparison to hemagglutination inhibition assay to monitor tiger antibody levels by Bayesian approach

BackgroundFeline panleukopenia virus (FPV) is an etiologic pathogen of feline panleukopenia that infects all members of Felidae including tigers (Panthera tigris). Vaccinations against FPV among wild felid species have long been practiced in zoos worldwide. However, few studies have assessed the tiger immune response post-vaccination due to the absence of a serological diagnostic tool. To address these limitations, this study aimed to develop an in-house indirect enzyme-linked immunosorbent assay (ELISA) for the monitoring of tiger antibody levels against the feline panleukopenia vaccine by employing the synthesized subunit capsid protein VP2. An in-house horseradish peroxidase (HRP) conjugated rabbit anti-tiger immunoglobulin G (IgG) polyclonal antibody (HRP-anti-tiger IgG) was produced in this study and employed in the assay. It was then compared to a commercial HRP-conjugated goat anti-cat IgG (HRP-anti-cat IgG). Sensitivity and specificity were evaluated using the Bayesian model with preferential conditional dependence between HRP-conjugated antibody-based ELISAs and hemagglutination-inhibition (HI) tests.ResultsThe posterior estimates for sensitivity and specificity of two indirect ELISA HRP-conjugated antibodies were higher than those of the HI test. The sensitivity and specificity of the indirect ELISA for HRP-anti-tiger IgG and HRP-anti-cat IgG were 86.5, 57.2 and 86.7%, 64.6%, respectively, while the results of the HI test were 79.1 and 54.1%. In applications, 89.6% (198/221) and 89.1% (197/221) of the tiger serum samples were determined to be seropositive by indirect ELISA testing against HRP-anti-tiger and HRP-anti-cat, respectively.ConclusionTo the best of our knowledge, the specific serology assays for the detection of the tiger IgG antibody have not yet been established. The HRP-anti-tiger IgG has been produced for the purpose of developing the specific immunoassays for tigers. Remarkably, an in-house indirect ELISA based on VP2 subunit antigen has been successfully developed in this study, providing a potentially valuable serological tool for the effective detection of tiger antibodies.

Development and use of indirect liquid-phase ELISA test system for detection of PRRS virus antigen during in-process control of raw materials intended for vaccine production

Porcine reproductive and respiratory syndrome (PRRS) being endemic and reported in the most countries in the world remains one of the most challenging diseases in pig industry. The main disease control measures include preventive vaccination and animal movement control within and outside the country as well as diagnostic testing of pigs in the population. Live and inactivated vaccines are used for specific prevention of porcine reproductive and respiratory syndrome. Complete and irreversible infectious agent inactivation with maximum epitope preservation and protective immunity in immunized animals are the main requirements for inactivated vaccines. Therefore, continuous improvement of methods for vaccine quality control at various vaccine production stages is of current importance. Results of development of the test system based on indirect liquid-phase enzyme-linked immunosorbent assay (ELISA) for PRRS virus antigen detection and activity testing in infectious and inactivated virus-containing cell cultures at intermediate stages of the vaccine production process are described in the paper. The test-system development process included purified and concentrated virus antigen as well as hyperimmune rabbit sera preparation. Specificity of purified and concentrated virus antigen was confirmed with real-time polymerase chain reaction. The developed test-system was shown to detect the virus antigen at initial infectivity titre of 4.87–7.21 lg TCID50/cm3 corresponding to ELISA titre (dilution) of 1:4 up to 1:64. Methodical Guidelines for detection of porcine reproductive and respiratory syndrome virus antigen with indirect liquid-phase enzyme-linked immunosorbent assay (ELISA) (2019) were developed based on the work results, commissioned and approved by the FGBI “ARRIAH” Scientific Board.