Protein-based Enzyme-linked Immunosorbent Assay Research Articles

Usefulness of receptor binding domain protein-based serodiagnosis of COVID-19

ObjectivesThis study evaluated the performance of recombinant receptor binding domain (RBD) protein-based enzyme-linked immunosorbent assays (RBD-ELISAs) for detecting anti-SARS-CoV-2 immunoglobulin (Ig) G and IgM antibodies. MethodsIn this study, 705 sera from SARS-CoV-2-infected individuals and 315 sera from healthy individuals were analyzed. ResultsThe RBD-ELISA IgG exhibited high specificity (99.1%) and moderate sensitivity (48.0%), with an overall diagnostic accuracy of 73.5%. RBD-ELISA IgM demonstrated specificity at 94.6% and sensitivity at 51.1%, with an accuracy of 72.8%. Both assays displayed improved performance when analyzing samples collected 15-21 days post-symptom onset, achieving sensitivity and accuracy exceeding 88% and 90%, respectively. Combining RBD-ELISA IgG and IgM in parallel analysis enhanced sensitivity to 98.6% and accuracy to 96.2%. Comparing these RBD-ELISAs with commercially available tests, the study found overlapping sensitivity and similar specificity values. Notably, the combined RBD-ELISA IgG and IgM showed superior performance. Cross-reactivity analysis revealed low false-positive rates (4.4% for IgG, 3.7% for IgM), primarily with viral infections. ConclusionThis research underscores the potential of RBD-based ELISAs for COVID-19 diagnosis, especially when assessing samples collected 15-21 days post-symptom onset and utilizing a parallel testing approach. The RBD protein's immunogenicity and specificity make it a valuable tool for serodiagnosis, offering an alternative to polymerase chain reaction-based methods, particularly in resource-limited settings.

Development and Application of a Nanobody-Based Competitive ELISA for Detecting Antibodies against Hepatitis E Virus from Humans and Domestic Animals.

Hepatitis E virus (HEV) is a zoonotic pathogen that is widespread worldwide. At present, most enzyme-linked immunosorbent assay (ELISA) kits only detect antibodies against human HEV. In this study, a nanobody-horseradish peroxidase (HRP) fusion protein-based competitive ELISA (cELISA) with more convenience and spectral characteristics for HEV antibody detection was developed and used to detect HEV IgG in various species. First, 6 anti-swine HEV capsid protein nanobodies were screened using phage display technology from an immunized Bactrian camel. Then, HEV-Nb67-HRP fusions were expressed and used as a probe for developing a cELISA. The cutoff value of the cELISA was 17.8%, and there was no cross-reaction with other anti-swine virus antibodies, suggesting that the cELISA had good specificity. The intra-assay and interassay coefficients of variation (CVs) were 1.33 to 5.06% and 1.52 to 6.84%, respectively. The cELISA and Western blot showed a higher coincidence rate (97.14%, kappa value = 0.927) than cELISA and indirect ELISA (95.00%, kappa value = 0.876) in clinical swine serum samples. Finally, the seroprevalence of HEV IgG in humans, pigs, rabbits, cows, and goats was 30.67%, 19.26%, 8.75%, 27.59%, and 18.08%, respectively, suggesting that cELISA may have a broader scale for mammalian HEV antibody detection. These results suggest that the newly developed cELISA was rapid, low-cost, reliable, and useful for the serological evaluation of current HEV. IMPORTANCE HEV is thought to be a zoonotic infection and is widespread worldwide; it is beneficial to establish a more convenient and spectral method for HEV antibody detection. In this study, a convenient, time-saving, reproducible, highly sensitive, specific, and novel nanobody-based cELISA was developed and can be used to detect IgG antibodies against mammalian HEV. It provides a new technique for serological evaluation and ELISA-based diagnosis of HEV infection.

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

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. 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%. 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.

Assessment of Immunologically Potent Carbohydrates.

