A novel S2-derived peptide-based ELISA for broad detection of antibody against infectious bronchitis virus.

Avian infectious bronchitis (IB) is a highly contagious disease caused by infectious bronchitis virus (IBV). Vaccination is an effective approach for controlling IBV. Therefore, reliable immune monitoring for IB is critical for poultry. In this study, a novel peptide derived from S2 protein was used to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of broadly cross-reactive antibodies against IBV. The peptide-based ELISA (pELISA) showed good specificity and sensitivity in detecting IBV antibodies against different serotypes. A semilogarithmic regression method for determining IBV antibody titers was also established. Antibody titers detected by pELISA and calculated with this equation were statistically similar to those evaluated by indirect fluorescence assay (IFA). Moreover, the comparison analysis showed a 96.07% compatibility between the pELISA and IDEXX ELISA. All these data demonstrate that the pELISA generated here can be as a rapid and reliable serological surveillance tool for monitoring IBV infection or vaccination.

Standardization of in-house anti-IgG and IgA ELISAs for the detection of COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). RT-PCR detection of viral RNA represents the gold standard method for diagnosis of COVID-19. However, multiple diagnostic tests are needed for acute disease diagnosis and assessing immunity during the COVID-19 outbreak. Here, we developed in-house anti-RBD IgG and IgA enzyme-linked immunosorbent assays (ELISAs) using a well-defined serum sample panel for screening and identification of human SARS-CoV-2 infection. We found that our in-house anti-SARS-CoV-2 IgG ELISA displayed a 93.5% sensitivity and 98.8% specificity whereas our in-house anti-SARS-CoV-2 IgA ELISA provided assay sensitivity and specificity at 89.5% and 99.4%, respectively. The agreement kappa values of our in-house anti-SARS-CoV-2 IgG and IgA ELISA assays were deemed to be excellent and fair, respectively, when compared to RT-PCR and excellent for both assays when compared to Euroimmun anti-SARS-CoV-2 IgG and IgA ELISAs. These data indicate that our in-house anti-SARS-CoV-2 IgG and IgA ELISAs are compatible performing assays for the detection of SARS-CoV-2 infection.

Rapid Quantitation of Various Therapeutic Monoclonal Antibodies Using Membranes with Fc-Specific Ligands.

Therapeutic monoclonal antibodies (mAbs) provide effective treatments for many diseases, including cancer, autoimmune disorders, and, lately, COVID-19. Monitoring the concentrations of mAbs is important during their production and subsequent processing. This work demonstrates a 5 min quantitation of most human immunoglobulin G (IgG) antibodies through capture of mAbs in membranes modified with ligands that bind to the fragment crystallizable (Fc) region. This enables binding and quantitation of most IgG mAbs. Layer-by-layer (LBL) adsorption of carboxylic acid-rich polyelectrolytes in glass-fiber membranes in 96-well plates allows functionalization of the membranes with Protein A or a peptide, oxidized Fc20 (oFc20), with high affinity for the Fc region of human IgG. mAb capture occurs in <1 min during the flow of solutions through modified membranes, and subsequent binding of a fluorophore-labeled secondary antibody enables quantitation of the captured mAbs using fluorescence. The intra- and inter-plate coefficients of variations (CV) are <10 and 15%, respectively, satisfying the acceptance criteria for many assays. The limit of detection (LOD) of 15 ng/mL is on the high end of commercial enzyme-linked immunosorbent assays (ELISAs) but certainly low enough for monitoring of manufacturing solutions. Importantly, the membrane-based method requires <5 minutes, whereas ELISAs typically take at least 90 min. Membranes functionalized with oFc20 show greater mAb binding and lower LODs than membranes with Protein A. Thus, the membrane-based 96-well-plate assay, which is effective in diluted fermentation broths and in mixtures with cell lysates, is suitable for near-real-time monitoring of the general class of human IgG mAbs during their production.

Biopanning of specific peptide for SARS-CoV-2 nucleocapsid protein and enzyme-linked immunosorbent assay-based antigen assay.

