tetano
Editor, Senior Moderator
Int J Nanomedicine
. 2021 Jul 9;16:4739-4753.
doi: 10.2147/IJN.S313140. eCollection 2021.
AuNP Coupled Rapid Flow-Through Dot-Blot Immuno-Assay for Enhanced Detection of SARS-CoV-2 Specific Nucleocapsid and Receptor Binding Domain IgG
Bijon Kumar Sil[SUP] 1 [/SUP], Mohd Raeed Jamiruddin[SUP] 2 [/SUP], Md Ahsanul Haq[SUP] 1 [/SUP], Mohib Ullah Khondoker[SUP] 3 [/SUP], Nowshin Jahan[SUP] 1 [/SUP], Shahad Saif Khandker[SUP] 1 [/SUP], Tamanna Ali[SUP] 1 [/SUP], Mumtarin Jannat Oishee[SUP] 1 [/SUP], Taku Kaitsuka[SUP] 4 [/SUP], Masayasu Mie[SUP] 5 [/SUP], Kazuhito Tomizawa[SUP] 6 [/SUP], Eiry Kobatake[SUP] 5 [/SUP], Mainul Haque[SUP] 7 [/SUP], Nihad Adnan[SUP] 8 [/SUP]
Affiliations
Abstract
Background: Serological tests detecting severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are widely used in seroprevalence studies and evaluating the efficacy of the vaccination program. Some of the widely used serological testing techniques are enzyme-linked immune-sorbent assay (ELISA), chemiluminescence immunoassay (CLIA), and lateral flow immunoassay (LFIA). However, these tests are plagued with low sensitivity or specificity, time-consuming, labor-intensive, and expensive. We developed a serological test implementing flow-through dot-blot assay (FT-DBA) for SARS-CoV-2 specific IgG detection, which provides enhanced sensitivity and specificity while being quick to perform and easy to use.
Methods: SARS-CoV-2 antigens were immobilized on nitrocellulose membrane to capture human IgG, which was then detected with anti-human IgG conjugated gold nanoparticle (hIgG-AuNP). A total of 181 samples were analyzed in-house. Within which 35 were further evaluated in US FDA-approved CLIA Elecsys SARS-CoV-2 assay. The positive panel consisted of RT-qPCR positive samples from patients with both <14 days and >14 days from the onset of clinical symptoms. The negative panel contained samples collected from the pre-pandemic era dengue patients and healthy donors during the pandemic. Moreover, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of FT-DBA were evaluated against RT-qPCR positive sera. However, the overall efficacies were assessed with sera that seroconverted against either nucleocapsid (NCP) or receptor-binding domain (RBD).
Results: In-house ELISA selected a total of 81 true seropositive and 100 seronegative samples. The sensitivity of samples with <14 days using FT-DBA was 94.7%, increasing to 100% for samples >14 days. The overall detection sensitivity and specificity were 98.8% and 98%, respectively, whereas the overall PPV and NPV were 99.6% and 99%. Moreover, comparative analysis between in-house ELISA assays and FT-DBA revealed clinical agreement of Cohen's Kappa value of 0.944. The FT-DBA showed sensitivity and specificity of 100% when compared with commercial CLIA kits.
Conclusion: The assay can confirm past SARS-CoV-2 infection with high accuracy within 2 minutes compared to commercial CLIA or in-house ELISA. It can help track SARS-CoV-2 disease progression, population screening, and vaccination response. The ease of use of the assay without requiring any instruments while being semi-quantitative provides the avenue of its implementation in remote areas around the globe, where conventional serodiagnosis is not feasible.
Keywords: AuNP; COVID-19; SARS-CoV-2; dot-blot immunoassay; gold nanoparticle; nucleocapsid; receptor binding domain; serosurveillance.
. 2021 Jul 9;16:4739-4753.
doi: 10.2147/IJN.S313140. eCollection 2021.
AuNP Coupled Rapid Flow-Through Dot-Blot Immuno-Assay for Enhanced Detection of SARS-CoV-2 Specific Nucleocapsid and Receptor Binding Domain IgG
Bijon Kumar Sil[SUP] 1 [/SUP], Mohd Raeed Jamiruddin[SUP] 2 [/SUP], Md Ahsanul Haq[SUP] 1 [/SUP], Mohib Ullah Khondoker[SUP] 3 [/SUP], Nowshin Jahan[SUP] 1 [/SUP], Shahad Saif Khandker[SUP] 1 [/SUP], Tamanna Ali[SUP] 1 [/SUP], Mumtarin Jannat Oishee[SUP] 1 [/SUP], Taku Kaitsuka[SUP] 4 [/SUP], Masayasu Mie[SUP] 5 [/SUP], Kazuhito Tomizawa[SUP] 6 [/SUP], Eiry Kobatake[SUP] 5 [/SUP], Mainul Haque[SUP] 7 [/SUP], Nihad Adnan[SUP] 8 [/SUP]
Affiliations
- PMID: 34267520
- PMCID: PMC8277418
- DOI: 10.2147/IJN.S313140
Abstract
Background: Serological tests detecting severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are widely used in seroprevalence studies and evaluating the efficacy of the vaccination program. Some of the widely used serological testing techniques are enzyme-linked immune-sorbent assay (ELISA), chemiluminescence immunoassay (CLIA), and lateral flow immunoassay (LFIA). However, these tests are plagued with low sensitivity or specificity, time-consuming, labor-intensive, and expensive. We developed a serological test implementing flow-through dot-blot assay (FT-DBA) for SARS-CoV-2 specific IgG detection, which provides enhanced sensitivity and specificity while being quick to perform and easy to use.
Methods: SARS-CoV-2 antigens were immobilized on nitrocellulose membrane to capture human IgG, which was then detected with anti-human IgG conjugated gold nanoparticle (hIgG-AuNP). A total of 181 samples were analyzed in-house. Within which 35 were further evaluated in US FDA-approved CLIA Elecsys SARS-CoV-2 assay. The positive panel consisted of RT-qPCR positive samples from patients with both <14 days and >14 days from the onset of clinical symptoms. The negative panel contained samples collected from the pre-pandemic era dengue patients and healthy donors during the pandemic. Moreover, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of FT-DBA were evaluated against RT-qPCR positive sera. However, the overall efficacies were assessed with sera that seroconverted against either nucleocapsid (NCP) or receptor-binding domain (RBD).
Results: In-house ELISA selected a total of 81 true seropositive and 100 seronegative samples. The sensitivity of samples with <14 days using FT-DBA was 94.7%, increasing to 100% for samples >14 days. The overall detection sensitivity and specificity were 98.8% and 98%, respectively, whereas the overall PPV and NPV were 99.6% and 99%. Moreover, comparative analysis between in-house ELISA assays and FT-DBA revealed clinical agreement of Cohen's Kappa value of 0.944. The FT-DBA showed sensitivity and specificity of 100% when compared with commercial CLIA kits.
Conclusion: The assay can confirm past SARS-CoV-2 infection with high accuracy within 2 minutes compared to commercial CLIA or in-house ELISA. It can help track SARS-CoV-2 disease progression, population screening, and vaccination response. The ease of use of the assay without requiring any instruments while being semi-quantitative provides the avenue of its implementation in remote areas around the globe, where conventional serodiagnosis is not feasible.
Keywords: AuNP; COVID-19; SARS-CoV-2; dot-blot immunoassay; gold nanoparticle; nucleocapsid; receptor binding domain; serosurveillance.