Carbohydrates have a great potential in generating structural and immunological diversities. Microbial pathogens often decorate their outmost surfaces with specific carbohydrate signatures. Carbohydrate antigens also differ significantly from protein antigens in physiochemical properties, especially in surface display of antigenic determinants in aqueous solutions. Technical optimization or modifications are often needed when we apply standard procedures for protein-based enzyme-linked immunosorbent assay (ELISA) to assess immunologically potent carbohydrates. We present here our laboratory protocols for performing carbohydrate ELISA and discuss several assay platforms that may be applied complementarily to explore the carbohydrate moieties that are critical for host immune recognition and induction of glycan-specific antibody responses.

Application of a truncated ORF2 protein-based ELISA for diagnosis of hepatitis E in an endemic area.

Enterically transmitted waterborne hepatitis E (HE) caused due to hepatitis E virus (HEV) prevails as a significant public health problem endemic to India. Due to short-term viremia/fecal excretion and poor in vitro transmissibility of HEV, HE diagnosis depends on detection of specific IgM antibodies in serum. Present study evaluated performances of two in-house and six commercial IgM detection enzyme-linked immunosorbent assays (ELISAs) using sera collected from volunteers/acute hepatitis patients (n = 716). The in-house ELISAs were based on complete and truncated open reading frame 2 (ORF2) proteins containing neutralizing epitope/s region of genotype 1 HEV (ORF2p, 1-660 amino acid (a.a.) and T1NEp, 458-607 a.a., respectively). The commercial ELISAs included Wantai (China), MP Diagnostics (MPD) (Singapore), DIA.PRO Diagnostics (Italy), MBS (Italy), abia (Germany), and ImmunoVision (USA). T1NE ELISA showed 97.0% positive percent agreement (PPA), 99.4% negative percent agreement (NPA), and 98.6% concordance (κ = 0.97, P = 0.0000) with ORF2 ELISA. ORF2, T1NE, Wantai, and MPD ELISAs agreed on results for 88% of sera tested. Two percent sera showed reactivity in each combination of three and two of aforementioned four ELISAs. Remaining 8% sera were single ELISA reactive. PPA and NPA value ranges were 76.3-99.0% and 84.8-99.5%, respectively. Pairwise concordances between all the eight ELISAs ranged from 88.0 to 100% (κ: 0.74-1.00). Both the in-house ELISAs agreed better with Wantai over MPD ELISA. In conclusion, both ORF2 and T1NE ELISAs were equally efficient in diagnosing HEV infections. T1NEp proved to be an excellent tool in HE sero-diagnosis and is worth exploring in development of simple rapid tests. KEY POINTS: • In-house ELISA based on bacterially expressed neutralizing epitope/s region protein • In-house ELISA based on complete ORF2 protein expressed in insect cells • Comparison of two in-house and six commercial anti-HEV IgM antibody detection ELISAs.

Establishment of an indirect ELISA method for antibody detection of porcine pseudorabies by recombinant gB, gC, and gD proteins.

Pseudorabies virus (PRV), as a neuroherpes virus, leads to heavy economic losses in the pig industry worldwide.This study was designed to establish recombinant PRV glycoprotein B (gB), C, and D proteins as PRV diagnostic antigens. The gB/C, gC/D, and gB/C/D fusion sequences were synthesized and inserted into pET-28a+ vector to generate the recombinant plasmids. The identified positive recombinant plasmids were transformed into BL21 Escherichiacoli.The results of thepolymerase chain reactionand enzyme digestion showed that the gB/C, gC/D, and gB/C/D fusion proteins were successfully expressed. An indirect sandwich ELISA was developed with the gB/C, gC/D, and gB/C/D as coating antigens. The results of indirect enzyme-linked immunosorbent assay(ELISA) analysis of 184 PRV-positive porcine sera showed that the positive coincidence rates of three recombinant proteins ELISAs relative to IDEXX kit were 98.25%, 95.32%, and 98.83%, and the negative coincidence rates were 85.71%, 75% and 100%, respectively. The inter and intra batch repeatability tests showed that the coefficient of variations of our kits were all less than 5%. Especially, the gB/C/D-ELISAhas the highest specificity and sensitivity among the ELISA methods developed in this study.We established a series expression system of gB/C, gC/D, and gB/C/D antigen epitope genes and Recombinant protein-based indirect ELISA, providing new ideas for PV diagnosis and vaccine development.

