key: cord-262602-on0w55x0
authors: Muruato, Antonio E.; Fontes-Garfias, Camila R.; Ren, Ping; Garcia-Blanco, Mariano A.; Menachery, Vineet D.; Xie, Xuping; Shi, Pei-Yong
title: A high-throughput neutralizing antibody assay for COVID-19 diagnosis and vaccine evaluation
date: 2020-05-22
journal: bioRxiv
DOI: 10.1101/2020.05.21.109546
sha: 
doc_id: 262602
cord_uid: on0w55x0

Virus neutralization remains the gold standard for determining antibody efficacy. Therefore, a high-throughput assay to measure SARS-CoV-2 neutralizing antibodies is urgently needed for COVID-19 serodiagnosis, convalescent plasma therapy, and vaccine development. Here we report on a fluorescence-based SARS-CoV-2 neutralization assay that detects SARS-CoV-2 neutralizing antibodies in COVID-19 patient specimens and yields comparable results to plaque reduction neutralizing assay, the gold standard of serological testing. Our approach offers a rapid platform that can be scaled to screen people for antibody protection from COVID-19, a key parameter necessary to safely reopen local communities.

infection. An ideal serological assay should measure neutralizing antibody levels, which should 3 9 predict protection from reinfection. Conventionally, neutralizing antibodies are measured by 4 0 plaque reduction neutralization test (PRNT). Although PRNT and ELISA results generally 4 1 corelate with each other, the lack of complete fidelity of ELISA continues to make PRNT the 4 2 gold-standard for determining immune protection 6,7 . However, due to its low throughput, PRNT 4 3 is not practical for large scale serodiagnosis and vaccine evaluation. This is a major gap for 4 4 COVID-19 surveillance and vaccine development.

To address the above gap, we developed a fluorescence-based assay that rapidly and using a high-content imaging reader (Fig. 1a) . Forty COVID-19 serum specimens from RT-PCR-5 4 confirmed patients and ten non-COVID-19 serum samples (archived before COVID-19 5 5 emergence) were analyzed using the reporter virus. After reporter viral infection, the cells turned serum dilution (Fig. 1c) , which allowed for determination of the dilution fold that neutralized 50% 6 0 of fluorescent cells (NT 50 ). The reporter assay rapidly diagnosed fifty specimens in less than 20 6 1 h: all forty COVID-19 sera (specimens 1-40) showed positive NT 50 of 80 to 5152, and all ten 6 2 non-COVID-19 sera (specimens 41-50) showed negative NT 50 of <20 for (Fig. 1d) .

To validate the reporter virus neutralization results, we performed the conventional 6 4 PRNT on the same set of patient specimens. In agreement with the reporter virus results, the 6 5 forty positive sera showed PRNT 50 of 40 to 3200, and the ten negative sera exhibited PRNT 50 of 6 6 <20 (Fig. 1d) . A strong correlation was observed between the reporter virus and PRNT results, 6 7 with a correlation efficiency R 2 of 0.9 (Fig. 1e) . The results demonstrate that when diagnosing 6 8 patient specimens, the reporter virus assay delivers neutralization results comparable to the 6 9 PRNT assay, the gold standard of serological testing.

Next, we evaluated the specificity of reporter neutralization assay using potentially cross-7 1 reactive sera and interfering substances (Table 1) . Two groups of specimens were tested for SARS-CoV-2 so that the assay could be performed at a BSL2 facility. Nevertheless, the 1 0 3 mNeonGreen reporter assay offers a rapid, high-throughput platform to test COVID-19 patient 1 0 4 sera not previously available.

Because neutralizing titer is a key parameter to predict immunity, the reporter 

Human sera and interfering substances. All suman serum specimens were obtained 1 7 7

at the University of Texas Medical Branch (UTMB). All specimens were de-identified from 1 7 8 patient information. A total of forty de-identified convalescent sera from COVID-19 patients 1 7 9

(confirmed with viral RT-PCR positive) were tested in this study. Ten non-COVID-19 sera, 1 8 0 collected before COVID-19 emergence 12,13 , were also tested in the reporter virus and PRNT 1 8 1 assays. For testing cross reactivity, a total of 138 de-identified specimens from patients with 1 8 2 antigens or antibodies against different viruses, bacteria, and parasites were tested in the 1 8 3 mNeonGreen SARS-COV-2 neutralization assay (Table 1) . For testing interfering substances, 1 8 4 nineteen de-identified serum specimens with albumin, elevated bilirubin, cholesterol, rheumatoid 1 8 5 factor, and autoimmune nuclear antibodies were tested in the reporter neutralization assay. All 1 8 6 human sera were heat-inactivated at 56°C for 30 min before testing. 

Severe Acute Respiratory Syndrome Coronavirus 2-Specific

(P/S; Gibco) were seeded in each well of black µCLEAR flat-bottom 96-well plate (Greiner Bio-1 9 0 one™). The cells were incubated overnight at 37°C with 5% CO 2 . On the following day, each 1 9 1 serum was 2-fold serially diluted in 2% FBS and 100 U/ml P/S DMEM, and incubated with Balanced Salt Solution; Gibco) were added to each well to stain cell nucleus. The plate was 1 9 7 sealed with Breath-Easy sealing membrane (Diversified Biotech), incubated at 37°C for 20 min, 1 9 8 and quantified for mNeonGreen fluorescence on Cytation TM 7 (BioTek). The raw images (2×2 1 9 9 montage) were acquired using 4× objective, processed, and stitched using the default setting.

UTMB has filed a patent on the reverse genetic system and reporter SARS-CoV-2.