Production of antigens expressed in Nicotiana benthamiana plant and Escherichia coli for the SARS-CoV-2 IgG antibody detection by ELISA.

The recent COVID-19 pandemic disclosed a critical shortage of diagnostic kits worldwide, emphasizing the urgency of utilizing all resources available for the development and production of diagnostic tests. Different heterologous protein expression systems can be employed for antigen production. This study assessed novel SARS-CoV-2 proteins produced by a transient expression system in Nicotiana benthamiana utilizing an infectious clone vector based on pepper ringspot virus (PepRSV). These proteins included the truncated S1-N protein (spike protein N-terminus residues 12-316) and antigen N (nucleocapsid residues 37-402). Two other distinct SARS-CoV-2 antigens expressed in Escherichia coli were evaluated: QCoV9 chimeric antigen protein (spike protein residues 449-711 and nucleocapsid protein residues 160-406) and QCoV7 truncated antigen (nucleocapsid residues 37-402). ELISAs using the four antigens individually and the same panel of samples were performed for the detection of anti-SARS-CoV-2 IgG antibodies. Sensitivity was evaluated using 816 samples from 351 COVID-19 patients hospitalized between 5 and 65 days after symptoms onset; specificity was tested using 195 samples collected before 2018, from domiciliary contacts of leprosy patients. Our findings demonstrated consistent test sensitivity, ranging from 85 % to 88 % with specificity of 97.5 %, regardless of the SARS-CoV2 antigen and the expression system used for production. Our results highlight the potential of plant expression systems as useful alternative platforms to produce recombinant antigens and for the development of diagnostic tests, particularly in resource-constrained settings.

An indirect ELISA for detecting anti-SARS-CoV-2 antibodies in human sera using a baculovirus-expressed recombinant nucleocapsid antigen.

This study focuses on the development and initial assessment of an indirect IgG enzyme-linked immunosorbent assay (ELISA) specifically designed to detect of anti-SARS-CoV-2 antibodies. The unique aspect of this ELISA method lies in its utilization of a recombinant nucleocapsid (N) antigen, produced through baculovirus expression in insect cells. Our analysis involved 292 RT-qPCR confirmed positive serum samples and 54 pre-pandemic healthy controls. The process encompassed cloning, expression, and purification of the SARS-CoV-2 N gene in insect cells, with the resulted purified protein employed in our ELISA tests. Statistical analysis yielded an Area Under the Curve of 0.979, and the optimized cut-off exhibited 92 % sensitivity and 94 % specificity. These results highlight the ELISA's potential for robust and reliable serological detection of SARS-CoV-2 antibodies. Further assessments, including a larger panel size, reproducibility tests, and application in diverse populations, could enhance its utility as a valuable biotechnological solution for diseases surveillance.

Development and evaluation of a Lateral flow immunoassay (LFIA) prototype for the detection of IgG anti-SARS-CoV-2 antibodies.

Lateral flow immunoassays (LFIA) for antibody detection represent cost-effective and user-friendly tools for serology assessment. This study evaluated a new LFIA prototype developed with a recombinant chimeric antigen from the spike/S and nucleocapsid/N proteins to detect anti-SARS-CoV-2 IgG antibodies. The evaluation of LFIA sensitivity and specificity used 811 serum samples from 349 hospitalized, SARS-CoV-2 RT-qPCR positive COVID-19 patients, collected at different time points and 193 serum samples from healthy controls. The agreement between ELISA results with the S/N chimeric antigen and LFIA results was calculated. The LFIA prototype for SARS-CoV-2 using the chimeric S/N protein demonstrated 85 % sensitivity on the first week post symptoms onset, reaching 94 % in samples collected at the fourth week of disease. The agreement between LFIA and ELISA with the same antigen was 92.7 %, 0.827 kappa Cohen value (95 % CI [0.765-0.889]). Further improvements are needed to standardize the prototype for whole blood use. The inclusion of the novel chimeric S + N antigen in the COVID-19 IgG antibody LFIA demonstrated optimal agreement with results from a comparable ELISA, highlighting the prototype's potential for accurate large-scale serologic assessments in the field in a rapid and user-friendly format.

Challenges and advances in serological and molecular tests to aid leprosy diagnosis.