Genetic and phenotypic correlations between Aleutian disease tests with body weight, growth, and feed efficiency traits in mink.

The ineffectiveness of vaccination, medicine, and culling strategy leads mink farmers to control Aleutian disease (AD) by selecting AD-resilient mink based on AD tests. However, the genetic background of AD tests and their correlations with economically important or AD-resilient traits are limited. This study estimated the genetic and phenotypic correlations between four AD tests and seven body weight (BW) traits, six growth parameters from the Richards growth model, and eight feed-related traits. Univariate models were used to test the significance (P < 0.05) of fixed effects (sex, color type, AD test year, birth year, and row-by-year), random effects (additive genetic, maternal genetic, and permanent environmental), and a covariate of age using ASReml 4.1. Likewise, pairwise bivariate analyses were conducted to estimate the phenotypic and genetic correlations among the studied traits. Both antigen- and virus capsid protein-based enzyme-linked immunosorbent assay tests (ELISA-G and ELISA-P) showed significant (P < 0.05) moderate positive genetic correlations (±SE) with maturation rate (from 0.36 ± 0.18 to 0.38 ± 0.19). ELISA-G showed a significant negative genetic correlation (±SE) with average daily gain (ADG, -0.37 ± 0.16). ELISA-P showed a significant positive moderate genetic correlation (±SE) with off-feed days (DOF, 0.42 ± 0.17). These findings indicated that selection for low ELISA scores would reduce the maturation rate, increase ADG (by ELISA-G), and minimize DOF (by ELISA-P). The iodine agglutination test (IAT) showed significant genetic correlations with DOF (0.73 ± 0.16), BW at 16 weeks of age (BW16, 0.45 ± 0.23), and BW at harvest (HW, -0.47 ± 0.20), indicating that selection for lower IAT scores would lead to lower DOF and BW16, and higher HW. These estimated genetic correlations suggested that the selection of AD tests would not cause adverse effects on the growth, feed efficiency, and feed intake of mink. The estimates from this study might strengthen the previous finding that ELISA-G could be applied as a reliable and practical indicator trait in the genetic selection of AD-resilient mink in AD-positive farms.

Seroprevalence of Getah virus in Pigs in Eastern China Determined with a Recombinant E2 Protein-Based Indirect ELISA.

Getah virus (GETV), in the genus Alphavirus and the family Togaviridae, has been detected throughout the world. GETV causes high morbidity and mortality in newborn piglets, entailing serious economic losses. Therefore, the experimental work on GETV detection is necessary. However, due to the influence of a variety of unavoidable factors, the ELISA test for the primary screening of animal diseases has low accuracy in detection results. Therefore, we optimized a recombinant E2 (rE2) protein-based enzyme-linked immunosorbent assay (ELISA) for the detection of GETV antibodies in pig serum. The E2 protein was successfully expressed and purified with SDS-PAGE. A Western blotting analysis of sera from infected pigs showed strong reaction with a viral antigen of ~46 KDa corresponding to the E2 glycoproteins. By using chessboard titration and comparing the P/N values, we found that the optimal concentration of coated antigen was found to be 24.5 μg/mL, and the optimal dilution of serum specimens was 1:100. The best working dilution of the horseradish peroxidase (HRP)-conjugated goat anti-pig immunoglobulin (IgG) was 1:5000. The optimal coating conditions were 12 h at 4 °C. The optimal incubation conditions for serum specimens, blocking, and reaction with the secondary antibody were all 1 h at 37 °C. We also investigated the seroprevalence of GETV in 133 serum specimens collected in Eastern China, and 37.59% of the samples tested positive for anti-GETV IgG antibodies, indicating that the seroprevalence of GETV is high in pig populations in China. The seroprevalence was significantly lower in spring (April; 24.24%, 16/66) than in autumn (October; 50.75%, 34/67), which suggested that the presence of anti-GETV antibodies in pigs was seasonal. In conclusion, we improved an rE2 ELISA that detected pig antibodies against GETV after experimental and natural infections. This should be useful in the diagnosis and surveillance of GETV infections.

