Generation and utility of a single-chain fragment variable monoclonal antibody platform against a baculovirus expressed recombinant receptor binding domain of SARS-CoV-2 spike protein.
Mol Immunol
; 141: 287-296, 2022 01.
Article
in English
| MEDLINE | ID: covidwho-1559780
ABSTRACT
As the second wave of COVID-19 launched, various variants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have emerged with a dramatic global spread amongst millions of people causing unprecedented case fatalities and economic shut-downs. That initiated a necessity for developing specific diagnostics and therapeutics along with vaccines to control such a pandemic. This endeavor describes generation of murine derived recombinant single-chain fragment variable (scFv) as a monoclonal antibody (MAb) platform targeting the receptor binding domain (RBD) of Spike protein of SARS-CoV-2. A specific synthesized RBD coding sequence was cloned and expressed in Baculovirus expression system. The recombinant RBD (rRBD) was ascertained to be at the proper encoding size of â¼ 600bp and expressed protein of the molecular weight of â¼ 21KDa. Purified rRBD was proved genuinely antigenic and immunogenic, exhibiting specific reactivity to anti-SARS-CoV-2 antibody in an indirect enzyme-linked immunosorbent assay (ELISA), and inducing strong seroconversion in immunized mice. The scFv phage display library against rRBD was successfully constructed, revealing â¼ 90 % recombination frequency, and great enriching factor reaching 88 % and 25 % in polyclonal Ab-based and MAb-based ELISAs, respectively. Typically, three unique scFvs were generated, selected, purified and molecularly identified. That was manifested by their accurate structure, close relation to the mouse immunoglobulin (Ig) superfamily, right anchored six complementarily-determining regions (CDRs) as three within variable heavy (vH) and variable light (vL) regions each, and proper configuration of the three-dimensional (3D) structure. Besides, their expression downstream in a non-suppressive amber codon of E. coli strain SS32 created a distinct protein band at an apparent molecular weight of â¼ 27KDa. Moreover, the purified scFvs showed authentic immunoreactivity and specificity to both rRBD and SARS-CoV-2 in western blot and ELISA. Accordingly, these developed scFvs platform might be a functional candidate for research, inexpensive diagnostics and therapeutics, mitigating spread of COVID-19.
Keywords
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
Receptors, Virus
/
Single-Chain Antibodies
/
Cell Surface Display Techniques
/
Spike Glycoprotein, Coronavirus
/
Angiotensin-Converting Enzyme 2
/
COVID-19 Serological Testing
/
SARS-CoV-2
/
COVID-19
/
Antibodies, Monoclonal
/
Antibodies, Viral
Type of study:
Diagnostic study
/
Observational study
/
Randomized controlled trials
Topics:
Vaccines
/
Variants
Limits:
Animals
Language:
English
Journal:
Mol Immunol
Year:
2022
Document Type:
Article
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