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread worldwide which triggered serious public health issues. The search for rapid and accurate diagnosis, effective prevention, and treatment is urgent. The nucleocapsid protein (NP) of SARS-CoV-2 is one of the main structural proteins expressed and most abundant in the virus, and is considered a diagnostic marker for the accurate and sensitive detection of SARS-CoV-2. Herein, we report the screening of specific peptides from the pIII phage library that bind to SARS-CoV-2 NP. The phage monoclone expressing cyclic peptide N1 (peptide sequence, ACGTKPTKFC, with C&C bridged by disulfide bonding) specifically recognizes SARS-CoV-2 NP. Molecular docking studies reveal that the identified peptide is bound to the "pocket" region on the SARS-CoV-2 NP N-terminal domain mainly by forming a hydrogen bonding network and through hydrophobic interaction. Peptide N1 with the C-terminal linker was synthesized as the capture probe for SARS-CoV-2 NP in ELISA. The peptide-based ELISA was capable of assaying SARS-CoV-2 NP at concentrations as low as 61 pg/mL (∼1.2 pM). Furthermore, the as-proposed method could detect the SARS-CoV-2 virus at limits as low as 50 TCID50 (median tissue culture infective dose)/mL. This study demonstrates that selected peptides are powerful biomolecular tools for SARS-CoV-2 detection, providing a new and inexpensive method of rapidly screening infections as well as rapidly diagnosing coronavirus disease 2019 patients.

[Competitive ELISA test system for the detection of antibodies to the rabies virus in animals (Rhabdoviridae: Lyssavirus)].

INTRODUCTION: The main approach to the rabies prevention is the vaccination of domestic and wild carnivores. For the routine evaluation the anti-rabies vaccination effectiveness, World Organization for Animal Health (OIE) recommends various enzyme-linked immunosorbent assays (ELISA).The aim of the study was to design and validate a competitive ELISA (cELISA) test system for the detection of antibodies to the rabies virus (RABV). MATERIALS AND METHODS: The development of the cELISA was carried out following the OIE recommendations. RESULTS: The repeatability of the cELISA results within one laboratory was satisfactory (coefficient of variation 7.95-13.61%). The coefficient of determination (CD) between the results of the virus neutralization reaction (FAVN) and cELISA was 0.988, p < 0.001. The lower threshold for antibody detection was less than 0.02 IU/ml. The cELISA did not demonstrate cross-reactivity against antibodies to canine distemper virus, parainfluenza virus, parvovirus, coronavirus, and canine adenovirus (types I and II). During the study of 137 dog blood sera, diagnostic specificity (DSp) and diagnostic sensitivity (DSe) for the cELISA were 83.1% and 94.9%, respectively, and CD between the cELISA and FAVN results was 0.968, p < 0.001. DISCUSSION: Indirect ELISA test systems for determining the level of antibodies to RABV are not sensitive enough compared to reference tests, unlike cELISA. The developed test system is not inferior for its DSp and DSe to the commercial cELISA BioPro ELISA Rabies Ab (DSp 66.7%, DSe 94.4%). CONCLUSION: The developed cELISA test system can be used to detect antibodies to RABV in the blood serum of dogs for evaluating the effectiveness of mass vaccination programs.

Beads- and oil-free single molecule assay with immuno-rolling circle amplification for detection of SARS-CoV-2 from saliva.

Digital enzyme linked immunosorbent assays (ELISA) can be used to detect various antigens such as spike (S) or nucleocapsid (N) proteins of SARS-CoV-2, with much higher sensitivity compared to that achievable using conventional antigen tests. However, the use of microbeads and oil for compartmentalization in these assays limits their user-friendliness and causes loss of assay information due to the loss of beads during the process. To improve the sensitivity of antigen test, here, we developed an oil- and bead-free single molecule counting assay, with rolling circle amplification (RCA) on a substrate. With RCA, the signal is localized at the captured region of an antigen, and the signal from a single antigen molecule can be visualized using the same immune-reaction procedures as in the conventional ELISA. Substrate-based single molecule assay was theoretically evaluated for kd value, and the concentration of capture and detection antibodies. As a feasibility test, biotin-conjugated primer and mouse IgG conjugates were detected even at femto-molar concentrations with this digital immuno-RCA. Using this method, we detected the N protein of SARS-CoV-2 with a limit of detection less than 1 pg/mL more than 100-fold improvement compared to the detection using conventional ELISA. Furthermore, testing of saliva samples from COVID-19 patients and healthy controls (n = 50) indicated the applicability of the proposed method for detection of SARS-CoV-2 with 99.5% specificity and 90.9% sensitivity.