Applying Machine Learning Algorithms for the Classification of Mink Infected with Aleutian Disease Using Different Data Sources.

Simple SummaryAleutian disease (AD) is a major infectious disease found in mink farms, and it causes financial losses to the mink industry. Controlling AD often requires a counterimmunoelectrophoresis (CIEP) method, which is relatively expensive for mink farmers. Therefore, predicting AD infected mink without using CIEP records will be important for controlling AD in mink farms. In the current study, we applied nine machine learning algorithms to classify AD-infected mink. We indicated that the random forest could be used to classify AD-infected mink (accuracy of 0.962) accurately. This result could be used for implementing machine learning in controlling AD in the mink farms.American mink (Neogale vison) is one of the major sources of fur for the fur industries worldwide, whereas Aleutian disease (AD) is causing severe financial losses to the mink industry. A counterimmunoelectrophoresis (CIEP) method is commonly employed in a test-and-remove strategy and has been considered a gold standard for AD tests. Although machine learning is widely used in livestock species, little has been implemented in the mink industry. Therefore, predicting AD without using CIEP records will be important for controlling AD in mink farms. This research presented the assessments of the CIEP classification using machine learning algorithms. The Aleutian disease was tested on 1157 individuals using CIEP in an AD-positive mink farm (Nova Scotia, Canada). The comprehensive data collection of 33 different features was used for the classification of AD-infected mink. The specificity, sensitivity, accuracy, and F1 measure of nine machine learning algorithms were evaluated for the classification of AD-infected mink. The nine models were artificial neural networks, decision tree, extreme gradient boosting, gradient boosting method, K-nearest neighbors, linear discriminant analysis, support vector machines, naive bayes, and random forest. Among the 33 tested features, the Aleutian mink disease virus capsid protein-based enzyme-linked immunosorbent assay was found to be the most important feature for classifying AD-infected mink. Overall, random forest was the best-performing algorithm for the current dataset with a mean sensitivity of 0.938 ± 0.003, specificity of 0.986 ± 0.005, accuracy of 0.962 ± 0.002, and F1 value of 0.961 ± 0.088, and across tenfold of the cross-validation. Our work demonstrated that it is possible to use the random forest algorithm to classify AD-infected mink accurately. It is recommended that further model tests in other farms need to be performed and the genomic information needs to be used to optimize the model for implementing machine learning methods for AD detection.

Development of robust, indigenous ELISA for detection of IgG antibodies against CoV-2N and S proteins: mass screening.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic has adversely affected humankind and caused millions of deaths globally since January 2020. Robust and quick serological tests such as antibody detection assays for SARS-CoV-2 provide relevant information and aid in the process of vaccine development and diagnostics, as well as in sero-epidemiological monitoring of antibody response to the virus. The receptor-binding domain (RBD) of spike and nucleocapsid protein are specific targets for detecting SARS-CoV-2 antibodies. Here, we present the development of a stable spike (S) and nucleocapsid (N) protein-based ELISA antibody detection test “CoroSuchak,” with 99% sensitivity, 98% specificity, cost-effective, and detection in a minimum time for serodiagnosis and mass screening of the population for antibodies against SARS-CoV-2. Blood samples were analyzed from 374 SARS-CoV-2 reverse transcription-polymerase chain reaction (RT-PCR) positive, 772 negative and asymptomatic, and 874 random groups of subjects. We found that the antibody titer was significantly higher (p < 0.0001) in infected and vaccinated group compared to the only vaccinated and only infected group. Using enzyme-linked immunosorbent assay (ELISA), we detected SARS-CoV-2 immunoglobulin G (IgG) antibodies in 118/123 (96%) infected individuals, 570/653 (87%) non-infected but vaccinated individuals, 231/237 (97%) individuals who were both infected and vaccinated, and 499/874 (57%) from randomly selected individuals from the first and second waves of the pandemic. Similarly in the third wave, 14/14 (100%) infected and 16/20 (80%) RT-PCR-negative but symptomatic subjects were detected. Thus, the highly sensitive and specific in-house developed ELISA antibody detection kit “CoroSuchak” is extremely useful to determine the seroprevalence of SARS-CoV-2 antibodies in the coronavirus-exposed population.Key points•Indigenous kit using a combination of spike and nucleocapsid proteins and peptide sequences.•High sensitivity and specificity to detect variants.•Highly sensitive for mass screening.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00253-022-12113-8.

