Both viral infection and vaccination affect the antibody repertoire of a person. Here, we demonstrate that the analysis of serum antibodies generates information not only on the virus type that caused the infection but also on the specific virus variant. We developed a rapid multiplex assay providing a fingerprint of serum antibodies against five different SARS-CoV-2 variants based on a microarray of virus antigens immobilized on the surface of a label-free reflectometric biosensor. We analyzed serum from the plasma of convalescent subjects and vaccinated volunteers and extracted individual antibody profiles of both total immunoglobulin Ig and IgA fractions. We found that Ig level profiles were strongly correlated with the specific variant of infection or vaccination and that vaccinated subjects displayed a larger quantity of total Ig and a lower fraction of IgA relative to the population of convalescent unvaccinated subjects.

Canine monocytic ehrlichiosis (CME) is the most common tick-borne disease affecting domestic dogs and other wild canids. It has a worldwide distribution and is associated with the presence of the brown dog tick. Few studies have been conducted in Mexico to identify and characterize Ehrlichia canis genetic variability. In the present study, 111 dogs of different sex, breed, and age from three geographic regions in Mexico were included. All of them had a previous history of tick infestation and/or the presence of one or more clinical signs compatible with CME. All dogs were tested by a commercial ELISA and nested PCR assay for the detection of E. canis. In addition, we analyzed the E. canis genetic diversity from the 16S rRNA gene sequences obtained in this study, along with 15 additional sequences described for E. canis in Mexico and obtained from GeneBank. Serological detection by commercial ELISA results showed overall infection rates of 85.58% (95/111), including 73.1% (30/41) in samples from Guerrero state; 75% (15/20) in Morelos; and 100% (50/50) in Chihuahua. On the other hand, molecular detection (nPCR assay) showed 31.5% (35/111) overall infection rate, with 41.4% (17/41) in Guerrero state; 55% (11/20) in Morelos; and 14% (7/50) in Chihuahua. We observed a high 16S rRNA gene sequence conservancy in most of the E. canis isolates in the three geographical areas from Mexico, including those analyzed in this research, suggesting a common geographic origin among isolates.

Current serological tests cannot differentiate between total immunoglobulin A (IgA) and dimeric IgA (dIgA) associated with mucosal immunity. Here, we describe two new assays, dIgA-ELISA and dIgA-multiplex bead assay (MBA), that utilize the preferential binding of dIgA to a chimeric form of secretory component, allowing the differentiation between dIgA and monomeric IgA. dIgA responses elicited through severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection were measured in (i) a longitudinal panel, consisting of 74 samples (n = 20 individuals) from hospitalized cases of coronavirus disease 2019 (COVID-19); (ii) a longitudinal panel, consisting of 96 samples (n = 10 individuals) from individuals with mild COVID-19; (iii) a cross-sectional panel with PCR-confirmed SARS-CoV-2 infection with mild COVID-19 (n = 199) and (iv) pre-COVID-19 samples (n = 200). The dIgA-ELISA and dIgA-MBA demonstrated a specificity for dIgA of 99% and 98.5%, respectively. Analysis of dIgA responses in the longitudinal panels revealed that 70% (ELISA) and 50% (MBA) of patients elicited a dIgA response by day 20 after PCR diagnosis with a SARS-CoV-2 infection. Individuals with mild COVID-19 displayed increased levels of dIgA within the first 3 weeks after diagnosis but responses appeared to be short lived, compared with sustained IgA levels. However, in samples from hospitalized patients with COVID-19 we observed high and sustained levels of dIgA, up to 245 days after PCR diagnosis. Our results suggest that severe COVID-19 infections are associated with sustained levels of plasma dIgA compared with mild cases.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes clinical syndromes typified as reproductive disorders in sows and respiratory diseases in piglets. PRRSV remains one of the most prevalent pathogens affecting the pig industry, because of its complex infection profile and highly heterogeneous genetic and recombination characteristics. Therefore, a rapid and effective PRRSV detection method is important for the prevention and control of PRRS. With extensive in-depth research on PRRSV detection methods, many detection methods have been improved and promoted. Laboratory methods include techniques based on virus isolation (VI), enzyme-linked immunosorbent assays (ELISA), indirect immunofluorescence assays (IFA), immunoperoxidase monolayer assays (IPMA), polymerase chain reaction (PCR), quantitative real-time PCR (qPCR), digital PCR (dPCR), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), clustered regularly interspaced short palindromic repeats (CRISPR), metagenomic next-generation sequencing (mNGS), and other methods. This study reviews the latest research on improving the main PRRSV detection methods and discusses their advantages and disadvantages.

