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Anal Biochem ; 631: 114360, 2021 10 15.
Article in English | MEDLINE | ID: covidwho-1474246


To monitor the levels of protecting antibodies raised in the population in response to infection and/or to immunization with SARS-CoV-2, we need a technique that allows high throughput and low-cost quantitative analysis of human IgG antibodies reactive against viral antigens. Here we describe an ultra-fast, high throughput and inexpensive assay to detect SARS-CoV-2 seroconversion in humans. The assay is based on Ni2+ magnetic particles coated with His tagged SARS-CoV-2 antigens. A simple and inexpensive 96 well plate magnetic extraction/homogenization process is described which allows the simultaneous analysis of 96 samples and delivers results in 7 min with high accuracy.

Antibodies, Viral/blood , COVID-19 Serological Testing/methods , COVID-19/diagnosis , Immunoglobulin G/blood , SARS-CoV-2/isolation & purification , Antibodies, Viral/immunology , Antigens, Viral/blood , Antigens, Viral/immunology , COVID-19/blood , COVID-19/immunology , COVID-19 Serological Testing/economics , Enzyme-Linked Immunosorbent Assay/economics , Enzyme-Linked Immunosorbent Assay/methods , Humans , Immunoglobulin G/immunology , Magnets/chemistry , Nickel/chemistry , SARS-CoV-2/immunology , Sensitivity and Specificity , Seroconversion , Time Factors
Braz J Microbiol ; 52(4): 2069-2073, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1338313


Serological assays are important tools to identify previous exposure to SARS-CoV-2, helping to track COVID-19 cases and determine the level of humoral response to SARS-CoV-2 infections and/or immunization to future vaccines. Here, the SARS-CoV-2 nucleocapsid protein was expressed in Escherichia coli and purified to homogeneity and high yield using a single chromatography step. The purified SARS-CoV-2 nucleocapsid protein was used to develop an indirect enzyme-linked immunosorbent assay for the identification of human SARS-CoV-2 seroconverts. The assay sensitivity and specificity were determined analyzing sera from 140 RT-qPCR-confirmed COVID-19 cases and 210 pre-pandemic controls. The assay operated with 90% sensitivity and 98% specificity; identical accuracies were obtained in head-to-head comparison with a commercial ELISA kit. Antigen-coated plates were stable for up to 3 months at 4 °C. The ELISA method described is ready for mass production and will be an additional tool to track COVID-19 cases.

COVID-19 , Coronavirus Nucleocapsid Proteins/immunology , Enzyme-Linked Immunosorbent Assay , Seroconversion , Antibodies, Viral/blood , COVID-19/diagnosis , COVID-19/immunology , Humans , Immunity, Humoral , Nucleocapsid Proteins/genetics , Phosphoproteins/immunology , Sensitivity and Specificity
ACS Sens ; 6(3): 703-708, 2021 03 26.
Article in English | MEDLINE | ID: covidwho-1047926


Immunological methods to detect SARS-CoV-2 seroconversion in humans are important to track COVID-19 cases and the humoral response to SARS-CoV-2 infections and immunization to future vaccines. The aim of this work was to develop a simple chromogenic magnetic bead-based immunoassay which allows rapid, inexpensive, and quantitative detection of human antibodies against SARS-CoV-2 in serum, plasma, or blood. Recombinant 6xHis-tagged SARS-CoV-2 Nucleocapsid protein was mobilized on the surface of Ni2+ magnetic beads and challenged with serum or blood samples obtained from controls or COVID-19 cases. The beads were washed, incubated with anti-human IgG-HPR conjugate, and immersed into a solution containing a chromogenic HPR substrate. Bead transfer and homogenization between solutions was aided by a simple low-cost device. The method was validated by two independent laboratories, and the performance to detect SARS-CoV-2 seroconversion in humans was in the same range as obtained using the gold standard immunoassays ELISA and Luminex, though requiring only a fraction of consumables, instrumentation, time to deliver results, and volume of sample. Furthermore, the results obtained with the method described can be visually interpreted without compromising accuracy as demonstrated by validation at a point-of-care unit. The magnetic bead immunoassay throughput can be customized on demand and is readily adapted to be used with any other 6xHis tagged protein or peptide as antigen to track other diseases.

Antibodies, Viral/blood , COVID-19 Serological Testing , COVID-19/diagnosis , Enzyme-Linked Immunosorbent Assay/methods , SARS-CoV-2/immunology , COVID-19/blood , COVID-19/immunology , Humans , Immunoglobulin G/immunology , Magnetic Phenomena