The N-terminal Subunit of the Porcine Deltacoronavirus Spike Recombinant Protein (S1) Does Not Serologically Cross-react with Other Porcine Coronaviruses.

Porcine deltacoronavirus (PDCoV), belonging to family Coronaviridae and genus Deltacoronavirus, is a major enteric pathogen in swine. Accurate PDCoV diagnosis relying on laboratory testing and antibody detection is an important approach. This study evaluated the potential of the receptor-binding subunit of the PDCoV spike protein (S1), generated using a mammalian expression system, for specific antibody detection via indirect enzyme-linked immunosorbent assay (ELISA). Serum samples were collected at day post-inoculation (DPI) −7 to 42, from pigs (n = 83) experimentally inoculated with different porcine coronaviruses (PorCoV). The diagnostic sensitivity of the PDCoV S1-based ELISA was evaluated using serum samples (n = 72) from PDCoV-inoculated animals. The diagnostic specificity and potential cross-reactivity of the assay was evaluated on PorCoV-negative samples (n = 345) and samples collected from pigs experimentally inoculated with other PorCoVs (n = 472). The overall diagnostic performance, time of detection, and detection rate over time varied across different S/P cut-offs, estimated by Receiver Operating Characteristic (ROC) curve analysis. The higher detection rate in the PDCoV group was observed after DPI 21. An S/P cut-off of 0.25 provided 100% specificity with no serological cross-reactivity against other PorCoV. These results support the use of S1 protein-based ELISA for accurate detection of PDCoV infections, transference of maternal antibodies, or active surveillance.

Development of Quantitative Spike and Nucleocapsid Protein-Based Enzyme-Linked Immunosorbent Assays (ELISAs) for Measurement of Antibodies against SARS-CoV-2

Abstract In the COVID-19 pandemic, serological assays will have important public health and clinical uses to monitor and respond to the pandemic. Assays to detect and quantify virus-specific antibodies are important to complement the RNA-based tests, improve our understanding of the pathogenesis of COVID-19, and inform vaccine development. The majority of commercial assays for measuring IgG to SARS-CoV-2 have been limited by a lack of quantitative information about antiviral antibodies in standardized units. Here, we have developed and commercialized several fully validated, quantitative ELISA kits using purified receptor-binding domain (RBD) or full-length of Spike S1, or Nucleocapsid proteins developed in BioLegend as the capture reagents. The SARS-CoV-2 Spike Protein antibody ELISA kits have both LEGEND MAXTM and RAPID MAXTM formats and are optimized to mitigate matrix effects and avoid false-positive results, and have a readout in standardized units (mg/mL). We have applied these sensitive and specific SARS-CoV-2 Spike Protein antibody ELISAs in an employee cohort study to monitor antibody immune response after first and second doses of 2 mRNA SARS-CoV-2 vaccines – BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna). We found that although all participants in this study are responsive to the first dose of immunization by both mRNA vaccines as evidenced by the elevation of anti-Spike S1 and RBD IgG levels above the pre-immunization baseline levels, more robust antibody responses to Spike S1 and RBD are observed after the second dose. In summary, our assays have good performance characteristics, have a quantitative readout in standardized units, and could be used by research and clinical laboratories that routinely use ELISA.

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 of a recombinant nucleocapsid protein-based ELISA for the detection of IgM and IgG antibodies to SARS-CoV-2.