Antibody (AB) testing or serotesting for reactive ABs against antigenic proteins is broadly used. Parallel examination of many antigens is of high interest to identify autoantibodies (AAB) or differential antigenic reactivities in many biological settings like allergy and infectious autoimmune, cancerous, or systemic disease. The resulting AAB profiles can be used for diagnosis, prognosis, and monitoring of such conditions. Protein microarrays have been used for AB profiling over the past decade but show some significant limitations which make them unsuitable for clinical applications. Alternative multiplexing platforms such as bead arrays were shown to provide a versatile tool for the confirmation and efficient analysis of high numbers of biological samples. Luminex\' bead-based xMAP technology combines advantages such as multiplexing and lower demand for sample volume and at the same time overcomes the challenges of microarrays. It works faster, shows better antigen stability, is more reproducible, and allows the analysis of up to 500 analytes in one sample well. In this chapter we introduce our established workflow for the use of the xMAP technology for AB profiling including an overview of the method principle and protocols for the covalent immobilization of proteins to the MagPlex beads, confirmation of protein coupling, the execution of a multiplexed bead-based protein immunoassay, and subsequent data handling.

The nucleic acid test is still the standard assessment for the diagnosis of coronavirus disease 2019 (COVID-19), which is caused by human infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In addition to supporting the confirmation of disease cases, serological assays are used for the analysis of antibody status and epidemiological surveys. In this study, a single Western blot strip (WBS) coated with multiple Escherichia coli (E. coli)-expressed SARS-CoV-2 antigens was developed for comprehensive studies of antibody profiles in COVID-19 patient sera. The levels of specific antibodies directed to SARS-CoV-2 spike (S), S2, and nucleocapsid (N) proteins were gradually increased with the same tendency as the disease progressed after hospitalization. The signal readouts of S, S2, and N revealed by the multi-antigen-coated WBS (mWBS)-based serological assay (mWBS assay) also demonstrated a positive correlation with the SARS-CoV-2 neutralizing potency of the sera measured by the plaque reduction neutralization test (PRNT) assays. Surprisingly, the detection signals against the unstructured receptor-binding domain (RBD) purified from E. coli inclusion bodies were not observed, although the COVID-19 patient sera exhibited strong neutralizing potency in the PRNT assays, suggesting that the RBD-specific antibodies in patient sera mostly recognize the conformational epitopes. Furthermore, the mWBS assay identified a unique and major antigenic epitope at the residues 1148, 1149, 1152, 1155, and 1156 located within the 1127-1167 fragment of the S2 subunit, which was specifically recognized by the COVID-19 patient serum. The mWBS assay can be finished within 14-16 min by using the automatic platform of Western blotting by thin-film direct coating with suction (TDCS WB). Collectively, the mWBS assay can be applied for the analysis of antibody responses, prediction of the protective antibody status, and identification of the specific epitope. KEY POINTS: • A Western blot strip (WBS) coated with multiple SARS-CoV-2 antigens was developed for the serological assay. • The multi-antigen-coated WBS (mWBS) can be utilized for the simultaneous detection of antibody responses to multiple SARS-CoV-2 antigens. • The mWBS-based serological assay (mWBS assay) identified a unique epitope recognized by the COVID-19 patient serum.

There is a massive demand to identify alternative methods to detect new cases of COVID-19 as well as to investigate the epidemiology of the disease. In many countries, importation of commercial kits poses a significant impact on their testing capacity and increases the costs for the public health system. We have developed an ELISA to detect IgG antibodies against SARS-CoV-2 using a recombinant viral nucleocapsid (rN) protein expressed in E. coli. Using a total of 894 clinical samples we showed that the rN-ELISA was able to detect IgG antibodies against SARS-CoV-2 with high sensitivity (97.5%) and specificity (96.3%) when compared to a commercial antibody test. After three external validation studies, we showed that the test accuracy was higher than 90%. The rN-ELISA IgG kit constitutes a convenient and specific method for the large-scale determination of SARS-CoV-2 antibodies in human sera with high reliability.