Development of an Indirect Enzyme-Linked Immunosorbent Assay Based on the Yeast-Expressed CO-26K-Equivalent Epitope-Containing Antigen for Detection of Serum Antibodies against Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea (PED) is a severe contagious intestinal disease caused by the porcine epidemic diarrhea virus (PEDV), which leads to high mortality in piglets. In this study, by analyzing a total of 53 full-length spike genes and COE domain regions of PEDVs, the conserved COE fragment of the spike protein from the dominant strain SC1402 was chosen as the target protein and expressed successfully in Pichia pastoris (P. pastoris). Furthermore, an indirect enzyme-linked immunosorbent assay (iELISA) based on the recombinant COE protein was developed for the detection of anti-PEDV antibodies in pig sera. The results showed that under the optimized conditions, the cut-off value of COE-based indirect ELISA (COE-iELISA) was determined to be 0.12. Taking the serum neutralization test as standard, the relative sensitivity of the COE-iELISA was 94.4% and specificity 92.6%. Meanwhile, no cross-reactivity to other porcine pathogens was noted with this assay. The intra-assay and inter-assay coefficients of variation were less than 7%. Moreover, 164 vaccinated serum samples test showed that overall agreement between COE-iELISA and the actual diagnosis result was up to 99.4%. More importantly, the developed iELISA exhibited a 95.08% agreement rate with the commercial ELISA kit (Kappa value = 0.88), which suggested that the expressed COE protein was an effective antigen in serologic tests and the established COE-iELISA is reliable for monitoring PEDV infection in pigs or vaccine effectiveness.

Capillary driven microfluidic sequential flow device for point-of-need ELISA: COVID-19 serology testing.

A capillary-driven microfluidic sequential flow device, designed for eventual at-home or doctor's office use, was developed to perform an enzyme-linked immunosorbent assay (ELISA) for serology assays. Serology assays that detect SARS-CoV-2 antibodies can be used to determine prior infection, immunity status, and/or individual vaccination status and are typically run using well-plate ELISAs in centralized laboratories, but in this format SARs-CoV-2 serology tests are too expensive and/or slow for most situations. Instead, a point-of-need device that can be used at home or in doctor's offices for COVID-19 serology testing would provide critical information for managing infections and determining immune status. Lateral flow assays are common and easy to use, but lack the sensitivity needed to reliably detect SARS-CoV-2 antibodies in clinical samples. This work describes a microfluidic sequential flow device that is as simple to use as a lateral flow assay, but as sensitive as a well-plate ELISA through sequential delivery of reagents to the detection area using only capillary flow. The device utilizes a network of microfluidic channels made of transparency film and double-sided adhesive combined with paper pumps to drive flow. The geometry of the channels and storage pads enables automated sequential washing and reagent addition steps with two simple end-user steps. An enzyme label and colorimetric substrate produce an amplified, visible signal for increased sensitivity, while the integrated washing steps decrease false positives and increase reproducibility. Naked-eye detection can be used for qualitative results or a smartphone camera for quantitative analysis. The device detected antibodies at 2.8 ng mL-1 from whole blood, while a well-plate ELISA using the same capture and detection antibodies could detect 1.2 ng mL-1. The performance of the capillary-driven immunoassay (CaDI) system developed here was confirmed by demonstrating SARS-CoV-2 antibody detection, and we believe that the device represents a fundamental step forward in equipment-free point-of-care technology.

Current methods for diagnosis of human coronaviruses: pros and cons.

The current global fight against coronavirus disease (COVID-19) to flatten the transmission curve is put forth by the World Health Organization (WHO) as there is no immediate diagnosis or cure for COVID-19 so far. In order to stop the spread, researchers worldwide are working around the clock aiming to develop reliable tools for early diagnosis of severe acute respiratory syndrome (SARS-CoV-2) understanding the infection path and mechanisms. Currently, nucleic acid-based molecular diagnosis (real-time reverse transcription polymerase chain reaction (RT-PCR) test) is considered the gold standard for early diagnosis of SARS-CoV-2. Antibody-based serology detection is ineffective for the purpose of early diagnosis, but a potential tool for serosurveys, providing people with immune certificates for clearance from COVID-19 infection. Meanwhile, there are various blooming methods developed these days. In this review, we summarise different types of coronavirus discovered which can be transmitted between human beings. Methods used for diagnosis of the discovered human coronavirus (SARS, MERS, COVID-19) including nucleic acid detection, gene sequencing, antibody detection, antigen detection, and clinical diagnosis are presented. Their merits, demerits and prospects are discussed which can help the researchers to develop new generation of advanced diagnostic tools for accurate and effective control of human coronavirus transmission in the communities and hospitals.