Coronavirus 2019 (COVID‐19) is a global concern for public health. Thus, early and accurate diagnosis is a critical step in management of this infectious disease. Currently, RT‐PCR is routine diagnosis test for COVID‐19, but it has some limitations and false negative results. enzyme‐linked immunosorbent assay (ELISA) against SARS‐CoV‐2 antigens seems to be an appropriate approach for serodiagnosis of COVID‐19. In the current study, an ELISA system, using a recombinant nucleocapsid (N) protein, was developed for the detection of IgM and IgG antibodies to SARS‐CoV‐2. The related protein was expressed, purified, and used in an ELISA system. Sera samples (67) for COVID‐19 patients, as well as sera samples from healthy volunteers (112), along with sera samples from non‐COVID‐19 patients were examined by the ELISA system. The expression and purity of the recombinant N protein were approved by SDS‐PAGE and Western blotting. The sensitivity of ELISA system was 91.04 and 92.53% for the detection of IgG and IgM antibodies, respectively. Moreover, the specificity of the developed ELISA system for IgG and IgM were 98.21 and 97.32%, respectively. Our developed ELISA system showed satisfactory sensitivity and specificity for the detection of antiSARS‐CoV‐2 IgM and IgG antibodies and could be used as a complementary approach for proper diagnosis of COVID‐19.

18 Genetic and Phenotypic Parameters for Aleutian Disease Tests and Their Correlations with Pelt Quality, Reproductive Performance, Packed-cell Volume, and Harvest Length in Mink

Abstract Aleutian disease (AD) is an untreatable immune complex disease in mink and brings tremendous economic losses to the mink industry globally. The ineffectiveness of culling, immunoprophylaxis, and medical treatment in controlling AD have urged mink farmers to select AD-resilient mink based on the AD tests. However, the genetic analysis of these tests and their correlations with AD-resilient traits have not been investigated. In this study, data on 5,824 mink were used to estimate the genetic and phenotypic parameters of four AD tests, including two systems of enzyme-linked immunosorbent assay (ELISA), counterimmunoelectrophoresis (CIEP), and iodine agglutination test (IAT), and their genetic and phenotypic correlations with pelt quality, reproductive performance, packed-cell volume (PCV), and harvest length (HL). Significance (P &amp;lt; 0.05) of fixed effects (sex, year, color type, the number of mating, and dam age), covariates (age at blood sampling and age at harvest), and random effects (additive genetic, permanent environmental, and maternal effects) were determined using univariate models. The genetic and phenotypic parameters for all traits were estimated under bivariate models using ASReml 4.1. Estimated heritabilities (±SE) were 0.39±0.05, 0.61±0.07, 0.11±0.07, and 0.26±0.05 for antigen-based ELISA (ELISA-G), virus capsid protein-based ELISA, CIEP, and IAT, respectively. The ELISA-G showed moderate repeatability (0.58±0.04) and significant (P &amp;lt; 0.05) negative genetic correlations (±SE) with reproductive performance traits (from -0.41±0.16 to -0.49±0.12), PCV (-0.53±0.09), and HL (-0.45±0.16). These results indicated that the selection of mink with a lower ELISA-G score could not only decrease the anti-AMDV antibody level and the extent of anemia but also improve the female reproductive performance and the harvest length of mink without causing adverse influences on the pelt quality. Hence, ELISA-G could be applied as an indicator for genetic selection of AD-resilient mink and help mink farmers reduce the adverse effects of AD.

