A urine-based ELISA with recombinant non-glycosylated SARS-CoV-2 spike protein for detecting anti-SARS-CoV-2 spike antibodies

Abstract

Serological assays have been widely used to detect anti-SARS-CoV-2 antibodies, which are generated from previous exposure to the virus or after vaccination. The presence of anti-SARS-CoV-2 Nucleocapsid antibodies was recently reported in patients´ urine using an in-house urine-based ELISA-platform, allowing a non-invasive way to collect clinical samples and assess immune conversion. In the current study, we evaluated and validated another in-house urine-based ELISA for the detection of anti-SARS-CoV-2 Spike antibodies. Three partial recombinant SARS-CoV-2 Spike proteins comprising the Receptor Binding Domain, expressed in eukaryotic or prokaryotic systems, were tested in an ELISA platform against a panel of over 140 urine and paired serum samples collected from 106 patients confirmed positive for SARS-CoV-2 by qRT-PCR. The key findings from our study were that anti-SARS-CoV-2 Spike antibodies could be detected in urine samples and that the prokaryotic expression of the rSARS-CoV-2 Spike protein was not a barrier to obtain relatively high serology efficiency for the urine-based assay. Thus, use of a urine-based ELISA assay with partial rSARS-CoV-2 Spike proteins, expressed in a prokaryotic system, could be considered as a convenient tool for screening for the presence of anti-SARS-CoV-2 Spike antibodies, and overcome the difficulties arising from sample collection and the need for recombinant proteins produced with eukaryotic expression systems.

B-Cell Epitopes-Based Chimeric Protein from SARS-CoV-2 N and S Proteins Is Recognized by Specific Antibodies in Serum and Urine Samples from Patients

“…Differing from the standard serum-based assay, the prokaryotic expression of the rSARS-CoV-2 S protein was not a barrier to obtain relatively high efficiency for the urine-based ELISA. In this context, the SARS-CoV-2 S protein expressed in the prokaryote system is still not suitable for the detection of anti-SARS-CoV-2 antibodies in sera samples [ 11 ]. In this sense, the N4S11-SC2 protein has demonstrated its potential as an alternative recombinant protein expressed in a prokaryotic system, showing the capability to recognize antibodies against N and S proteins of SARS-CoV-2 in serum or urine samples.…”

“…However, we observed no humoral reactivity in serum samples from individuals collected before 2019, sounding that the N4S11-SC2 protein may be specific for SARS-CoV-2 in our in-house ELISA. These same control samples were previously tested using N and S antigens with results of high specificity [ 11 , 12 ]. The patients enrolled in our study had not received any vaccinations at the time of sample collection, so it can be reasonably inferred that the antibodies detected were predominantly produced in response to exposure to the virus.…”

“…The prokaryotic recombinant proteins, N and S (Prok2-S1) were validated by Ludolf et al [ 12 ] and Ramos et al [ 11 ]. These antigens were used as controls to compare the performance of chimeric protein by using serum and urine samples ( n = 72 each), which were obtained after eight days PSO.…”

“…Although considered less invasive than swabs, the sample’s collection presents a significant rate of complication, since it can be unpleasant and requires a trained phlebotomist. By contrast, the collection of urine to detect specific antibodies is less costly and samples are easy to store and thus could be convenient for clinical and epidemiological studies [ 11 ]. Urine-based tests to detect antibodies have been recently suggested as a non-invasive, simple and safe alternative to detect anti-SARS-CoV-2 N and S antibodies.…”

“…Meanwhile, the same assay using the S protein purified from a prokaryotic system showed sensitivity and specificity of 89.0% and 97.0%, respectively. Interestingly, when sera samples collected from the same patients were tested against the prokaryotic S antigen, results showed sensitivity of 40.0% and specificity of 98.0%, highlighting the difficulty to obtain antigens with high accuracy derived of prokaryotic systems of purification [ 11 , 12 ].…”