The detection of IgG class antibodies against SARS-CoV-2 nucleocapsid protein by application of nanoparticles.

OBJECTIVE: Enzyme-linked immunosorbent assay (ELISA) is a widely used biochemical analytical method for the detection of a biomarker, through a specific antigen-antibody reaction. A common with ELISA is the amount of concrete biomarkers falling below the detection limit. Thus, the approach that will contribute to enhanced sensitivity of enzyme-linked immunosorbent assay is of great importance for medical practice. To address this issue, we used nanoparticles to improve the detection limit of traditional ELISA. MATERIALS AND METHODS: 80 samples were used, for which the presence of IgG antibodies against SARS-CoV-2 nucleocapsid protein were already determined qualitatively.  We tested the samples using an in vitro ELISA kit [SARS-CoV-2 IgG ELISA, COVG0949 (NovaTec, Leinfelden-Echterdingen, Germany)]. Additionally, we tested the same sample with the same ELISA kit but with the addition of 50 nm diameter citrate-capped silver nanoparticles. The reaction was performed, and data were calculated according to manufacturer guidelines. To measure ELISA results absorbance (optical density - OD) at 450 nm was read. RESULTS: Greater absorbance values have been revealed in case of silver nanoparticles application (66 cases, 82.5%, p<0.05). ELISA with application of nanoparticles classified 19 equivocal cases as positive and 3 equivocal ones as negative, 1 negative case as equivocal. CONCLUSIONS: Our findings suggest that nanoparticles can be used to improve the sensitivity of ELISA method and increase the detection limit. Thus, it is logical and desirable to enhance the sensitivity of ELISA method by application of nanoparticles; the approach is low cost and with a positive impact on accuracy.

Enzyme-Linked Immunosorbent Assay: Types and Applications.

Enzyme-linked immunosorbent assay (ELISA) is an immunological assay widely used in basic science research, clinical application studies, and diagnostics. The ELISA technique relies on the interaction between the antigen (i.e., the target protein) versus the primary antibody against the antigen of interest. The presence of the antigen is confirmed through the enzyme-linked antibody catalysis of the added substrate, the products of which are either qualitatively detected by visual inspection or quantitatively using readouts from either a luminometer or a spectrophotometer. ELISA techniques are broadly classified into direct, indirect, sandwich, and competitive ELISA-all of which vary based on the antigens, antibodies, substrates, and experimental conditions. Direct ELISA relies on the binding of the enzyme-conjugated primary antibodies to the antigen-coated plates. Indirect ELISA introduces enzyme-linked secondary antibodies specific to the primary antibodies bound to the antigen-coated plates. Competitive ELISA involves a competition between the sample antigen and the plate-coated antigen for the primary antibody, followed by the binding of enzyme-linked secondary antibodies. Sandwich ELISA technique includes a sample antigen introduced to the antibody-precoated plate, followed by sequential binding of detection and enzyme-linked secondary antibodies to the recognition sites on the antigen. This review describes ELISA methodology, the types of ELISA, their advantages and disadvantages, and a listing of some multifaceted applications both in clinical and research settings, including screening for drug use, pregnancy testing, diagnosing disease, detecting biomarkers, blood typing, and detecting SARS-CoV-2 that causes coronavirus disease 2019 (COVID-19).

Process development and characterization of recombinant nucleocapsid protein for its application on COVID-19 diagnosis.

