key: cord-0816958-v2yt0715
authors: Sil, B. K.; Oishee, M. J.; Haq, M. A.; Jahan, N.; Ali, T.; Khandker, S. S.; Kobatake, E.; Mie, M.; Khondoker, M. U.; Jamiruddin, M. R.; Adnan, N.
title: Development And Performance Evaluation of A Rapid In-House ELISA for Retrospective Serosurveillance of SARS-CoV-2
date: 2020-12-11
journal: nan
DOI: 10.1101/2020.12.10.20244350
sha: 0e6eacd3320038a63b98044407d919b781fda50d
doc_id: 816958
cord_uid: v2yt0715

Background In the ongoing pandemic situation of COVID-19, serological tests can complement the molecular diagnostic methods, and can be one of the important tools of sero-surveillance and vaccine evaluation. Aim To develop and evaluate a rapid SARS-CoV-2 specific IgG ELISA with increased sensitivity and specificity. Methods In order to develop the ELISA, three panels of samples (n=184) have been used: panel 1 (n=19) and panel 2 (n=60) were collected from RT-PCR positive patients within 14 and after 14 days respectively following the onset of clinical signs of disease whereas panel 3 consisted of negative samples (n=105) collected either from healthy donors during pre-pandemic era, pandemic era or from RT-PCR negative healthy individuals. As a capturing agent full-length SARS-CoV-2 specific recombinant nucleocapsid was immobilized and blocked using blocking agent. In total of 30 samples from the panels have been tested with Elecsys Anti-SARS-CoV-2 for selecting positive and negative controls, as well as comparator assay. The threshold cut-off point, inter-assay and intra assay variations were determined. Results The assay time was set at a total of 30 minutes with the sensitivity of 84% (95% confidence interval, CI, 60.4%, 96.6%) and 98% (95% CI, 91.1%, 100.0%), for panel 1 and 2 respectively, with overall 94.9% sensitivity (95% CI 87.5%, 98.6%). Moreover, the clinical specificity is 97.1% (95% CI, 91.9%, 99.4%) with no cross-reaction with dengue sample. The overall positive and negative predictive values are 96.2% (95% CI 89.2%, 99.2%) and 96.2% (95% CI, 90.6% 99.0%) respectively. In-house ELISA demonstrated 100% positive and negative percent agreement with ROCHE (Elecsys; Anti-SARS-CoV-2), with a Cohens kappa value of 1.00 (very strong agreement), while comparing 13 positive and 17 negative confirmed cases. Conclusion The assay is rapid and can be applied as one of the early and retrospective sero-monitoring tools in all over the affected areas.

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The copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101 https://doi.org/10. /2020 Introduction 55 The current situation of the world is all about the war between the visible and invisible. Life has 56 to adopt a new normality due to the advent of an acute respiratory disease, . The 57 disease, emerged in December 2019 in China, has been evolved as a public health threat due to its 58 global spread (2), morbidity and mortality rate. The etiological agent of this is SARS-CoV-2, a 59 positive strand RNA virus, belonging to the beta-coronavirus family (3). The disease presents an 60 unprecedented spectrum in clinical manifestations ranging from asymptomatic, mild or quasi-61 common-cold symptoms to severe complications requiring immediate medical intervention (4-6).

Droplet, airborne, orofecal and fomite transmission of this virus as well as direct contact with 63 symptomatic and asymptomatic individuals contribute to the rampant spread of the disease (5, 7).

