Coupled beads corresponded to bead regions 18 (S), 33 (RBD), 73 (NP), 45 (internal control IC45), and 66 (human IgG). Beads were also coated with purified human IgG (catalog no. The S protein was coupled at 10 pM, while the NP and RBD were coupled at 100 pM (i.e., equimolar coupling). Protein coupling to magnetic microsphere beads was performed according to the manufacturer’s recommendations using an xMAP antibody coupling (AbC) kit (Luminex, Austin, TX). Multiplex SARS-CoV-2 IgG microsphere immunoassay development. Where possible, RT-PCR cycle threshold ( C T) values from multiple RT-PCR tests were obtained and assessed alongside the serological response for the patients included in this study. Thus, values representing days from symptom onset were calculated using the collection date for each serum sample. Given the need to assess for the serological response within a known time frame (days from symptom onset), all patients in this study had symptomatic manifestations of COVID-19. Information collected included patient demographics (sex and age), clinical course (including the estimated date of symptom onset, days of hospitalization, intensive care unit admission, and death), reported symptoms (including fever, cough, and shortness of breath), and comorbidities (including history of smoking, coronary artery disease, chronic obstructive pulmonary disease, and diabetes). Patient charts were reviewed by at least two people from a four-person team, and discrepancies were resolved by a third person. Taking the data together, this study described the performance of an assay built on a flexible and high-throughput serological platform that proved adaptable to the emergence of a novel infectious agent. Using soluble ACE2, inhibition of antibody binding was demonstrated for S and RBD, and not for NP. Considerable interperson variation was observed with a subset of extensively sampled intensive care unit (ICU) patients. This revealed the relative rise, peak (S, 23.8 RBD, 23.6 NP, 16.7 ), and decline of the antibody response.
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A larger collection ( n = 534) of discarded sera was profiled from patients ( n = 140) whose COVID-19 course was characterized through chart review.
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With samples obtained at ≤5 days from symptom onset, the 3Flex assay was more sensitive (48.0% versus 32.0%), but the two assays performed comparably using serum obtained ≥21 days from symptom onset. Sensitivity was measured and compared to that of the Abbott Architect SARS-CoV-2 IgG assay using serum samples from 125 unique patients equally binned ( n = 25) into 5 time intervals (≤5, 6 to 10, 11 to 15, 16 to 20, and ≥21 days from symptom onset). Specificity was assessed using 213 prepandemic samples. Here, we describe a multiplex fluorescent microsphere-based assay, 3Flex, that can detect antibodies to three major SARS-CoV-2 antigens-spike (S) protein, the spike ACE2 receptor-binding domain (RBD), and nucleocapsid (NP). Serological tests can be used diagnostically and for surveillance, but their usefulness depends on their throughput, sensitivity, and specificity. The coronavirus disease 2019 (COVID-19) pandemic has highlighted the challenges inherent to the serological detection of a novel pathogen such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).