tetano
Editor, Senior Moderator
Clin Chim Acta
. 2023 Mar 3;117279.
doi: 10.1016/j.cca.2023.117279. Online ahead of print.
Designing and developing a sensitive and specific SARS-CoV-2 RBD IgG detection kit for identifying positive human samples
Ehsan Raoufi[SUP] 1 [/SUP], Fatemeh Hosseini[SUP] 2 [/SUP], Bahman Onagh[SUP] 3 [/SUP], Mohammad Salehi-Shadkami[SUP] 4 [/SUP], Marjan Mehrali[SUP] 4 [/SUP], Monireh Mohsenzadegan[SUP] 5 [/SUP], Jim Q Ho[SUP] 6 [/SUP], Banafsheh Bigdelou[SUP] 7 [/SUP], Mohammad Reza Sepand[SUP] 7 [/SUP], Thomas J Webster[SUP] 8 [/SUP], Steven Zanganeh[SUP] 7 [/SUP], Mohammad M Farajollahi[SUP] 9 [/SUP]
Affiliations
Abstract
Background: More than 3 y into the coronavirus 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to undergo mutations. In this context, the Receptor Binding Domain (RBD) is the most antigenic region among the SARS-CoV-2 Spike protein and has emerged as a promising candidate for immunological development. We designed an IgG-based indirect enzyme-linked immunoassay (ELISA) kit based on recombinant RBD, which was produced from the laboratory to 10 liter industry scales in Pichia pastoris.
Methods: A recombinant-RBD comprising 283 residues (31kDa) was constructed after epitope analyses. The target gene was initially cloned into an Escherichia coli TOP10 genotype and transformed into Pichia pastoris CBS7435 muts for protein production. Production was scaled up in a 10 liter fermenter after a 1 liter shake-flask cultivation. The product was ultrafiltered and purified using ion-exchange chromatography. IgG-positive human sera for SARS-CoV-2 were employed by an ELISA test to evaluate the antigenicity and specific binding of the produced protein.
Results: Bioreactor cultivation yielded 4 g/l of the target protein after 160 h of fermentation, and ion-exchange chromatography indicated a purity > 95%. A human serum ELISA test was performed in 4 parts, and the ROC area under the curve (AUC) was > 0.96 for each part. The mean specificity and sensitivity of each part was 100% and 91.5%, respectively.
Conclusion: A highly specific and sensitive IgG-based serologic kit was developed for improved diagnostic purposes in patients with COVID-19 after generating an RBD antigen in Pichia pastoris at laboratory and 10 liter fermentation scales.
Keywords: ELISA; Pichia pastoris; Receptor-binding domain; SARS-CoV-2; bioengineering; scale-up fermentation.
. 2023 Mar 3;117279.
doi: 10.1016/j.cca.2023.117279. Online ahead of print.
Designing and developing a sensitive and specific SARS-CoV-2 RBD IgG detection kit for identifying positive human samples
Ehsan Raoufi[SUP] 1 [/SUP], Fatemeh Hosseini[SUP] 2 [/SUP], Bahman Onagh[SUP] 3 [/SUP], Mohammad Salehi-Shadkami[SUP] 4 [/SUP], Marjan Mehrali[SUP] 4 [/SUP], Monireh Mohsenzadegan[SUP] 5 [/SUP], Jim Q Ho[SUP] 6 [/SUP], Banafsheh Bigdelou[SUP] 7 [/SUP], Mohammad Reza Sepand[SUP] 7 [/SUP], Thomas J Webster[SUP] 8 [/SUP], Steven Zanganeh[SUP] 7 [/SUP], Mohammad M Farajollahi[SUP] 9 [/SUP]
Affiliations
- PMID: 36871661
- DOI: 10.1016/j.cca.2023.117279
Abstract
Background: More than 3 y into the coronavirus 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to undergo mutations. In this context, the Receptor Binding Domain (RBD) is the most antigenic region among the SARS-CoV-2 Spike protein and has emerged as a promising candidate for immunological development. We designed an IgG-based indirect enzyme-linked immunoassay (ELISA) kit based on recombinant RBD, which was produced from the laboratory to 10 liter industry scales in Pichia pastoris.
Methods: A recombinant-RBD comprising 283 residues (31kDa) was constructed after epitope analyses. The target gene was initially cloned into an Escherichia coli TOP10 genotype and transformed into Pichia pastoris CBS7435 muts for protein production. Production was scaled up in a 10 liter fermenter after a 1 liter shake-flask cultivation. The product was ultrafiltered and purified using ion-exchange chromatography. IgG-positive human sera for SARS-CoV-2 were employed by an ELISA test to evaluate the antigenicity and specific binding of the produced protein.
Results: Bioreactor cultivation yielded 4 g/l of the target protein after 160 h of fermentation, and ion-exchange chromatography indicated a purity > 95%. A human serum ELISA test was performed in 4 parts, and the ROC area under the curve (AUC) was > 0.96 for each part. The mean specificity and sensitivity of each part was 100% and 91.5%, respectively.
Conclusion: A highly specific and sensitive IgG-based serologic kit was developed for improved diagnostic purposes in patients with COVID-19 after generating an RBD antigen in Pichia pastoris at laboratory and 10 liter fermentation scales.
Keywords: ELISA; Pichia pastoris; Receptor-binding domain; SARS-CoV-2; bioengineering; scale-up fermentation.