The havoc brought down by the pandemic demands early diagnosis during the acute phase of 65 infection. Viral RNA detection by real time RT-PCR is the gold standard for early diagnosis (8) . 66 This immediate step identifies acute illness facilitating disease management and restricting rapid 67 spread to some extent. However, collection of samples from suitable sites, sample transportation, 68 the constraints of efficient and trained personnel as well as well-equipped facilities and increased 69 false negative results of RT-PCR at later phase of infection disqualifies its sole implementation in 70 the field of SARS-CoV2 diagnostics (9, 10). Although other molecular based methods such as 71 isothermal amplification techniques or CRISPR-based technology are implemented and suggested, 72 are yet to be well-practised considering the cost-effectiveness (11, 12) . 73 Though serological tests are not yet suggested for case detection by World Health Organization, 74 in order to reveal the scenario of the prevalent and past episodes, serological assay has to be of 75 prime importance (13, 14) . Retrospective serosurveillance, not only enlightens with current 76 immune status of the exposed individuals, but also facilitates therapeutic action by selecting 77 . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101 https://doi.org/10. /2020 5 convalescent plasma donor as well as to study the plausible outcome from the vaccine shoot 78 focused on neutralizing antibody (15). Mass screening of a population is required to move towards 79 relaxing COVID-19 restrictions. Decisions like 'back to the work' and school require timely 80 seroprevalence study. All these aspects necessitate highly sensitive and specific immunoassay (16, 81 17).

The key structural proteins of SARS-CoV2 include Nuclecapsid protein (NCP), Spike (S), Envelop 

The nucleoprotein of SARS-CoV-2 is highly immunogenic and detection of antibody against this 88 protein is found to be more sensitive compared to Spike (S) or RBD (20, 21). This study 89 characterizes an in-house ELISA targeting IgG antibody against full-length SARS-CoV-2 90 nucleocapsid protein (NCP). To our knowledge, it is the first indigenous ELISA that is rapid with 91 thirty minutes incubation time and possessing higher sensitivity and specificity. Three panels 92 comprising of a total of 184 samples have been considered for the development of this ELISA and 93 a comparative study has been done with Elecsys Anti-SARS-CoV-2 which is based on 94 chemiluminescent assay.

Recombinant nucleocapsid protein specific to SARS-CoV-2 was obtained from Sino Biologicals, 99 China, and used as capturing agent. Goat anti-human IgG conjugated with HRP (Native Antigen, 100 . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101/2020.12.10.20244350 doi: medRxiv preprint 6 UK) was used to detect human IgG which formed an immune-complex with coated SARS-CoV-2 101 specific antigen. 3,3′,5,5′-Tetramethylbenzidine (TMB) (Dojindo Molecular Technologies, USA) 102 was used as colour developer suitable for peroxidase substrate (Wako, Japan) while 1.5 M H2SO4 103 (Sigma-Aldrich, Germany) was used to stop the colour developed by TMB-peroxidase and read at 104 450 nm using ELISA plate reader (Thermo-Fisher Scientific, USA).

Sample selection and panel composition 106 The assay was developed and evaluated using panels of serum samples as per the FDA guidance CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101/2020.12.10.20244350 doi: medRxiv preprint 7 A 96-well flat-bottom immunoplate (Extra Gene, USA) was coated with 100 µl/well of SARS-124 CoV-2 specific recombinant nucleoprotein (0.125 µg/well) in coating buffer (sodium bi-carbonate, 125 pH >9) and incubated either at 37 °C for an hour or overnight at 4 °C. The unbound antigen was 126 then decanted followed by blocking with 100 µl/well of blocking buffer (PBS, 0.1% Tween-20, 127 2% BSA) and incubated at 37 °C for an hour. Following incubation wells were washed three times 128 with ELISA wash buffer (50 mm Tris, 0.05% Tween 20, 0.1% SDS, 0.8% NaCl, distilled water) 129 and used for the assay. 100l of test serum at 1:100 dilution in diluent buffer (PBS, 0.1% Tween20, 1% BSA) was added 132 into each well and incubated at 37 °C for 15 minutes. One positive, two negative and two plate 133 controls (no serum was added) were added in each plate. After incubation, the contents of the 134 wells were aspirated and the plate was washed 5 times using ELISA wash buffer. 100µl of 135 optimized goat anti-human IgG conjugated with HRP was added to each well and then incubated 136 for 10 minutes at 37 °C. Following incubation, the plate was washed 5 times and 100l TMB was 137 added into each well and incubated for 5 minutes at room temperature. Further colour 138 development, 100l 1.5M Sulfuric Acid (H2SO4) was used as stop solution and the optical density 139 (OD) was measured at 450 nm using a Multiplex micro plate ELISA reader. CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101/2020.12.10.20244350 doi: medRxiv preprint 8 that showed the best signal to noise ratio (S/N) with acceptable background has been selected. The . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101/2020.12.10.20244350 doi: medRxiv preprint Statistical analysis 170 Panel 1 and 2 were used to determine sensitivity and panel 3 for assessing specificity and cross 171 reactivity. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value 172 (NPV) and area under curve (AOC) with 95% confidence interval were estimated to see the 173 effectiveness of this ELISA assay. Cohen's Kappa test was used to evaluated the test agreement. 