COVID-19 pandemic was caused by the severe acute respiratory syndrome coronavirus 2 (Sars-CoV-2). The nucleocapsid (N) protein from Sars-CoV-2 is a highly immunogenic antigen and responsible for genome packing. Serological assays are important tools to detect previous exposure to SARS-CoV-2, complement epidemiological studies, vaccine evaluation and also in COVID-19 surveillance. SARS-CoV-2 N (r2N) protein was produced in Escherichia coli, characterized, and the immunological performance was evaluated by enzyme-linked immunosorbent assay (ELISA) and beads-based array immunoassay. r2N protein oligomers were evidenced when it is associated to nucleic acid. Benzonase treatment reduced host nucleic acid associated to r2N protein, but crosslinking assay still demonstrates the presence of higher-order oligomers. Nevertheless, after RNase treatment the higher-order oligomers reduced, and dimer form increased, suggesting RNA contributes to the oligomer formation. Structural analysis revealed nucleic acid did not interfere with the thermal stability of the recombinant protein. Interestingly, nucleic acid was able to prevent r2N protein aggregation even with increasing temperature while the protein benzonase treated begin aggregation process above 55 °C. In immunological characterization, ELISA performed with 233 serum samples presented a sensitivity of 97.44% (95% Confidence Interval, CI, 91.04%, 99.69%) and a specificity of 98.71% (95% CI, 95.42%, 99.84%) while beads-based array immunoassay carried out with 217 samples showed 100% sensitivity and 98.6% specificity. The results exhibited an excellent immunological performance of r2N protein in serologic assays showing that, even in presence of nucleic acid, it can be used as a component of an immunoassay for the sensitive and specific detection of SARS-CoV-2 antibodies.

Development of an enzyme-linked immunosorbent assay based on viral antigen capture by anti-spike glycoprotein monoclonal antibody for detecting immunoglobulin A antibodies against porcine epidemic diarrhea virus in milk.

BACKGROUND: Porcine epidemic diarrhea (PED), caused by PED virus (PEDV), is a severe enteric disease burdening the global swine industry in recent years. Especially, the mortality of PED in neonatal piglets approaches 100%. Maternal antibodies in milk, particularly immunoglobulin A (IgA) antibodies, are of great importance for protection neonatal suckling piglets against PEDV infection as passive lactogenic immunity. Therefore, appropriate detection methods are required for detecting PEDV IgA antibodies in milk. In the current study, we prepared monoclonal antibodies (mAbs) against PEDV spike (S) glycoprotein. An enzyme-linked immunosorbent assay (ELISA) was subsequently developed based on PEDV antigen capture by a specific anti-S mAb. RESULTS: The developed ELISA showed high sensitivity (the maximum dilution of milk samples up to 1:1280) and repeatability (coefficient of variation values < 10%) in detecting PEDV IgA antibody positive and negative milk samples. More importantly, the developed ELISA showed a high coincidence rate with a commercial ELISA kit for PEDV IgA antibody detection in clinical milk samples. CONCLUSIONS: The developed ELISA in the current study is applicable for PEDV IgA antibody detection in milk samples, which is beneficial for evaluating vaccination efficacies and neonate immune status against the virus.

Lateral Flow Microarray-Based ELISA for Cytokines.

Cytokines are well known to be involved in numerous biological responses with diverse mechanisms of action, including the inflammatory process. The so-called "cytokine storm" has recently been associated with cases of severe COVID-19 infection.Lateral flow microarray (LFM) devices have been constructed for multiplex detection of cytokines. The LFM-cytokine rapid test involves the immobilization of an array of capture anti-cytokine antibodies. Here, we describe the methods to create and use multiplex lateral flow-based immunoassays based upon the enzyme-linked immunosorbent assay (ELISA).

A review post-vaccination SARS-CoV-2 serological test: Method and antibody titer response.

The development of the Coronavirus disease 2019 (COVID-19) vaccine is one of the most important efforts in controlling the pandemic. Serological tests are used to identify highly reactive human donors for convalescent plasma therapy, measuring vaccine efficacy and durability. This review article presents a review of serology tests and how antibody titers in response to vaccines have been developed. Some of the serological test methods discussed are Plaque Reduction Neutralization Test (PRNT), Enzyme-Linked Immunosorbent Assay (ELISA), Lateral flow immunoassay (LFIA), chemiluminescent immunoassay (CLIA), and Chemiluminescent Micro-particle Immunoassay (CMIA). This review can provide an understanding of the application of the body's immune response to vaccines to get some new strategies for vaccines.

Performance estimation of two in-house ELISA assays for COVID-19 surveillance through the combined detection of anti-SARS-CoV-2 IgA, IgM, and IgG immunoglobulin isotypes.