Detections of Rainbow Trout Antibodies to Infectious Pancreatic Necrosis Virus and Viral Hemorrhagic Septicemia Virus via a Recombinant Protein-Based Enzyme-Linked Immunosorbent Assay

Infectious Pancreatic Necrosis Virus (IPNV) and Viral hemorrhagic septicemia virus (VHSV) cause significant losses in the aquaculture industry. There have been few reports of the use of screening rainbow trout for antibodies against IPNV and VHSV as an epidemiological tool. Several ELISAs using a whole virus or recombinant IPNV and VHSV proteins have been described. In this study, a recombinant protein-based enzyme-linked immunosorbent assay (ELISA) for the detection of IPNV and VHSV antibodies in rainbow trout (Oncorhynchus mykiss) was evaluated. To develop recombinant protein-based enzyme-linked immunosorbent assays, a fragment containing the entire length of the gG gene of VHSV and VP2 of IPNV was amplified by PCR using the viruses' genomic RNA and cloned in pET-28a(+) plasmid. Recombinant structural viral proteins (rVP2 and rgG) were expressed in the Escherichia coli BL21 (DE3). The rgG was extracted and purified. 96-well plates were coated with VP2 and gG separately. For VHSV, Assay could detect until 1/15625 dilution in VHSV positive fish serum. For IPNV, Assays could detect until 1/3125 dilution in IPNV positive fish serum. These results show rgG and rVP2, used in ELISA, are more sensitive than virus neutralization tests.

Genetic and phenotypic parameters for Aleutian disease tests and their correlations with pelt quality, reproductive performance, packed-cell volume, and harvest length in mink.

Aleutian disease (AD), caused by the Aleutian mink disease virus (AMDV), is a major health concern that results in global economic losses to the mink industry. The unsatisfactory outcome of the culling strategy, immunoprophylaxis, and medical treatment in controlling AD have urged mink farmers to select AD resilient mink based on several detection tests, including enzyme-linked immunosorbent assay (ELISA), counterimmunoelectrophoresis (CIEP), and iodine agglutination test (IAT). However, the genetic analysis of these AD tests and their correlations with pelt quality, reproductive performance, packed-cell volume (PCV), and harvest length (HL) have not been investigated. In this study, data on 5,824 mink were used to estimate the genetic and phenotypic parameters of four AD tests, including two systems of ELISA, CIEP, and IAT, and their genetic and phenotypic correlations with two pelt quality, five female reproductive performance, PCV, and HL traits. Significances (P < 0.05) of fixed effects (sex, year, dam age, and color type), covariates (age at harvest and blood sampling), and random effects (additive genetic, permanent environmental, and maternal effects) were determined under univariate models using ASReml 4.1 software. The genetic and phenotypic parameters for all traits were estimated under bivariate models using ASReml 4.1 software. Estimated heritabilities (±SE) were 0.39 ± 0.06, 0.61 ± 0.07, 0.11 ± 0.07, and 0.26 ± 0.05 for AMDV antigen-based ELISA (ELISA-G), AMDV capsid protein-based ELISA, CIEP, and IAT, respectively. The ELISA-G also showed a moderate repeatability (0.58 ± 0.04) and had significant negative genetic correlations (±SE) with reproductive performance traits (from -0.41 ± 0.16 to -0.49 ± 0.12), PCV (-0.53 ± 0.09), and HL (-0.45 ± 0.16). These results indicated that ELISA-G had the potential to be applied as an indicator trait for genetic selection of AD resilient mink in AD endemic ranches and therefore help mink farmers to reduce the adverse effects caused by AD.

Comparable Accuracies of Nonstructural Protein 1- and Envelope Protein-Based Enzyme-Linked Immunosorbent Assays in Detecting Anti-Dengue Immunoglobulin G Antibodies.

Background: Dengue virus (DENV) infection remains a global public health concern. Enzyme-linked immunosorbent assays (ELISAs), which detect antibodies targeting the envelope (E) protein of DENV, serve as the front-line serological test for presumptive dengue diagnosis. Very few studies have determined the serostatus by detecting antibodies targeting the nonstructural protein 1 (NS1), which can function as diagnostic biomarkers to distinguish natural immunity from vaccine-induced immunity. Methods: We used community-acquired human serum specimens, with the serostatus confirmed by focus reduction microneutralization test (FRμNT), to evaluate the diagnostic performances of two NS1-based ELISA methods, namely, immunoglobulin G antibody-capture ELISA (NS1 GAC–ELISA) and indirect NS1 IgG ELISA, and compared the results with an E-based virus-like particle (VLP) GAC–ELISA. Results: NS1-based methods had comparable accuracies as VLP GAC–ELISA. Although the sensitivity in detecting anti-NS1 IgM was poor, indirect NS1 IgG ELISA showed similar limits of detection (~1–2 ng/mL) as NS1 GAC–ELISA in detecting anti-NS1 IgG. Combining the results from two or more tests as a composite reference standard can determine the DENV serostatus with a specificity reaching 100%. Conclusion: NS1-based ELISAs have comparable accuracies as VLP GAC–ELISA in determining dengue serostatus, which could effectively assist clinicians during assessments of vaccine eligibility.