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The copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101/2020.12.10.20244350 doi: medRxiv preprint As mentioned before, the intra-assay and inter-assay variation have been evaluated, with 05 198 replicates of positive and negative controls on the same day in a plate for the former, and testing 199 the controls in 15 different days for the later. Coefficient of variation (CV) depicts reproducibility 200 and precision and analysis showed CV was <25% for the controls used in inter assay and <10% 201 for intra assay (Table 2) . (Table 3 and Fig 1A) .

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The copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101/2020.12.10.20244350 doi: medRxiv preprint 13 approved commercial antibody immunoassay (Table 5 ). The test agreement between Elecsys and 239 in-house ELISA was 100%. . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

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On the context of the current pandemic situation, several serological tests based on either 247 chemiluminescence, lateral flow, neutralization or immunosorbent assay have been developed and 248 approved by FDA for emergency use (19, 22, 23) . Eleven of these 58 serological tests are mounted 249 on the enzyme linked immunosorbent assay targeting either IgM, IgG or total antibody (19).

The biologics, biosimilars, and bio-diagnostics developed by different biopharmaceuticals and in-house assay kits to meet the demand are essential to manage the wreckage. The emerging 261 condition underpins our endeavour to develop an indigenous IgG-ELISA specific to 262 an approach to creating an opportunity to satisfy national and global demand.

This assay is mounted upon nucleocapsid as an antigen which provides increased sensitivity 264 compared to either Spike-1 (S1) or Receptor Binding Domain (RBD) for detecting early phase of 265 infection due to its primordial inception (21, 33, 34) . High expression of NCP protein of 266 coronaviruses has already been reported during infection (35), which is not only B-cell immunogen 267 but also evoke cellular immune response in SARS infected patients (36, 37) . Also, spike (S) gene 268 . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101 https://doi.org/10. /2020 of SARS-CoV-2 has 76% similarities with that of SARS-CoV-1, which exhibits non-synonymous 269 mutations as the disease evolves over time (38) (39) (40) . On the other hand, the nucleocapsid protein is 270 more conserved having 90% amino acid homology with SARS-CoV-1 (39) which affected South-271 East Asian countries comparatively to a lesser extent (41).

For tropical and sub-tropical dengue-endemic countries (42) developing serological tests specific 273 to COVID-19 is quite challenging, for serological and symptomatological overlap between the 274 diseases in question (43, 44) . Misdiagnosis of COVID-19 as dengue due to serological cross-275 reactivity has already been reported in Indonesia (45, 46) . Singapore (47) and Thailand (48) and 276 vice-versa for rapid COVID-19 antibody kits in India (49). To address the concern, our assay is 

Diagnostic settings, handling a surge of samples, require a time-saving, convenient test method.

The strength of our indigenous system lies here, assay time being optimized at total 30 minutes, 288 while currently available ELISA kits such as "Euroimmun Anti-SARS-CoV-2 ELISA (IgG)" or 289 "BioRad Platelia SARS-CoV-2 Total Ab" require total of 120 minutes of incubation to perform 290 their assay (19) . Spectrum bias, which may affect sensitivity calculation, is circumvented by 291 . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101/2020.12.10.20244350 doi: medRxiv preprint 16 longitudinal antibody analysis of individuals whose sera have been exploited as positive controls.