The main objective of this study was to estimate the performance, under local epidemiological conditions, of two in-house ELISA assays for the combined detection of anti-SARS-CoV-2 IgA, IgM, and IgG immunoglobulins. A total of 94 serum samples were used for the assessment, where 44 corresponded to sera collected before the pandemic (free of SARS-CoV-2 antibodies), and 50 sera were collected from confirmed COVID-19 patients admitted to the main public hospital in the city of Valdivia, southern Chile. The Nucleocapsid (Np) and the receptor-binding domain (RBD) proteins were separately used as antigens (Np and RBD ELISA, respectively) to assess their diagnostic performance. A receiver operating characteristic (ROC) analysis was performed to estimate the optical density (OD) cut-off that maximized the sensitivity (Se) and specificity (Sp) of the ELISA assays. Np ELISA had a mean Se of 94% (95% CI = 83.5-98.8%) and a mean Sp of 100% (95% CI = 92.0-100%), with an OD 450 nm positive cut-off value of 0.88. On the other hand, RBD ELISA presented a mean Se of 96% (95% CI = 86.3-99.5%) and a mean Sp of 90% (95% CI = 78.3-97.5%), with an OD 450 nm positive cut off value of 0.996. Non-significant differences were observed between the Se distributions of Np and RBD ELISAs, but the latter presented a significant lower Sp than Np ELISA. In parallel, collected sera were also analyzed using a commercial lateral flow chromatographic immunoassay (LFCI), to compare the performance of the in-house ELISA assays against a commercial test. The LFCI had a mean sensitivity of 94% (95% CI = 87.4-100%) and a mean specificity of 100% (95% CI = 100-100%). When compared to Np ELISA, non-significant differences were observed on the performance distributions. Conversely, RBD ELISA had a significant lower Sp than the LFCI. Although, Np ELISA presented a similar performance to the commercial test, this was 2.5 times cheaper than the LFCI assay (labor cost not considered). Thus, the in-house Np ELISA could be a suitable alternative tool, in resource limited environments, for the surveillance of SARS-CoV-2 infection, supporting further epidemiological studies.

Production of a Monoclonal Antibody to the Nucleocapsid Protein of SARS-CoV-2 and Its Application to ELISA-Based Detection Methods with Broad Specificity by Combined Use of Detector Antibodies.

The coronavirus disease 2019 pandemic, elicited by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is ongoing. Currently accessible antigen-detecting rapid diagnostic tests are limited by their low sensitivity and detection efficacy due to evolution of SARS-CoV-2 variants. Here, we produced and characterized an anti-SARS-CoV-2 nucleocapsid (N) protein-specific monoclonal antibody (mAb), 2A7H9. Monoclonal antibody 2A7H9 and a previously developed mAb, 1G10C4, have different specificities. The 2A7H9 mAb detected the N protein of S clade, delta, iota, and mu but not omicron, whereas the 1G10C4 antibody recognized the N protein of all variants under study. In a sandwich enzyme-linked immunosorbent assay, recombinant N protein bound to the 1G10C4 mAb could be detected by both 1G10C4 and 2A7H9 mAbs. Similarly, N protein bound to the 2A7H9 mAb was detected by both mAbs, confirming the existence of dimeric N protein. While the 1G10C4 mAb detected omicron and mu with higher efficiency than S clade, delta, and iota, the 2A7H9 mAb efficiently detected all the strains except omicron, with higher affinity to S clade and mu than others. Combined use of 1G10C4 and 2A7H9 mAb resulted in the detection of all the strains with considerable sensitivity, suggesting that antibody combinations can improve the simultaneous detection of virus variants. Therefore, our findings provide insights into the development and improvement of diagnostic tools with broader specificity and higher sensitivity to detect rapidly evolving SARS-CoV-2 variants.

Diagnostic performance of a novel antigen-capture ELISA for the detection of SARS-CoV-2.