Detection of SARS-CoV-2-specific antibodies via rapid diagnostic immunoassays in COVID-19 patients

BackgroundEfficient monitoring and control of coronavirus disease 2019 (COVID-19) require access to diagnostic tests, and serological diagnostic testing is desirable. In the current study, antibodies were investigated in patients recently diagnosed with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.MethodsCross-sectional data were obtained from 245 patients in whom SARS-CoV-2 infection had been confirmed via real-time reverse transcriptase-polymerase chain reaction between March and October 2020. Serum samples were acquired between 2 and 60 days following the onset of COVID-19 symptoms or the first detection of SARS-CoV-2 in asymptomatic patients. All specimens were tested simultaneously using an IgM/IgG rapid diagnostic test (RDT), IgG nucleocapsid protein-based chemiluminescent microparticle immunoassay (CMIA), IgG, and IgA spike protein-based enzyme-linked immunosorbent assays (ELISAs). Blood donor samples obtained in 2018 were used as negative controls.ResultsThe sensitivity and specificity of the RDT IgG were compared with the IgG immunoassays as standards. The RDT IgG exhibited 97.5% sensitivity and 89.4% specificity compared with a CMIA IgG, 98.4% sensitivity, and 78.8% specificity compared with an ELISA IgG. IgM, IgG, and IgA seropositivity rates were low between 1 and 2 weeks after COVID-19 symptom onset or the detection of SARS-CoV-2 RNA. IgM seropositivity rate began decreasing after 4 weeks, whereas IgG and IgA seropositivity rate remained at appreciable levels over the 8-week study period. No cross-reactivity with seasonal coronaviruses was detected.ConclusionsIgG RDT alone or combined with molecular diagnostic tests may be useful for identifying recent SARS-CoV-2 infection.

Development of an Immunodiagnostic Test for Screening Human Brucellosis Cases Using the Whole-Cell Antigens of Brucella abortus

BackgroundHuman brucellosis is an important zoonotic disease of public health and often remains neglected owing to lack of sensitive and efficient diagnostic methods. This study evaluates diagnostic utility of in-house designed enzyme-linked immunosorbent assay (ELISA) using whole-cell antigens of Brucella abortus (B. abortus) S19 against the commercially available kits.MethodsA prospective cohort study involving different populations within the Vidarbha regions of Maharashtra, India was conducted through camps organised from May 2009 to October 2015. A total of 568 serum samples were collected from high-risk people recruited as study cohorts based on inclusion criteria, additional risk factors and clinical symptoms. Samples were evaluated by indirect ELISA using the whole-cell antigens of B. abortus. The results were compared with the commercially available IgG detection ELISA kit to ascertain the specificity and sensitivity of the developed test.ResultsFever, body ache, joint pain, lower back pain, loss of appetite and weight loss were major symptoms associated with the disease. With the cut-off of > 0.8, the positivity of brucellosis infection was at 12.32% (70/568) compared to 9.33% (53/568) as detected by the commercial kit. The in-house developed ELISA method yielded a sensitivity of 87.5% and specificity of 99.18% as compared to the commercial kits (sensitivity −80.30% and specificity −99.6%).DiscussionThe B. abortus S19-derived whole-cell protein-based ELISA is rapid and cost-effective and can be used for screening brucellosis infection in lieu of the commercially available ELISA kits.