A Customized Novel Blocking ELISA for Detection of Bat-Origin Swine Acute Diarrhea Syndrome Coronavirus Infection.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a newly discovered emerging alphacoronavirus. SADS-CoV shares over 90% genome sequence identity with bat alphacoronavirus HKU2. SADS-CoV was associated with severe diarrhea and high mortality rates in piglets. Accurate serological diagnosis of SADS-CoV infection is key in managing the emerging SADS-CoV. However, thus far there have been no effective antibody-based diagnostic tests for diagnose of SADS-CoV exposure. Here, monoclonal antibody (MAb) 6E8 against SADS-CoV N protein accurately recognized SADS-CoV infection. Then, MAb 6E8 was utilized as a blocking antibody to develop blocking ELISA (bELISA). We customized the rN coating antigen with concentration 0.25 µg/mL. According to receiver operator characteristic curve analysis, the cutoff value of the bELISA was determined as 38.19% when the max Youden index was 0.955, and specificity was 100%, and sensitivity was 95.5%. Specificity testing showed that there was no cross-reactivity with other serum positive swine enteric coronaviruses, such as porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), porcine rotavirus (PoRV), and porcine sapelovirus (PSV). In conclusion, we customized a novel and high-quality blocking ELISA for detection of SADS-CoV infection, and the current bELISA will be linked to a clinical and epidemiological assessment of SADS-CoV infection. IMPORTANCE SADS-CoV was reported to be of high potential for dissemination among various of host species. Accurate serological diagnosis of SADS-CoV infection is key in managing the emerging SADS-CoV. However, thus far there have been no effective antibody-based diagnostic tests for diagnose of SADS-CoV exposure. We customed a novel and high-quality bELISA assay for detection of SADS-CoV N protein antibodies, and the current bELISA will be linked to a clinical and epidemiological assessment of SADS-CoV infection.

Development of a Nucleocapsid Protein-Based Blocking ELISA for the Detection of Porcine Deltacoronavirus Antibodies.

Porcine deltacoronavirus (PDCoV) is an emerging enteropathogen which mainly causes diarrhea, dehydration and death in nursing piglets, threatening the global swine industry. Moreover, it can infect multiple animal species and humans. Hence, reliable diagnostic assays are needed to better control this zoonotic pathogen. Here, a blocking ELISA was developed using a recombinant nucleocapsid (N) protein as the coating antigen paired with an N-specific monoclonal antibody (mAb) as the detection antibody. The percent inhibition (PI) of the ELISA was determined using 384 swine serum samples, with an indirect immunofluorescence assay (IFA) as the reference method. Through receiver operating characteristic analysis in conjunction with Youden's index, the optimal PI cut-off value was determined to be 51.65%, which corresponded to a diagnostic sensitivity of 98.79% and a diagnostic specificity of 100%. Of the 330 serum samples tested positive via IFA, 326 and 4 were tested positive and negative via the ELISA, respectively, while the 54 serum samples tested negative via IFA were all negative via the ELISA. The overall coincidence rate between the two assays was 98.96% (380/384). The ELISA exhibited good repeatability and did not cross-react with antisera against other swine pathogens. Overall, this is the first report on developing a blocking ELISA for PDCoV serodiagnosis.

Correlation between the atellica SARS-COV-2 IGG assay results and the presence of SARS-COV-2 neutralizing antibodies

Background-aim: Antibodies against SARS-CoV-2 detected by routine immunoassays ensure the existence of antibodies binding the virus, but not necessarily the elimination of the infection. Instead, neutralizing antibodies protect against SARS-CoV-2. Virus neutralization test remains the gold standard, but other neutralization assays have been developed to estimate the neutralizing potential against SARS-CoV-2, among them the FDA approved cPass SARS-CoV-2 Neutralization Antibody Detection (cPass) assay. We studied the correlation between the results obtained with SARS-CoV-2 IgG (Siemens Healthineers) and cPass (GenScript) assays in 218 patients. Methods: SARS-CoV-2 IgG is a 2-step sandwich immunoassay automated on Atellica analyzer. It is an assay based on indirect chemiluminescent technology used for the qualitative and quantitative detection of IgG antibodies (sCOVG) against SARS-CoV-2. cPass assay is a blocking ELISA detection assay using the HRP-conjugated recombinant receptor binding domain (RBD) from the viral spike protein and the human Angiotensin-Converting Enzyme 2 (ACE2). The interaction between HRP-RBD and ACE2 will be blocked if neutralizing antibodies against SARS-CoV-2 RBD are present in the sample. Results higher than 30% indicate the presence of neutralizing antibodies. Result: We observed that the presence of SARS-CoV-2 neutralizing antibodies correlated with sCOVG results, observing that neutralizing antibodies using the manufacturer cutoff (>30%) were present in 5% of samples with sCOVG <1, 83% of samples with sCOVG between 1 and 2, 96% of samples with sCOVG between 2,01 and 10, and 100% of samples with sCOVG >10. On the other hand we studied the agreement between the titers obtained for both tests using Passing Bablok regression analysis, obtaining a regression coefficient of 0.881. Finally, we compared the accuracy of the results of the sCOVG and the cPass tests. The area under the curve obtained in the comparison between both tests was 0,969. Furthermore, we observed a concordance between both tests considering the respective cut-off points (≥1 for sCOVG and ≥ 30 for cPass assay) in 96% of patients included in our study. Conclusions: We concluded that the Atellica SARS-CoV-2 IgG assay correlates well with the detection of neutralizing antibodies.