BACKGROUND AND AIMS: The coronavirus disease 2019 (COVID-19) pandemic is a serious health problem worldwide. Early virus detection is essential for disease control and management. Viral antigen detection by ELISA is a cost-effective, rapid, and accurate antigen diagnostic assay which could facilitate early viral detection. METHOD: An antigen-capture sandwich ELISA was developed using novel nucleocapsid (NP)-specific mouse monoclonal antibodies (MAbs). The clinical performance of the assay was assessed using 403 positive and 150 negative respiratory samples collected during different SARS-CoV-2 variants outbreaks in Iran. RESULTS: The limit of detection of our ELISA assay was found to be 43.3 pg/ml for recombinant NP. The overall sensitivity and specificity of this assay were 70.72% (95% CI: 66.01-75.12) and 100% (95% CI: 97.57-100), respectively, regardless of Ct values and SARS-CoV-2 variants. There was no significant difference in our assay sensitivity for the detection of Omicron subvariants compared to Delta variant. Assay sensitivity for the BA.5 Omicron subvariant was calculated as 91.89% (95% CI: 85.17-96.23) for samples with Ct values < 25 and 82.70% (95% CI: 75.19-88.71) for samples with Ct values < 30. CONCLUSION: Our newly developed ELISA method is reasonably sensitive and highly specific for detection of SARS-CoV-2 regardless of the variants and subvariants of the virus.

3D-printed capillaric ELISA-on-a-chip with aliquoting.

Sandwich immunoassays such as the enzyme-linked immunosorbent assay (ELISA) have been miniaturized and performed in a lab-on-a-chip format, but the execution of the multiple assay steps typically requires a computer or complex peripherals. Recently, an ELISA for detecting antibodies was encoded structurally in a chip thanks to the microfluidic chain reaction (Yafia et al. Nature, 2022, 605, 464-469), but the need for precise pipetting and intolerance to commonly used surfactant concentrations limit the potential for broader adoption. Here, we introduce the ELISA-on-a-chip with aliquoting functionality that simplifies chip loading and pipetting, accommodates higher surfactant concentrations, includes barrier channels that delay the contact between solutions and prevent undesired mixing, and that executed a quantitative, high-sensitivity assay for the SARS-CoV-2 nucleocapsid protein in 4×-diluted saliva. Upon loading the chip using disposable pipettes, capillary flow draws each reagent and the sample into a separate volumetric measuring reservoir for detection antibody (70 µL), enzyme conjugate (50 µL), substrate (80 µL), and sample (210 µL), and splits washing buffer into 4 different reservoirs of 40, 40, 60, and 20 µL. The excess volume is autonomously drained via a structurally encoded capillaric aliquoting circuit, creating aliquots with an accuracy of >93%. Next, the user click-connects the assay module, comprising a nitrocellulose membrane with immobilized capture antibodies and a capillary pump, to the chip which triggers the step-by-step, timed flow of all aliquoted solutions to complete the assay in 1.5 h. A colored precipitate forming a line on a nitrocellulose strip serves as an assay readout, and upon digitization, yielded a binding curve with a limit of detection of 54 and 91 pg mL-1 for buffer and diluted saliva respectively, vastly outperforming rapid tests. The ELISA chip is 3D-printed, modular, adaptable to other targets and assays, and could be used to automate ELISA in the lab; or as a diagnostic test at the point of care with the convenience and form factor of rapid tests while preserving the protocol and performance of central laboratory ELISA.

Guest edited collection serological study of SARS-CoV-2 antibodies in japanese cats using protein-A/G-based ELISA.

BACKGROUND: Little is known about the epidemic status of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in cats in Japan due to insufficiently reliable seroepidemiological analysis methods that are easy to use in cats. RESULTS: We developed a protein-A/G-based enzyme-linked immunosorbent assay (ELISA) to detect antibodies against SARS-CoV-2 in cats. The assay was standardized using positive rabbit antibodies against SARS-CoV-2. The ELISA results were consistent with those of a conventional anti-feline-immunoglobulin-G (IgG)-based ELISA. To test the protein-A/G-based ELISA, we collected blood samples from 1,969 cats that had been taken to veterinary clinics in Japan from June to July 2020 and determined the presence of anti-SARS-CoV-2 antibodies. Nine cats were found to have SARS-CoV-2 S1-specific IgG, of which 4 had recombinant receptor-binding domain-specific IgG. Of those 9 samples, one showed neutralizing activity. Based on these findings, we estimated that the prevalence of SARS-CoV-2 neutralizing antibodies in cats in Japan was 0.05% (1/1,969 samples). This prevalence was consistent with the prevalence of neutralizing antibodies against SARS-CoV-2 in humans in Japan according to research conducted at that time. CONCLUSIONS: Protein-A/G-based ELISA has the potential to be a standardized method for measuring anti-SARS-CoV-2 antibodies in cats. The infection status of SARS-CoV-2 in cats in Japan might be linked to that in humans.