There is a massive demand to identify alternative methods to detect new cases of COVID-19 as well as to investigate the epidemiology of the disease. In many countries, importation of commercial kits poses a significant impact on their testing capacity and increases the costs for the public health system. We have developed an ELISA to detect IgG antibodies against SARS-CoV-2 using a recombinant viral nucleocapsid (rN) protein expressed in E. coli. Using a total of 894 clinical samples we showed that the rN-ELISA was able to detect IgG antibodies against SARS-CoV-2 with high sensitivity (97.5%) and specificity (96.3%) when compared to a commercial antibody test. After three external validation studies, we showed that the test accuracy was higher than 90%. The rN-ELISA IgG kit constitutes a convenient and specific method for the large-scale determination of SARS-CoV-2 antibodies in human sera with high reliability.

Introduction Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific humoral immune persistence has been proposed to be affected by patients\' characteristics. Moreover, available conflicting assay results are needed to be settled through comparative research with defined clinical specimens. Methods This prospective study investigated SARS-CoV-2-specific antibodies among 43 adults and 34 children at a mean of 12 weeks after the onset of COVID-19 symptoms using six serological assays and compared their performance. We used two Euroimmun (Euroimmun, Luebeck, Germany), two automated Roche Elecsys (Basel, Switzerland), and two rapid immuno-chromatographic Ecotest (Matrix Diagnostics, Assure Tech. (Hangzhou) Co., L, China) assays to investigate SARS-CoV-2 antibodies. Results The findings showed that the Roche Elecsys anti-S total test yielded the best positivity/sensitivity (children 94.1% and adults 93.0%; p = 0.877) while five immunoglobulin IgG targeting assays had similar positivity/sensitivity between children (88.2% to 94.1%) and adults (88.4% to 93.0%) (p > 0.05). Although IgM positivity was relatively low (p < 0.001), it was found in the majority of our pediatric and adult patients (67.6% and 86.0%, respectively; p = 0.098). SARS-CoV-2 S IgG titers were found to be higher among males in pediatric and adult groups compared to females (p = 0.027 and p = 0.041, respectively). Furthermore, we observed significantly higher antibody titers among pneumonia patients (p = 0.001). Conclusion Overall, we concluded SARS-CoV-2 antibody persistence over an average of 12 weeks after the onset of COVID-19 symptoms. While automated Roche Elecsys total antibody assays yielded the best sensitivity (> 90%) and five assays targeting IgG had acceptable performance. Patients with pneumonia and males have higher antibody titers. The effect of antibody persistence on re-infections should be monitored in longitudinal studies.

Quantification of the anti-SARS-CoV-2 antibody response has proven to be a prominent diagnostic tool during the COVID-19 pandemic. Antibody measurements have aided in the determination of humoral protection following infection or vaccination and will likely be essential for predicting the prevalence of population level immunity over the next several years. Despite widespread use, current tests remain limited in part, because antibody capture is accomplished through the use of complete spike and nucleocapsid proteins that contain significant regions of overlap with common circulating coronaviruses. To address this limitation, a unique epitope display platform utilizing monovalent display and protease-driven capture of peptide epitopes was used to select high affinity peptides. A single round of selection using this strategy with COVID-19 positive patient plasma samples revealed surprising differences and specific patterns in the antigenicity of SARS-CoV-2 proteins, especially the spike protein. Putative epitopes were assayed for specificity with convalescent and control samples, and the individual binding kinetics of peptides were also determined. A subset of prioritized peptides was used to develop an antibody diagnostic assay that showed low cross reactivity while detecting 37% more positive antibody cases than a gold standard FDA EUA test. Finally, a subset of peptides were compared with serum neutralization activity to establish a 2 peptide assay that strongly correlates with neutralization. Together, these data demonstrate a novel phage display method that is capable of comprehensively and rapidly mapping patient viral antibody responses and selecting high affinity public epitopes for the diagnosis of humoral immunity.