A rapid novel strategy for screening of antibody phage libraries for production, purification, and functional characterization of amber stop codons containing single‐chain antibody fragments

Perween, Reshma; Ahmed, Shubbir; Shrivastava, Tripti; Parray, Hilal Ahmad; Singh, Balwant; Pindari, Kamal S; Sharma, Chandresh; Shukla, Shivangi; Sinha, Subrata; Panchal, Anil Kumar; Kumar, Rajesh

doi:10.1002/btpr.3136

Cited by 11 publications

(8 citation statements)

References 15 publications

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“…Tomlinson I + J libraries are widely used, based on scFv format with diversity of 18 amino acids incorporated in CDRs 2 and 3 in positions involved in antigen binding, and are highly diverse in the natural repertoire. Many studies used this library, and their antibody showed high specificity and binding affinity to the targets. − …”

Section: Discussionmentioning

confidence: 99%

Selection and Characterization of a Single-Chain Variable Fragment against Porcine Circovirus Type 2 Capsid and Impedimetric Immunosensor Development

et al. 2021

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Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated disease (PCVAD) that causes huge global economic losses for the swine industry. Effective strategies or rapid detection of PCV2 in pig are essential to control PCVAD. Here, single-chain variable fragments (scFvs) were selected and characterized against the PCV2 capsid using phage display technology. Phage scFv clones were selected from the human scFv phagemid library (Tomlinson I + J) for direct panning against the PCV2 capsid. Eighty-four monoclonal phage scFvs were individually tested for binding to the PCV2 capsid by ELISA. Eight scFv clones showed significant binding to the PCV2 capsid and only three clones (clone nos. 13, 37, and 81) contained both VHCDRs and VLCDRs in the sequence. Clone scFv no. 81 had the highest reactivity to the PCV2 capsid and was constructed in the pET22b (+) expression vector. The recombinant was transformed to Escherichia coli BL21(DE3) for expression and purification. The scFv showed appropriate affinity to the PCV2 capsid by western blot analysis. Kinetics of scFv and the PCV2 capsid were determined using surface plasmon resonance and showed binding affinity in the nanomolar range (K D = 57.2 nM). Our scFv was first applied in the development of an impedimetric immunosensor for PCV2 capsid detection, and results showed that impedance increased with increasing PCV2 capsid expression with limit of detection = 114 nM. Findings demonstrated that our scFv has potential for use as a receptor for biosensor devices.

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Section: Discussionmentioning

confidence: 99%

Selection and Characterization of a Single-Chain Variable Fragment against Porcine Circovirus Type 2 Capsid and Impedimetric Immunosensor Development

et al. 2021

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“…The cells were transiently transfected with the expressing plasmids. The supernatant was collected after 5–6 days, and the soluble protein was purified using Ni-NTA affinity chromatography with Ni 2+ ions immobilised on a resin by covalent attachment to nitrilotriacetic acid (NTA) (QIAGEN, Germany), as described (Parray et al 2020 ; Perween et al 2021 ).…”

Section: Methodsmentioning

confidence: 99%

Characterization of a broadly cross reactive tetravalent human monoclonal antibody, recognizing conformational epitopes in receptor binding domain of SARS-CoV-2

et al. 2022

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We used human semi-synthetic phage antibody gene libraries to select anti-SARS-CoV-2 RBD scFv antibody fragment and subsequent characterization of this novel tetravalent monoclonal antibody targeting conformational epitopes in the receptor binding domain of SARS-CoV-2. Binding studies suggest that II62 tetravalent antibody cross-reacts with RBD protein of SARS-CoV2 and its different variants of concerns. The epitope mapping data reveals that II62 tetravalent antibody targets an epitope that does not directly interferes with RBD: ACE2 interaction. Neutralization studies with live authentic SARS-CoV2 virus suggests that increase in valency of II62 mAb from monovalent to tetravalent doesn’t perturbate virus interactions with the ACE2 expressing host cells in cytopathic effect-based (CPE) assay. Supplementary Information The online version contains supplementary material available at 10.1007/s13205-022-03272-6.

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“…Recombinant proteins were expressed using the Expi 293 F expression system, from a codon-optimized nucleic sequence of RBD-His, Spike-His, HA-His, following the methodology published earlier [14] , [15] , [16] . Briefly, the culture supernatant was harvested 5-7 days post-transfection and purified by Ni-NTA affinity chromatography followed by dialysis in phosphate buffer saline (pH 7.4) as described in our previous papers [14] , [15] , [16] , [17] . The N protein of SARS-CoV-2 was expressed in the bacterial expression system and was purified by Ni-NTA affinity chromatography.…”

Section: Methodsmentioning

confidence: 99%

Non-neutralizing SARS CoV-2 directed polyclonal antibodies demonstrate cross-reactivity with the HA glycans of influenza virus

Murugavelu

Perween

Shrivastava

et al. 2021

International Immunopharmacology

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The spike protein of the SARS-CoV-2 virus is the foremost target for the designing of vaccines and therapeutic antibodies and also acts as a crucial antigen in the assessment of COVID-19 immune responses. The enveloped viruses; such as SARS-CoV-2, Human Immunodeficiency Virus-1 (HIV-1) and influenza, often hijack host-cell glycosylation pathways and influence pathobiology and immune selection. These glycan motifs can lead to either immune evasion or viral neutralization by the production of cross-reactive antibodies that can lead to antibody-dependent enhancement (ADE) of infection. Potential cross-protection from influenza vaccine has also been reported in COVID-19 infected individuals in several epidemiological studies recently; however, the scientific basis for these observations remains elusive. Herein, we show that the anti-SARS-CoV2 antibody cross-reacts with the Hemagglutinin (HA) protein. This phenomenon is common to both the sera from convalescent SARS-CoV-2 donors and spike immunized mice, although these antibodies were unable to cross-neutralize, suggesting the presence of a non-neutralizing antibody response. Epitope mapping suggests that the cross-reactive antibodies are targeted towards glycan epitopes of the SARS-CoV-2 spike and HA. Overall, our findings address the cross-reactive responses, although non-neutralizing, elicited against RNA viruses and warrant further studies to investigate whether such non-neutralizing antibody responses can contribute to effector functions such as antibody-dependent cellular cytotoxicity (ADCC) or ADE.

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A rapid novel strategy for screening of antibody phage libraries for production, purification, and functional characterization of amber stop codons containing single‐chain antibody fragments

Cited by 11 publications

References 15 publications

Selection and Characterization of a Single-Chain Variable Fragment against Porcine Circovirus Type 2 Capsid and Impedimetric Immunosensor Development

Selection and Characterization of a Single-Chain Variable Fragment against Porcine Circovirus Type 2 Capsid and Impedimetric Immunosensor Development

Characterization of a broadly cross reactive tetravalent human monoclonal antibody, recognizing conformational epitopes in receptor binding domain of SARS-CoV-2

Non-neutralizing SARS CoV-2 directed polyclonal antibodies demonstrate cross-reactivity with the HA glycans of influenza virus

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