(51). Also the positive panels comprises of multiple samples from three patients who exhibited 293 higher antibody titer for a long period which is mentioned in a previous study (52).

Certain limitations in our assay development exist that are to be addressed. Firstly, for cross 295 reactivity test, no known respiratory sample was assessed, and secondly, the cohort sample size 296 was actually inadequate to draw conclusions on samples collected within 0-14 days of symptom 297 onset.

In conclusion, this in-house ELISA demonstrates its usefulness for the early detection as well as 299 for serosourveillence of SARS-CoV-2 IgG that developed against nucleocapsid proteins. This 300 assay is easy to perform, cost-effective and results can be interpreted at 30 minutes of test run.

Moreover, this test showed comparable level of performance against commercial FDA approved 302 electrochemiluminescence immunoassay and detected IgG from SARS-CoV-2 infected patients.

Hence, this SARS-CoV-2 NCP-IgG Rapid ELISA could be equally applied as one of the COVID-304 19 early sero-monitoring tools all over the COVID-19 pandemic countries. The authors express their thanks all the participants who were willing participate in the study. They 308 also thank their team members for their support work during the current pandemic situation. Md. Ahsanul H. performed the data curation and statistical analysis. Mohib Ullah K., Eiry 314 . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

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The copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101 https://doi.org/10. /2020 

Natural history of COVID-19 and current knowledge on treatment 331 therapeutic options

SARS-CoV-2: an Emerging Coronavirus that Causes a Global Threat

Severe Acute Respiratory Syndrome 335

Coronavirus-2 (SARS-CoV-2): An Update

Prevention and treatment of the common cold: making sense of the 337 evidence

COVID-19 for the 339 cardiologist: a current review of the virology, clinical epidemiology, cardiac and other clinical 340 manifestations and potential therapeutic strategies. JACC: Basic to Translational Science

Information about the new coronavirus disease (COVID-19). Radiol Bras

COVID-19) current status and future perspectives: a narrative review. 345 International journal of environmental research and public health

COVID-19 diagnostic approaches: different 11

An ultrasensitive, rapid, and portable coronavirus 356 SARS-CoV-2 sequence detection method based on CRISPR-Cas12

Loop-mediated 358 isothermal amplification (Lamp): A rapid, sensitive, specific, and cost-effective point-of-care test 359 for coronaviruses in the context of covid-19 pandemic

Laboratory testing for coronavirus disease ( COVID-19) in suspected 363 human cases: interim guidance

Effectiveness of convalescent plasma 365 therapy in severe COVID-19 patients

COVID-19 Serosurveillance May Facilitate Return-368 to-Work Decisions. The American journal of tropical medicine and hygiene

Children are not COVID-19 super spreaders: time to go back to 41

Disease surveillance using online news: dengue and 439

Zika in tropical countries

Coronavirus disease of 441 2019: a mimicker of dengue infection? SN Comprehensive Clinical Medicine

Covid-19 and dengue

Double punches for dengue-endemic countries in Asia. Reviews in medical virology

Serological cross-reaction and co-infection of dengue and COVID-19 in Asia

Kembuan GJ. Dengue serology in Indonesian COVID-19 patients: Coinfection or 448 serological overlap? IDCases

Covert COVID-19 and false-450 positive dengue serology in Singapore. The Lancet Infectious Diseases

Nurse Infected with Covid-19 from a Provisional Dengue Patient

Dengue antibodies can cross-react 455 with SARS-CoV-2 and vice versa-Antibody detection kits can give false-positive results for both 456 viruses in regions where both COVID-19 and Dengue

Antibody responses to

roads to the same destination. VirusDisease. 2020:1-9. 

. CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprintThe copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101 school. Archives of disease in childhood. 2020.

. CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprintThe copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101 

. CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprintThe copyright holder for this this version posted December 11, 2020. 

. CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprintThe copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101 https://doi.org/10. /2020 23 458 SARS-CoV-2 in patients with COVID-19. Nature medicine. 2020:1-4.

. CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprintThe copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101 . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprintThe copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101 https://doi.org/10. /2020