Leprosy is a neglected chronic infectious disease caused by obligate intracellular bacilli, Mycobacterium leprae and Mycobacterium lepromatosis. Despite multidrug therapy (MDT) success, leprosy accounts for more than 200,000 new cases yearly. Leprosy diagnosis remains based on the dermato-neurologic examination, but histopathology of skin biopsy and bacilloscopy of intradermal scraping are subsidiary diagnostic tests that require expertise and laboratory infrastructure. This minireview summarizes the state of the art of serologic tests to aid leprosy diagnosis, highlighting enzyme-linked immunosorbent assay (ELISA) and point-of-care tests (POCT) biotechnologies. Also, the impact of the postgenomic era on the description of new recombinantly expressed M. leprae-specific protein antigens, such as leprosy Infectious Disease Research Institute (IDRI) diagnostic (LID)-1 is summarized. Highly specific and sensitive molecular techniques to detect M. leprae DNA as the quantitative polymerase chain reaction (qPCR) and the loop-mediated isothermal amplification (LAMP) are briefly reviewed. Serology studies using phenolic glycolipid-I (PGL-I) semi-synthetic antigens, LID-1 fusion antigen, and the single fusion complex natural disaccharide-octyl (NDO)-LID show high sensitivity in multibacillary (MB) patients. However, serology is not applicable to paucibacillary patients, as they have weak humoral response and robust cell-mediated response, requiring tests for cellular biomarkers. Unlike ELISA-based tests, leprosy-specific POCT based on semi-synthetic PGL-I antigens and NDO-LID 1 antigen is easy to perform, cheaper, equipment-free, and can contribute to early diagnosis avoiding permanent incapacities and helping to interrupt M. leprae transmission. Besides its use to help diagnosis of household contacts or at-risk populations in endemic areas, potential applications of leprosy serology include monitoring MDT efficacy, identification of recent infection, especially in young children, as surrogate markers of disease progression to orient adult chemoprophylaxis and as a predictor of type 2 leprosy reactions. Advances in molecular biology techniques have reduced the complexity and execution time of qPCR confirming its utility to help diagnosis while leprosy-specific LAMP holds promise as an adjunct test to detect M. leprae DNA.

A novel highly specific biotinylated MAC-ELISA for detection of anti-SARS-CoV-2 nucleocapsid antigen IgM antibodies during the acute phase of COVID-19.

The gold standard for diagnosing COVID-19 in the acute phase is RT-qPCR. However, this molecular technique can yield false-negative results when nasopharyngeal swab collection is not conducted during viremia. To mitigate this challenge, the enzyme-linked immunosorbent assay (ELISA) identifies anti-SARS-CoV-2 IgM antibodies in the initial weeks after symptom onset, facilitating early COVID-19 diagnosis. This study introduces a novel and highly specific IgM antibody capture ELISA (MAC-ELISA), which utilizes biotinylated recombinant SARS-CoV-2 nucleocapsid (N) antigen produced in plants. Our biotinylated approach streamlines the procedure by eliminating the requirement for an anti-N-conjugated antibody, circumventing the need for peroxidase-labeled antigens, and preventing cross-reactivity with IgM autoantibodies such as rheumatoid factor. Performance evaluation of the assay involved assessing sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy using 682 RT-qPCR-positive samples, categorized by weeks relative to symptoms onset. Negative controls included 205 pre-pandemic serum samples and 46 serum samples from patients diagnosed with other diseases. Based on a cut-off of 0.087 and ROC curve analysis, the highest sensitivity of 81.2% was observed in the 8-14 days post-symptom (dps) group (2nd week), followed by sensitivities of 73.8% and 68.37% for the 1-7 dps (1st week) and 15-21 dps groups (3rd week), respectively. Specificity was consistently 100% across all groups. This newly developed biotinylated N-MAC-ELISA offers a more streamlined and cost-effective alternative to molecular diagnostics. It enables simultaneous testing of multiple samples and effectively identifies individuals with false-negative results.

Practical use of tobravirus-based vector to produce SARS-CoV-2 antigens in plants.

A plant-based heterologous expression system is an attractive option for recombinant protein production because it is based on a eukaryotic system of high feasibility, and low biological risks. Frequently, binary vector systems are used for transient gene-expression in plants. However, plant virus vector-based systems offer advantages for higher protein yields due to their self-replicating machinery. In the present study, we show an efficient protocol using a plant virus vector based on a tobravirus, pepper ringspot virus, that was employed for transient expression of severe acute respiratory syndrome coronavirus 2 partial gene fragments of the spike (named S1-N) and the nucleocapsid (named N) proteins in Nicotiana benthamiana plants. Purified proteins yield of 40-60 µg/g of fresh leaves were obtained. Both proteins, S1-N and N, showed high and specific reactivities against convalescent patients' sera by the enzyme-linked immunosorbent assay format. The advantages and critical points in using this plant virus vector are discussed.

Antibody- and nucleic acid-based lateral flow immunoassay for Listeria monocytogenes detection.

Listeria monocytogenes is an invasive opportunistic foodborne pathogen and its routine surveillance is critical for protecting the food supply and public health. The traditional detection methods are time-consuming and require trained personnel. Lateral flow immunoassay (LFIA), on the other hand, is an easy-to-perform, rapid point-of-care test and has been widely used as an inexpensive surveillance tool. In recent times, nucleic acid-based lateral flow immunoassays (NALFIA) are also developed to improve sensitivity and specificity. A significant improvement in lateral flow-based assays has been reported in recent years, especially the ligands (antibodies, nucleic acids, aptamers, bacteriophage), labeling molecules, and overall assay configurations to improve detection sensitivity, specificity, and automated interpretation of results. In most commercial applications, LFIA has been used with enriched food/environmental samples to ensure detection of live cells thus prolonging the assay time to 24-48 h; however, with the recent improvement in LFIA sensitivity, results can be obtained in less than 8 h with shortened and improved enrichment practices. Incorporation of surface-enhanced Raman spectroscopy and/or immunomagnetic separation could significantly improve LFIA sensitivity for near-real-time point-of-care detection of L. monocytogenes for food safety and public health applications.